The animals were housed in individual stainless steel cages with

The animals were housed in individual stainless steel cages with free access to a standard sodium diet (Guabi Rat Chow, Paulinia, SP, Brazil), water and 0.3 M NaCl solution. The positions of the bottles containing water and 0.3 M NaCl were rotated daily to avoid place preference. Rats were maintained in a room whose temperature was controlled at 23 ± 2 °C and humidity at in a 12-h light/dark cycle with lights on 7:30 a.m. The animals were randomly divided into two groups: the control group (CN) and the periodontal disease group (PD). Under general anaesthesia (a mixture of ketamine (80 mg/kg of

body weight (b.w.), Cristália, Brazil) and xylazine (7 mg/kg of b.w., Agener, Brazil)) injected subcutaneously, a sterile silk Roscovitine cost ligature (strength 4/0) was tied in the cervical region of the mandibular first molars teeth bilaterally in the PD group using a technique that was previously described.9 The ligatures served UK-371804 cost as a retention device for oral micro-organisms. Ingestion of 0.3 M NaCl and water (ml/24 h) was measured 3 and 16 days after experimental ligature-induced periodontal disease in order to verify the systemic conditions of the animals. On the 15th day after ligature placement, control rats (without ligature) and rats with PD were anaesthetised with i.p. injection of ketamine (80 mg/kg of b.w.) combined with xylazine (7 mg/kg of b.w.)

and placed in a stereotaxic instrument (Kopf, Tujunga, CA, USA). The skull was levelled between bregma and lambda. Stainless steel guide-cannulas (12 mm × 0.6 mm outer diameter (o.d.)) were implanted bilaterally into the LPBN using the following coordinates: 9.2 mm caudal to bregma, 2.2 mm lateral to the midline and 3.8 mm below the dura mater. The tips of the cannulas were positioned 2 mm above each LPBN. The cannulas were fixed to the cranium using dental acrylic resin and jeweller screws and were filled with 30-gauge metal obturators

between tests. After the surgery, only control rats received a prophylactic dose of the antibiotic penicillin (30,000 IU). All animals were allowed to recover for 5 days before starting ingestion tests and during this period they had free access to standard sodium diet, water and 0.3 M NaCl solution. Tryptophan synthase Bilateral injections into the LPBN were made using 5-μl Hamilton syringes connected to 30-gauge injection cannulas by means of polyethylene tubing (PE-10). At the time of testing, the obturators were removed and the injection cannula (2 mm longer than the guide cannula) was carefully inserted into the guide cannula. For bilateral injections, the first injection was performed on one side, the needle was removed and repositioned on the contralateral side and then the second injection was given. Therefore, injections were given ∼1 min apart. The injection volume into the LPBN was 0.2 μl on each site. The obturators were replaced after the injections, and the rats were put back into their cages.

Eight hours after injection, severity of mucus secretion, loss of

Eight hours after injection, severity of mucus secretion, loss of turgor, matting of spines, and tissue necrosis ranged from low to medium. There was an increase in severity of these signs after 24 h. Even at 0.25× the standard concentration, severity of

mucus secretion, loss of turgor, matting of spines, selleck and tissue necrosis ranged from medium to severe after 24 h and resulted in 80% mortality. All sea stars were dead 48 h after injection. There was 0% mortality at the TCBS standard concentration (5 g l−1) and also when this concentration was doubled. Disease signs were not exhibited except for localized swelling and tissue necrosis at the site of injection. Twelve days after exposure to A. planci injected with oxgall (8 g l−1, 4 g l−1), peptone (20 g l−1), and TCBS (44 g l−1), none of the fish, corals, and mobile invertebrates exhibited any signs of disease. No signs of bacterial disease such as cloudy eyes, fin rot, pop eyes and changes in skin color were observed in any of the fish tested. There were also no spots, bands, or discoloration observed in corals that were constantly in contact with floating A. planci particles in the water. It is important

to mention that corals anti-EGFR antibody were not attacked by A. planci and there was minimal movement of the starfish one hour after injection with oxbile. Mobile invertebrates remained active each night and there was no loss of spines observed in sea urchins and no lesions in sea stars and sea cucumbers ( Fig. 3). Bile derivatives (i.e. oxgall, bile salts) have consistently resulted in high mortality rates in previous studies (Rivera-Posada

et al., 2012) and in this study. Bile salts are added in media culture formulations to inhibit the growth of gram-positive bacteria and isolate resistant strains. Bile is a natural digestive enzyme produced by all vertebrates to aid in the digestion of lipophilic nutrients. In addition, bile is an important route of elimination of environmental toxins, carcinogens, hormones, drugs and their metabolites and may control the growth of bacteria in the small intestine (Nathanson and Boyer, 1991). Two well-known mechanisms of cell death are triggered by bile acids: necrosis at higher concentrations and apoptosis at lower concentrations (Palmeira and Rolo, 2004 and Rolo et al., 2004). Several Histone demethylase studies indicate that impairment of mitochondrial oxidative phosphorylation is an early and critical event in the mechanism of bile acid cytotoxicity. Apoptosis induction is dependent on the bile acid, its concentration, or its conjugation state. Toxic bile acid-induced apoptosis involves both extrinsic (death receptor-mediated apoptosis) and intrinsic (direct targeting to mitochondria) apoptotic pathways. Bile acids induce alterations in membrane fluidity associated with impairment of mitochondrial respiration and mitochondrial depolarization.

2 × 104 M−1 cm−1 ( Murphy and Kehrer, 1989) The total protein co

2 × 104 M−1 cm−1 ( Murphy and Kehrer, 1989). The total protein content of lymphocytes was measured by the method of Bradford (Bradford, 1976), using BSA as standard. All data are expressed as mean values and standard errors of at least three independent experiments. Data were analyzed by one-way ANOVA followed by the Tukey’s post hoc test. The software employed for statistical

analyses was GraphPad Prism (version4; GraphPad Software, San Diego, CA, USA). The Selleckchem HSP inhibitor functional activity of lymphocytes was assayed by their capacity to proliferate in response to a specific stimulation. Fig. 1 shows the MTT assay results after stimulation with Con A (a T lymphocytes mitogen) or LPS (a B lymphocytes mitogen)

for 48 h. FA at 0.3 mM ERK inhibitor in vitro increased both basal (without stimulation) and LPS-stimulated proliferative capacity of human lymphocytes by 38% and 30%, respectively as compared with non stimulated control group. The addition of astaxanthin to cells treated with FA caused a decrease in the proliferation of lymphocytes in basal, Con A and LPS-stimulated conditions by 43%, 26% and 30%, respectively as compared with 0.3 mM of FA mixture. Intracellular Ca2+ mobilization was significantly enhanced by the mixture of FA in human lymphocytes (about 31-fold) when compared to the control group (Fig. 2). The increase in Ca2+ levels was sustained during 20 min of kinetic monitoring. Treatment with ASTA was unable to prevent the calcium increase induced by FA. BSA (0.2%) addition was able to partially decrease calcium mobilization probably by chelating free FA. To measure intracellular superoxide anion, hydrogen peroxide and nitric oxide production, cells were acutely treated with the FA mixture with or without ASTA as indicated in the material and methods section. As shown in Fig. 3A, the treatment of human lymphocytes with the FA mixture increased the intracellular superoxide

anion levels by 135% as compared with the PMA-control group and as assessed by using Amine dehydrogenase DHE probe. The addition of ASTA to FA-treated cells promoted a reduction of 20% in superoxide production. Treatment of PMA-control cells with DPI, a NADPH-oxidase inhibitor, totally inhibited superoxide anion production, whereas sodium azide (SA) partially inhibited superoxide anion production. DPI addition in cells treated with fatty acid mixture partially decreased (20%) the superoxide anion production (Fig. 3A). A similar pattern was observed when DCFH-DA probe was used as a general ROS probe (Fig. 3B). An increase of threefold in total ROS production was observed in lymphocytes treated with the FA mixture as compared with PMA-control group. ASTA-treatment decreased the ROS production induced by FA in 20%. Addition of BSA, used as a FA chelating agent, reduced the ROS production in about 32%.

To successfully select those residues in the active site, a theor

To successfully select those residues in the active site, a theoretical model of RgPAL was constructed through homology modeling using RtPAL (PDB ID: 1T6J) as the template. As shown in Fig. 2, all of the residues that were

the superimposed with RtPAL showed an RMSD of 0.224 Å ( Fig. 2A), and the Ramachandran plot suggests that 94.9%, 3.2%, and 1.9% of the residues in derived model are in acceptable region, marginal Epigenetic activity region and disallowed region, respectively ( Fig. 2B), These finding indicated that the model is reasonable and could be used in further molecular docking simulation. Using the AutoDock global–local evolutionary algorithm, we searched for those sites with the lowest free energy of binding between the ligand and the enzyme. As shown in Fig. 3, the active site cavity of RgPAL was bisected into

two regions ( Fig. 3A): one binds the amide group adjacent to the aromatic ring and the other binds the carboxyl group of the substrate. The phenyl ring of the substrate is roughly orthogonal to the plane of the MIO, and the methylidene of the MIO points to C2 of the aromatic ring ( Fig. 3A and B). In the carboxyl group binding pocket, the Arg361 residue is 3.2 Å from the carboxyl group of the substrate, and this residue might play a role in PD332991 the binding of the carboxylate moiety of the substrate through a salt bridge. The Tyr358 residue is 2.7 Å from the β-H of substrate, which is close enough to act as the β-H abstracted base ( Fig. 3C). The Glu491 residue is the closet residue to the amino group of the substrate (2.8 Å, Fig. 3C) and might accept the amino group of substrate as the enzyme base, which is consistent with the results reported by Bartsch [1]. The Tyr358, Arg361 and Glu491 are highly conserved in PAL ( Fig. 1). In the aromatic ring binding pocket, the His136 residue points to the phenyl ring of the substrate. The imidzaole group

of His136 is parallel to the phenyl ring and might generate a π–π interaction. Moreover, the imidazole of His136 and the adjacent amide group of Gln137 which points to the phenyl ring within a distance of 4.5 Å, form a hairpin motif to clamp the phenyl ring ( Fig. 3B and C). To verify the function of the hairpin, the His136, Gln137 were deleted (RgPAL-Δ136H, RgPAL-Δ137Q) and mutated to negative (RgPAL-H136E, Grape seed extract RgPAL-Q137E) and positive charges (RgPAL-H136K, RgPAL-Q137K) as well as uncharged amino acids (RgPAL-H136F, RgPAL-Q137L), respectively. The mutant and wild type RgPAL proteins appeared a single band of about 75 kDa on SDS-PAGE ( Fig. 4). The activities of RgPAL-Δ136H and RgPAL-Δ137Q were not detected (data not shown), suggesting that the residues at the two sites were essential for catalysis. The RgPAL-H136K, RgPAL-Q137K and RgPAL-H136E lost the enzymatic activity (data not shown), and the RgPAL-H136F, RgPAL-Q137L sharply decreased the activity ( Fig. 5). Compared with those mutants, the activity of RgPAL-Q137E decreased slightly ( Fig. 5).

79 nmol/min) it is more than twice that showed by the isoforms Lm

79 nmol/min) it is more than twice that showed by the isoforms LmTX-I and LmTX-II (6.1 nmol/min and 5.7 nmol/min, respectively). Both data clearly

indicate that exits some degree of structural differences between these proteins. Along with the biochemical Palbociclib supplier data, the molecular mass of LmrTX (14,277.50 Da) is different from LmTX-I and LmTX-II (14,245.4 and 14,186.2, respectively). The molecular mass difference found is in accordance with the aminoacid composition, which shown variation in the number of Pro, Thr and Ala residues. Despite the biochemical and structural differences between LmrTX and the isoforms LmTX-I and LmTX-II, the high degree of identity suggest that this toxin

could exert similar pharmacologic activities, i.e neurotoxic activity ex vivo. Phospholipase enzymes can exert their anticoagulant effects by the hydrolysis and physical destruction of the membrane surface required Ruxolitinib for the formation of coagulation complexes or by their interaction with blood coagulation proteins and not phospholipid hydrolysis (Kini, 2005). APTT is used to measure the integrity of components of the intrinsic pathaway and PT measures the integrity of the extrinsic pathaway. LmrTx interfered only with APTT, prolonging this time. The protein could be acting in the enzymatic cleavage of the available phospholipids required to intrinsic pathaway, since it was seen that chemical modification of histidine residues neutralized its anticoagulant activity. Based on the comparison of the three dimensional structure of class II PLA2 enzymes, three independent groups supported the predicted anticoagulant site (Carredano et al., 1998; Zhao et al., 2000; Singh et al., 2001). This region shows conformational

similarity and the presence of positively charged residue free for intermolecular interactions at the corner of molecule corresponding to the stretch of residues 55–67 seems to be a common feature of most of the anticoagulant PLA2 enzymes (Carredano et al., 1998; Zhao et al., 2000; Singh et al., 2001). For the RVV-VD, a PLA2 from the venom of Russell’s viper (Vipera russeli russeli), a strong Montelukast Sodium anticoagulant PLA2 of this class, this region has several lysine residues ( Carredano et al., 1998). In LmrTx this region has not been fully determined, only two residues positively charged in this segment was showed, which are favorably oriented to induce the anticoagulant effect. When performing chemical modification of histidine residues (alkylation with p-bromophenacyl bromide), LmrTX showed a reduction in its catalytic activity in 89% and there was an inactivation of the anticoagulant activity. The present study supports that anticoagulant activity in vitro of LmrTX is dependent on its catalytic activity.

A controlled rate freezer (EF600-103, Asymptote, Cambridge, UK) w

A controlled rate freezer (EF600-103, Asymptote, Cambridge, UK) was modified to achieve either NS or PS during cryopreservation by the addition of two modules designed to take polypropylene scintillation vials (Sigma, Z376825, 16 mm × 57 mm). One module was made of aluminum, the other of acetal (Fig. 1); these materials are good and poor conductors of heat respectively. These modules Sirolimus mouse were fixed to the flat cooling plate of the EF600-103. Thermocouples (K type) we used to measure the temperature at the base, middle, and upper sample volume inside the vial, (0 mm, 20 mm, and 40 mm from base respectively)

the thermocouples were connected to a Pico Logger (Pico-technology). For small volume PS or NS studies, 5 ml aliquots of ELS were harvested and mixed 1:1 with a freezing solution (24% Me2SO, 76% Viaspan v/v) precooled to 4 °C, and once AZD6244 price equilibrated (15 min), 80% of the excess CPA supernatant was removed, giving a final volume of 6 ml of 12% Me2SO,

38% Viaspan, and 50% ELS in culture medium, by volume. Icestart beads (1% w/v) (Asymptote) – sterile insoluble granules – which induce ice nucleation close to the equilibrium melting temperature of the mixture, were added and these sank by gravity to the base of the vial. These vials and the CRF were cooled to 4 °C before 5 vials (containing 6 ml each) were placed in the aluminum module, while 5 were placed into the acetal module (see aminophylline Fig. 1). The EF600-103 was programmed to cool at 1 °C/min from 4 °C to −80 °C. The samples were held in the EF600-103 at −80 °C for 1 h after

the cooling cycle was complete, before being transferred to a −80 °C freezer for 7 days. The samples were warmed rapidly during 330 s in a 37 °C water bath until all the ice had melted (yielding an approximate warming rate of 15 °C/min). The Me2SO was diluted out of solution during a 10 min stepwise process with prepared chilled culture medium, with residual ice start granules remaining at the bottom of the tube and easily avoided during decanting. The samples were re-cultured in a 5% CO2 humidified incubator at 37 °C. To observe physical changes in the structure of the samples, CryoSEM was carried out. Samples recovered from storage at −80 °C were warmed slightly (25 s in a 37 °C water bath) to loosen the ice matrix from the container wall, allowing the bulk frozen samples to be removed rapidly and transferred onto dry ice (−78 °C) without re-warming. These were wrapped in foil and stored on dry ice before being transferred to a −80 °C freezer. Under liquid nitrogen, each sample was held in a metal bracket and split horizontally using a blade, giving a circular cross-section. This was transferred into a cryo scanning electron microscope (FEI XL30 FEGSEM with a Quorum pp2000 cryo-stage) and etched at −80 °C, before being coated in a thin layer (∼20 nm) of gold.

At the same time, residual colonic innate immunity cells, such as

At the same time, residual colonic innate immunity cells, such as neutrophils and macrophages, of WT + DSS mice regressed to WT control baseline levels ( Figure 2B). The adaptive immunity colonic mucosa cells, including Treg, however, did not fully regress (WT vs WT + DSS, P = .048; Figure 2B). This result,

which is in line with gross pathology observation of MLN enlargement at 7 months after DSS treatments, suggests that subtle alterations in local gut adaptive immunity networks may persist for a particularly click here long period after the restoration of colonic mucosa architecture and the regression of colitis. In an effort to explain why uPA−/− + DSS mice develop colonic polypoid adenomas in the long term, while WT + DSS ones do not, we next examined the colon of mice at the early time point of 1 week after DSS treatment. We found that WT and uPA−/−controls showed normal colon histology, whereas their DSS-treated counterparts had the typical DSS-associated ulcerative colitis. At this early time point, DSS-treated mice had numerous foci of epithelial dysplasia, characterized by the same histopathologic and immunohistochemical features as those described in polyps (Figure 3A). Colonic

dysplastic foci of uPA−/− + DSS mice, however, were in a more advanced stage of the dysplasia/preneoplasia sequence than those of WT + DSS mice (P = .0001; Figure 3, A and B). A total of 2-minute polyps were found in 2 uPA−/− + DSS mice (2 of 24) and 1-minute polyp was found in the WT + DSS mice (1 of 23). DSS-induced ulcerative lesions, located mostly at the last part of the descending colon and the rectum, consistently presented a larger surface epithelium deficit in the uPA−/− + Nintedanib in vitro Aldol condensation DSS mice compared to the same lesions

of the WT + DSS mice (P < .0001; Figure 3C). In the non-ulcerative parts of the gut mucosa, however, colitis in both groups of DSS-treated mice was characterized by comparable levels of inflammatory cell infiltration (P = .1098; Figure 3D). To examine whether the tumor-promoting uPA deficiency is associated with a different inflammatory cell composition of DSS colitis, we labeled in situ and then quantified selected critical inflammatory cell types in the colonic mucosa. We found that the numbers of MPO + neutrophils were significantly higher in both the ulcerative lesions (P = .0052; Figure 4A) and the remaining colonic mucosa (P = .0079; Figure W4A) of uPA−/− + DSS mice compared to topographically matching areas of WT + DSS mice. The presence of neutrophils was unremarkable in both uPA−/− and WT untreated controls ( Figure W4A). Likewise, F4/80 + macrophages were significantly more in the non-ulcerated colonic mucosa of the uPA−/− + DSS compared to the WT + DSS mice (P = .0011; Figure 4B). CD3 + lymphocytes, however, were less in the ulcerative lesions (P = .0039; Figure 4C) and in the colonic lamina propria (P = .0282; Figure W4B) of uPA−/− + DSS mice than those counted in the corresponding areas of WT + DSS mice.

, 2009) Chequerboards were composed of either 3 × 3 or 4 × 4 gri

, 2009). Chequerboards were composed of either 3 × 3 or 4 × 4 grids with the height/width of individual grid squares being

kept constant (subtending .5° of visual angle at a viewing distance of 50 cm). Each chequerboard comprised a pattern of white and black squares, constructed so as to avoid obvious patterns and many squares of the same colour being adjacent to one another (see Table 4). Each chequerboard pattern was paired once with itself and once with another pattern that differed by a single square. Palbociclib cost This produced a total of 48 pairs, with each pair consisting of chequerboards being presented one above the other at the centre of the screen. Each pair of chequerboards was preceded by a fixation point presented for 1000 msec. Participants were asked to decide whether the chequerboards in each pair were the same or different as quickly and accurately as possible by verbal response. The pairs remained on screen LY2109761 until a response was given and there was a 1000 msec inter-trial interval. One block of 6 practice

trials preceded 2 blocks of 24 test trials. Each block contained an equal number of 3 × 3 and 4 × 4 chequerboards. Table 4. Performance on tests of visuoperceptual function. In order to gather a sizeable body of reading responses, all participants were requested to read aloud 3 corpora yielding a total of 250 words. Each corpus was as follows: 1. Brown and Ure words ( Brown and Ure, 1969): 72 words taken from the Brown and Ure (1969) corpus, which was composed of a subset of words at

three levels of length (4, 6 and 8 letters) matched on two levels of frequency and two levels of concreteness. All words were presented in Arial Unicode MS for an unlimited duration within a rectangular fixation box at the centre of the screen; letter height corresponded to a visual angle of 1.2° from a viewing distance of 50 cm. A series of letter processing tasks were administered, with all stimuli presented within a central fixation box to ameliorate the effects of visual disorientation: 1. Letter naming – all participants were requested to read the letters of the alphabet, excluding I, J, O, Q, W and X, in upper case. Letter height corresponded to a visual angle of 1.2° from a viewing distance of 50 cm. In each flanking condition, target letter identification was probed under two spatial conditions, condensed and spaced. The distance between the target letter and flankers was .875 mm in the condensed condition and 8.75 mm in the spaced condition, with the height of stimuli corresponding to a visual angle of 1.0°. The same combination of flankers was used for each target letter under both spatial conditions. The stimuli were presented in blocks of 6 items with the same spacing between the target letter and flankers, with blocks being administered in an ABBA design.

, 2010, Okano et al , 2012, Roberts and Wallis, 2000, Sasaki et a

, 2010, Okano et al., 2012, Roberts and Wallis, 2000, Sasaki et al., 2009, Sawamoto et al., 2011 and Yamazaki and Watanabe, 2009). It is known that expression patterns of several genes are different between mice and marmoset brain. Moreover, when marmosets vocalize, neural activity dependent gene expression is observed in the marmoset homologue of human Broca’s area ( Simoes et al., 2010).

Marmoset vocalization reflects developmental changes in the acoustic structure of species-specific communicative sounds, produced in social settings ( Pistorio et al., 2006). Thus, for all of these reasons, the common marmoset is a suitable animal model for biological approaches to studying BTK inhibitor human language. In this study, we have for the first time, compared genes related to human speech and dyslexia in primates using the common marmoset brain as a model for the human brain. The common marmosets used in this study were derived from a breeding colony at the Support Unit for Animal Resources Development, RIKEN BSI Research Resources Center, or from a colony at the Central Institute for Experimental Animals (CIEA). Experiments were performed in two (one male, one female) neonatal (postnatal day (P) 0), and four (two male, two female) adult marmosets (over 18 months of age). These ages were used to investigate gene expression changes during development because

the common marmoset Dabrafenib ic50 displays vocalizations at P0 that change

during development. All experimental protocols were approved by the institutional animal care and use committee at RIKEN and CIEA. All interventions and animal care procedures were performed in accordance with the Laboratory Animal these Welfare Act, Guide for the Care and Use of Laboratory Animals (National Institutes of Health), and the Guidelines and Policies for Animal Surgery provided by the Animal Study Committees of RIKEN and CIEA. RNA was isolated from an adult female common marmoset brain using the RNeasy Lipid Tissue Mini kit (Qiagen, Tokyo, Japan). cDNA fragments of speech disorder-related genes were synthesized from adult common marmoset brain RNA by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific primers (Table 1). cDNA fragments of dyslexia-related genes were synthesized from cDNA clones provided by the DNA Bank of the RIKEN BioResource Center (Ibaraki, Japan) (Tatsumoto et al., 2013) by PCR. Primers were designed using Primer3 ( to assess common marmoset and human nucleotide sequences. PCR products were examined on 1.0% agarose gels, excised from gels, and then cloned into pGEMTeasy plasmids (Promega, Madison, WI, USA). Cloned plasmids were transformed into DH5 alpha-competent Escherichia coli cells, and transformed E. coli colonies selected by growth on ampicillin (100 μg/mL) agar plates.

Slices were cut in ice-cold sucrose-based solution (in mM: 248 su

Slices were cut in ice-cold sucrose-based solution (in mM: 248 sucrose, 1.3 MgSO4, 5 KCl, 2.4 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 10 d-glucose, pH 7.4, bubbled with 95% O2/5% CO2) and stored in standard Krebs–Henseleit solution (in mM: 124 NaCl, 1.3 MgSO4, 5 KCl, 2.4 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 10 d-glucose, pH 7.4, bubbled with 95% O2/5% CO2) at room temperature prior to patch-clamp recording. Current-clamp recordings were made with patch-pipettes (thick-walled borosilicate glass, coated with Sylgard 184, fire-polished) and an Axopatch 200B amplifier in fast current-clamp mode (Axon Instruments,

Union City, CA), from slices superfused with Krebs–Henseleit solution at ~ 23 °C, in keeping with previous patch-clamp studies of granule cells at a similar temperature (Brickley et al., 2001, Brickley et al., 2007, Cathala et al., 2003 and Pugh and Jahr, 2011). Pipettes contained,

in mM: 126 KCH3SO3, 4 KCl, 10 HEPES, 4 MgATP, 5 EGTA, 4 NaCl, 0.5 CaCl2, pH 7.2 with KOH, and had resistances of 4.5–8.5 MΩ. Constant current injections were applied once every 5 s, from − 10 pA in + 2 pA steps. Recordings of voltage were low-pass EX 527 cost filtered at 10 kHz (4 pole Bessel filter on the amplifier), acquired at 62.5 kHz with a Cambridge Electronic Design (CED) power 1401 A/D interface and Signal software (CED, Cambridge, UK), and analyzed with Signal software and Origin software (Microcal, Northampton, MA). Membrane potentials were corrected for a calculated junction potential of 8.8 mV. Action potential

(AP) parameters were measured for the first three APs elicited at or just above rheobase (the current injection required for PIK-5 initiation of APs) and averaged. Voltage-threshold and maximum rates of fall and rise were measured using phase-plane plots (supplementary Signal script, Steven Clifford, CED) (Bean, 2007). The first three APs evoked near rheobase were averaged for each cell, and these were averaged across cells to generate the ‘average wild-type AP’ and the ‘average Ts65Dn AP’. The input capacitance (Cin) of each cell was measured in two ways. One measure was calculated from the time-constant of a single exponential function fitted to the voltage deflection generated by a negative current injection (− 10 or − 8 pA) ( D’Angelo et al., 1995). A second measure was taken from amplifier settings used to cancel current transients generated by 5 mV jumps in voltage-clamp mode, as in several previous patch-clamp studies of granule cells ( Brickley et al., 2001 and Cathala et al., 2003). GCs of all ages behave as a single electrical compartment and the measured Cin encompasses capacitances of the soma and dendrites ( Cathala et al., 2003). The Cin calculated from fits to voltage-changes caused by negative current injections was used to express current as current-density (pA/pF).