11% with zidovudine monotherapy/single-dose nevirapine) [57]. The randomized studies above are two of few studies that have been able to look at individual PIs. One additional analysis from the APR of 955 live births exposed to lopinavir/ritonavir reported a

PTD rate of 13.4% [58]. A retrospective study from the UK reported a PTD GDC-0068 mw rate of 10% in 100 women taking ritonavir-boosted atazanavir in pregnancy, of whom 67% had conceived on their regimen [34]. The data regarding HAART, individual components of HAART and PTD remain conflicting. Some studies suggest that PIs, in particular ritonavir-boosted PIs, are associated with an increased risk of PTD but this is not confirmed by others. There is a

need for a randomized study of sufficient power to explore these issues further and the Promoting Maternal and Infant Survival Everywhere (PROMISE) study (NCT01061151), with 6000 women either randomly allocated to a PI-based combination regimen or zidovudine monotherapy will hopefully provide some answers to these important questions. 5.2.4 No routine dose alterations are recommended for ARVs during pregnancy if used at adult licensed doses with the exception of darunavir, which should be dosed twice daily. Grading: 1C Consider third-trimester TDM particularly if combining tenofovir AZD2281 in vivo and atazanavir. Grading: 1C If dosing off licence, consider switching to standard dosing throughout pregnancy or regular TDM. Grading: 1C Physiological changes that occur even during the first trimester of pregnancy may affect the kinetics of drug absorption, distribution, metabolism and elimination, thereby affecting the drug dosing. Gastrointestinal transit time becomes prolonged; body water and fat increase throughout gestation and there are accompanying increases in cardiac output, ventilation, and liver and renal blood flow; plasma protein concentrations Adenosine decrease, notably albumin

and α1 acid glycoprotein; renal sodium reabsorption increases; and changes occur in the metabolic enzyme pathway in the liver, including changes in cytochrome P450. Caution should be exercised if women fall pregnant on unlicensed doses and consideration given to performing TDM to assess trough levels, or reverting to licensed dosing, often twice per day, during pregnancy. The pharmacokinetics of most NRTIs (zidovudine [59], stavudine [60], lamivudine [61], abacavir [62]) are not significantly affected by pregnancy and dose adjustment is not required. Renal excretion of didanosine is increased in pregnancy, but dose alteration is probably not required [63]. Tenofovir concentrations in the third trimester were reported to be reduced by about 15% compared with postpartum, but trough levels are adequate [64] although in a population-based study of tenofovir use, pregnant women appear to have 39% more clearance than non-pregnant women [65].

The ribosomal protein database of 16 type strains of the Sphingomonadaceae constructed by sequencing S10 and spc operons using these designed primers was compared with MALDI mass spectra. The results revealed that nine ribosomal subunit proteins coded in the S10 and spc operons, L18, L22, L24, L29, L30, S08, S14, S17, and S19, were commonly detectable subunits by MALDI-TOF

MS analysis of the Sphingomonadaceae (Table 3, Fig. 1). To evaluate these nine selected ribosomal selleck inhibitor subunit proteins, phylogenetic analysis based on their amino acid sequences, the S10-GERMS method, was compared with that based on 16S rRNA gene sequences (Fig. 2). Each phylogenetic tree formed four genera clusters of the Sphingomonadaceae, respectively, and almost the same clusters with slight differences in their details. The most marked difference

was the phylogenetic position between Sphingomonas jaspsi NBRC 102120T and Sphingomonas wittichii Galunisertib NBRC 105917T. As the phylogenetic positions based on the 16S rRNA gene sequence showed that these two type strains were assigned into different clusters, more research into the Sphingomonadaceae may be required. Seven strains of genus Sphingopyxis and one strain of genus Sphingobium identified based on the 16S rRNA gene sequence were isolated as APEOn-degrading bacteria; therefore, nine selected biomarkers and the ribosomal protein database of the Sphingomonadaceae were applied very for bacterial identification of the APEOn-degrading bacteria by MALDI-TOF MS. The results demonstrated that the biomarkers were significantly useful for bacterial classification using the rapid MALDI-TOF MS method to identify APEOn-degrading bacteria (Table 3, Fig. 1). The 16S rRNA sequence identity between APEOn-degrading bacteria strain BSN20 and S. terrae NBRC 15098T was 99.9%, and the difference in the 16S rRNA gene sequence was only one base; however, comparison of their MALDI mass spectra revealed a mass difference of subunit S14, whose m/z was 11513.6 or 11527.6, respectively (Fig. 3a and b). Therefore, the S10-GERMS method could successfully discriminate S. terrae,

implying that it is a significantly useful tool for bacterial discrimination at the strain level, even though there was only one base difference in the 16S rRNA gene. Similarly, three strains of S. terrae, NBRC 15593, NBRC 15598, and NBRC 15599, were discriminated by the S10-GERMS method at the strain level (Fig. 3c–e). Strain NBRC 15593, isolated as polyethylene glycol-degrading bacteria, was registered as S. macrogoltabidus in NBRC. In this study, the 16S rRNA gene sequence and MALDI mass spectra of strain BSN20 were identical to strain NBRC 15593; however, as the MALDI mass spectra were not identical to that of S. macrogoltabidus NBRC 15033T, strains BSN20 and NBRC 15593 were identified as S. terrae.

Slides were incubated in a wet chamber in the dark at room temperature for 1 h, washed three times with PBS-FCS and once with PBS. They were then fixed a second time with 4% formaldehyde-PBS for 15 min at 4 °C, mounted in VectaShield media containing 4′-6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, PF-562271 cell line CA), covered with

a 1-mm coverslip and sealed with nail polish. A similar protocol was used for B. burgdorferi cells that had been fixed with 50 μL of 60% methanol for 10 min, before being washed and reacted with the primary and secondary antibodies as described above. Stained cells were visualized using a Zeiss Inverted Axiovert 200 motorized microscope with a × 100 PlanApo 1.4 oil PH3 objective and Zeiss filter sets 31, 34 and 38 for AlexaFluor 594, 488 and DAPI, respectively. The pictures were taken using a Zeiss Axiocam MRM cool CCD camera and were analyzed using axiovision 4.3 software. Unabsorbed anti-rBmpA Ig had a dot immunobinding titer of 1 : 10 000 with 10 ng

of rBmpA or rBmpB and reacted minimally with rBmpC or rBmpD. After absorption with rBmpB, anti-rBmpA Ig had a titer of 1 : 100 with 1 and 10 ng selleckchem of rBmpA and did not react with similar quantities of rBmpB, rBmpC or rBmpD (Fig. 1a). Absorbed anti-rBmpA at a 1 : 100 dilution detected a single immunoreactive spot consistent with BmpA at 39 kDa, pI 5.0, in 2D-NEPHGE gels of B.

burgdorferi lysates (Fig. 1b). This dilution of this reagent was used for all subsequent immunoblotting. Fractionation of intact B. burgdorferi cells with Triton X-114 showed that both immunoreactive BmpA and FlaB were present in the detergent-insoluble fraction containing periplasmic core proteins (Fig. 2a, lanes 2), while only BmpA was present in the detergent phase of the Triton X-114-soluble fraction containing the outer-membrane proteins (Fig. 2a, lanes 4). A small amount of BmpA was also detected in the aqueous phase of the Triton X-114-soluble fraction (Fig. 2a, lanes 3). Detection of BmpA in the detergent phase of Triton X-114 fractionation is consistent with its being located in Regorafenib solubility dmso the outer membranes of B. burgdorferi (Brusca & Radolf, 1994; Skare et al., 1995). While the detection of immunoreactive BmpA in the Triton X-114-insoluble fraction might imply that some BmpA is associated with periplasmic cellular proteins and the cytoplasmic membrane, this fraction also includes intact cells with the outer membranes still attached (Crother et al., 2003). These data suggest that BmpA, unlike FlaB, is a lipoprotein, and most probably located in the outer membrane of B. burgdorferi. To provide additional data on BmpA localization, intact B. burgdorferi cells were incubated with increasing concentrations of proteinase K in the absence or presence of Triton X-100.

(2004, 2008). Lancefield serotyping (Lancefield, 1933) was performed using Pastorex Strep (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer’s protocol. Biochemical and enzymatic characterizations were performed using the API 20 STREP® and the API ZYM® systems (bioMerieux, Marcy-l’Etoile, France), respectively. All the isolates were cultured on blood agar (Columbia

agar base; Becton Dickinson) containing 5% sheep blood (Nippon Bio-Test Laboratories) at 37 °C for 24 h, and fresh colonies were evaluated according to the manufacturer’s instructions. learn more The antimicrobial susceptibility of the strains was determined using the disk diffusion method on Muller–Hinton agar (Difco Laboratories, Detroit, MI). The following R428 molecular weight chemotherapeutic agents (microgram per disk) were used in the disk diffusion method: oxytetracycline (30) (Eiken Chemical

Co. Ltd, Tokyo, Japan), erythromycin (15) (Oxoid, UK), florfenicol (30) (Oxoid), lincomycin (10) (Oxoid), and ampicillin (10) (Oxoid). The strains were considered resistant to oxytetracycline if the diameter of the inhibition zone around the disk was less than 19 mm (Constable & Morin, 2002). The presence of tet(L), tet(O), tet(S), and tet(M) genes that encode tetracycline resistance was investigated for all the resistant isolates by PCR according to the method reported previously (Agersøet al., 2002). Internal fragments representing 85% of the sodA gene of 23 fish isolates were amplified using the universal primer set and sequenced according to the method reported by Nomoto et al. (2008). The nucleotide sequences were analyzed using bioedit version 7.0 (Hall, 1999). The phylogenetic analysis was carried out using the neighbor-joining method using mega version 3 (Kumar

et al., 2004). The restriction enzyme-digested chromosomal Y-27632 2HCl DNA was analyzed by BSFGE, a modified pulsed-field gel electrophoresis (PFGE) technique (Madinabeitia et al., 2009). Streptococcus dysgalactiae strains were cultured on THA at 37 °C for 24 h, and the preparation of genomic DNA and DNA digestion with the restriction enzyme ApaI were carried out according to the previously described method (Nomoto et al., 2006). Macrorestriction fragments digested by ApaI were separated using a 1% agarose horizontal gel using the BSFGE system (Genofield; Atto, Tokyo, Japan). The biased sinusoidal electric field was applied for 20 h at DC 48 V and AC 288 V at a frequency of 0.005 Hz (initial) and 0.330 Hz (final). After gel electrophoresis, the gel was stained and visualized under UV light. The macrorestriction patterns were then calibrated and analyzed using the gene profiler software package along with treecon software (version 4.05; Scanalytics Inc., Fairfax, VA).

Sequences were compared to A. fumigatus Af293 genomic sequence (Nierman et al., mTOR inhibitor 2005) using the blast function on the cadre database (Mabey Gilsenan et al., 2009).

Both flanking regions were located in the genomic sequence and used to pinpoint the insertion site. Colony radial growth experiments were carried out as described previously (Robson et al., 1995) using 2% glucose in agar plates containing Vogel’s salts. Four thousand transformants were isolated and screened for altered susceptibility to ITR. After overlay with ITR containing agar, 19 transformants that displayed either continued or completely arrested growth were selected, of which eight had at least a fourfold difference in ITR susceptibility relative to the parental strain (Table 2). All eight transformants displayed normal growth rate colony morphology and sporulation compared to the parental strain. These eight transformants were selected for further analysis. The eight transformants (termed REMI-11, REMI-14D, REMI-56, REMI-85, REMI-101, REMI-102, REMI-103 and REMI-116) were characterised further to determine the nature of the REMI insertion. PCR using primers directed against the AmpR gene in pUC19 confirmed that all of them had at least one integrated copy of pPyrG. Restriction digestion followed by Southern hybridisation with the pUC19 vector fragment of pPyrG

was carried out to determine the nature of the plasmid integrations. XhoI digests established whether or not ‘perfect’ Interleukin-2 receptor REMI that retained the XhoI sequence at the site of insertion had selleck screening library occurred: a single 4.8 kb hybridising band, which represents pPyrG, indicated such an event (Fig. 1). REMI-11, REMI-56 and REMI-101 all give 4.8 kb bands expected from a single insertion. REMI-85,

REMI-14D, REMI-103 and REMI-102 give single bands larger than 4.8 kb and REMI-116 gives two bands. This data were combined with sequence from the insertion site and flanking regions to determine whether the REMI event had occurred at a genomic XhoI site. In REMI-85, REMI-14D, REMI-102, REMI-104 and REMI-116, the rescued plasmids had partial XhoI sites flanking the insertion suggesting that integration occurred in an imperfect manner. REMI-11, REMI-56 and REMI-101 all contained intact XhoI sites at the insertional locus. Combining the Southern blot data and the flanking sequence, we were able to categorise the REMI insertion into perfect or imperfect (Table 2) and determine the insertional copy number. 7/8 RMI isolates had one single plasmid insertion in the genome, three which were perfect REMI. One of them, REMI-116, had multiple insertions and was not investigated further. The site of plasmid insertion was successfully determined by plasmid rescue in all REMI transformants.

Mozambique Selisistat chemical structure has recently released nationwide community prevalence survey data suggesting pockets of high HIV prevalence in central and southern Mozambique [15]. The Manhiça study area is likely to be representative of other semi-rural Mozambican populations with intensive migration to and from high HIV prevalence areas in South Africa, and thus the findings are not generalizable to all areas of the country. Despite the evidence suggesting that a plateau has been reached in HIV incidence

in Manhiça, the incidence among pregnant women remains unacceptably high from a public health standpoint. Many factors may contribute to this high HIV incidence, including migration, a high prevalence of sexually transmitted infections, high numbers of concurrent sexual partnerships and insufficient health care services. There is an urgent need for the current HIV prevention and treatment programmes to be expanded and for

access to them to be improved. We are grateful to all the women who participated in the studies, thus allowing this analysis to be carried out. Financial support for the prevalence studies was provided by the Institut Català d’Oncologia (Barcelona), Hospital buy BIBF 1120 Clinic (Barcelona), the CISM (Mozambique), which receives core funding from the Spanish Agency for InternationalCooperation (AECI) and the Spanish Fondo de Investigación Sanitaria (FIS01/1236; PI070233), the Banco de Bilbao, Vizcaya, and the Argentaria Foundation (grant number BBVA 02–0). The VCT clinic and day hospital receives core funding from the Agencia Catalana de Cooperacio al Desenvolupament.

D.N. was supported by either a grant from the Spanish Ministry of Education and Science (Ramon y Cajal). S.P.H was partially financed by the EU-FP7 Pregvax Project. “
“Acquired immune deficiency appears to be associated with serious non-AIDS (SNA)-defining conditions such as cardiovascular disease, liver and renal insufficiency and non-AIDS-related malignancies. We analysed the incidence of, and factors associated with, several SNA events in the LATINA retrospective cohort. Cases of SNA events were recorded among cohort patients. Three controls were selected for each case from cohort members at risk. Conditional logistic models were fitted to estimate the effect of traditional risk factors as well as HIV-associated factors on non-AIDS-defining conditions. Among 6007 patients in follow-up, 130 had an SNA event (0.86 events/100 person-years of follow-up) and were defined as cases (40 with cardiovascular events, 54 with serious liver failure, 35 with non-AIDS-defining malignancies and two with renal insufficiency). Risk factors such as diabetes, hepatitis B and C virus coinfections and alcohol abuse showed an association with events, as expected.

Using comprehensive routinely collected surveillance data, we present quality of care measures for persons diagnosed with HIV infection at the national level for the first time. Almost all (97%) adults diagnosed with HIV infection in 2011 were linked to HIV care within 3 months, and 88% within 4

weeks. Furthermore, among adults diagnosed in 2010, 85% were retained in care in 2011 and 92% of those diagnosed late were receiving treatment. Collectively, these findings indicate that the NHS provides high-quality selleck compound care to persons newly diagnosed with HIV infection in the UK. Importantly, there was little variation of linkage to care, retention and treatment coverage by sociodemographic characteristics and exposure category. There was no evidence of health inequalities with regard to access to and retention Everolimus in HIV care in the UK. These findings are strikingly different from those of studies carried out in the USA, which show lower rates of linkage to and retention in care following diagnosis, with important inequalities in access

to health care [15]. Despite excellent HIV care, in 2011 almost half of adults diagnosed with HIV infection had a CD4 count at or below the threshold at which treatment should have been initiated. Patients diagnosed late have an 8-fold increased risk of mortality within a year of diagnosis compared with those diagnosed promptly. Reducing late diagnosis is also a public health priority, as HIV diagnosis provides awareness of infection and access to drugs to reduce viral load. Late HIV diagnosis is a key indicator for monitoring the success of testing interventions and is included in the Public Health Outcome Framework for England [16]. PI-1840 Heterosexual men had the highest rate of late diagnosis compared

with other risk groups. This is probably a consequence of the impact of the universal offer of an HIV test during antenatal care and targeted testing campaigns aimed at MSM. The proportions of late diagnoses in both pregnant women and MSM have declined slightly over the past decade [1]. Nevertheless, an estimated 1000 MSM (a third of diagnoses] in 2011 were diagnosed late. The elevated proportion of late diagnoses among black men and women is largely the result of the high numbers of new diagnoses reported among adults of sub-Saharan origin, who acquired their infection before arriving in the UK [17]. Our analyses indicate that the reduction of late HIV diagnoses requires urgent investment to increase testing coverage and frequency among groups at highest risk of HIV infection. In addition, we demonstrate exceptionally high 1-year mortality rates among persons diagnosed late. These data highlight the importance of early ART, with the magnitude of the benefit of ART being greatest among older adults [18]. BHIVA Standards of Care guidelines recommend linkage to HIV care within 14 days of HIV diagnosis [6].

Thus, SgII is present in LDCV and non-LDCV compartments of various neural cells. The wide subcellular selleck chemicals localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non-LDCV compartments. “
“Neurons in the visual cortex

exhibit heterogeneity in feature selectivity and the tendency to generate action potentials synchronously with other nearby neurons. By examining visual responses from cat area 17 we found that, during gamma oscillations, there was a positive correlation between each unit’s sharpness of orientation tuning, strength of oscillations, and propensity towards synchronisation with other units. Using a computational

buy Epigenetic inhibitor model, we demonstrated that heterogeneity in the strength of rhythmic inhibitory inputs can account for the correlations between these three properties. Neurons subject to strong inhibition tend to oscillate strongly in response to both optimal and suboptimal stimuli and synchronise promiscuously with other neurons, even if they have different orientation preferences. Moreover, these strongly inhibited neurons can exhibit sharp orientation selectivity provided that the inhibition they receive is broadly tuned relative to their excitatory inputs. These results predict that the strength and orientation tuning of synaptic inhibition are heterogeneous across area 17 neurons, which could have important implications for these neurons’ sensory processing capabilities. Furthermore, although our experimental recordings were conducted in the visual cortex, our model and simulation results can apply more generally to any brain region with analogous neuron types in which heterogeneity in the strength of rhythmic

inhibition can arise during gamma oscillations. Parvulin “
“Hypoglossal motoneurons (HMs) are known to be under ‘permanent’ bicuculline-sensitive inhibition and to show ‘transient’ synaptic γ-aminobutyric acid (GABA)A and glycine inhibitory responses. The present paper describes a permanent bicuculline-sensitive current that should contribute to their tonic inhibition. This current was recorded in brainstem slices superfused without any exogenous agonist and remained detectable with tetrodotoxin. It could also be blocked by the other GABAA antagonists picrotoxin (PTX) and 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide) (SR95531; gabazine), but persisted in the presence of a specific blocker of α5-containing GABAA receptors. Addition of 2 μm 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP), known to preferentially activate GABAA receptors devoid of a γ-subunit, induced a sustained anionic current that could be further enhanced by neurosteroids such as allopregnanolone (100 nm).

5, 100 mM NaCl, 10 mM MgSO4 and 0.01% gelatin solution). Contaminated bacterial DNA was eliminated by DNase I. Phage nucleic

acids were extracted with the phenol : chloroform method and dissolved in TE buffer (Sambrook & Russell, 2001). The types of nucleic acids were identified by agarose gel electrophoresis and treated with DNase or RNase enzymes. For double-stranded DNA, 1 μg of DNA was digested with BamHI, HindIII, EcoRI, PstI or XhoI restriction endonucleases using the manufacturer’s recommended conditions HCS assay (Promega, Wisconsin) and the patterns were observed by agarose gel electrophoresis. The MOI that gave the highest phage titer was determined with some modification (Lu et al., 2003). Burkholderia pseudomallei P37 at mid-log phase was transfected with a selected phage at three different MOIs, 0.01, 0.1 and 1 PFU CFU−1. Phage titers were determined by the drop plate method. Phage-free cultures containing only bacteria and bacterial-cell-free cultures containing only phages were used as controls in all experiments. All assays were performed

in duplicate. The bacterial challenge test was performed by growing B. pseudomallei Ibrutinib clinical trial P37 in 300 mL nutrient broth in the presence of 3.6 mM CaCl2 at 37 °C and the phage solution was added at 5 h after inoculation with 0.1 MOI at the mid-log phase (O’Flynn et al., 2004). The numbers of bacteria were measured every hour for 16 h using OD550 nm and the plate count technique in triplicate. The phage that could lyse a broader range of B. pseudomallei, but not other related bacteria except B. mallei, and also provide a high titer was selected to study using the one-step growth method to obtain the latent period,

the eclipse period and the burst size (Pajunen et al., 2000). Ten milliliters of a mid-log phase of B. pseudomallei P37 was centrifuged and resuspended in a 0.25 volume of fresh nutrient broth/CaCl2 (c. 109 CFU mL−1). The phage at an MOI of 0.0005 was added and allowed to adsorb for 5 min at 37 °C and then centrifuged at 10 000 g Isoconazole for 5 min. The pellet was resuspended in 10 mL nutrient broth and incubated at 37 °C for 2 h. Three hundred microliters of culture were taken at 10-min intervals, half of which was treated with 1% (v/v) chloroform to release the intracellular phage and plated on the bacterial lawn for the latent period determination. The other half was immediately diluted and plated to measure the phage titer. The experiment was repeated three times. Twenty-one soil samples from 140 tested that yielded positive plaques on B. pseudomallei P37 lawn (plaque sizes were approximately 0.1–1.0 mm in diameter) were chosen for repropagation. After repropagation, only six isolates, named ST2, ST7, ST70, ST79, ST88 and ST96, clearly lysed the bacterium in liquid culture.

The aim of the current Etoposide mw study is to further investigate the possible interactions between antipsychotic treatment, estrogen and the dopaminergic system in a rodent

model, by using female, D-amphetamine sulphate (AMPH)-sensitized rats. Behaviors elicited by AMPH sensitization are thought to reflect some of the positive and cognitive symptoms of schizophrenia (Tenn et al., 2003; Featherstone et al., 2007). These changes are further thought to correspond to nucleus accumbens (NAcc) DA transmission changes in both rodents and non-human primates (Tenn et al., 2003; Castner et al., 2005; Peleg-Raibstein et al., 2008). In a previous study, locomotor activity was recorded in response to an acute injection of AMPH in male rats receiving chronic antipsychotic treatment over a period of 12 days (Samaha et al., 2007). Chronic continuous antipsychotic treatment became progressively ineffective at blocking AMPH-induced locomotion, with the higher doses resulting in a potentiated response to AMPH 5 days after treatment cessation. In the current study, we administered the typical antipsychotic haloperidol (HAL), at the lower concentration of the chronic regimen used by Samaha et al. (2007) which is still shown to reflect effective doses in humans (Kapur et al., 2000; Samaha et al., 2007, 2008) to either AMPH-sensitized or

non AMPH-sensitized female rats. Cetuximab clinical trial These ovariectomized (OVX) rats received either chronic low alone, or chronic low plus phasic high 17β-estradiol (E2) replacement to simulate two different estrogen levels during different phases of the estrous cycle in young females (Quinlan et al., 2008). Following an AMPH challenge, locomotor activity was recorded and NAcc DA and its metabolites were measured using in vivo microdialysis. It has been suggested that

antipsychotic administration may lead to DA receptor supersensitivity, which could lead to a almost rebound effect when drug administration is discontinued (Antelman et al., 1986; Samaha et al., 2008); such a rebound was observed in male rats following discontinuation of continuous HAL at a higher concentration than used here (Samaha et al., 2007). To examine this phenomenon in females, HAL administration was discontinued for 1 week, after which locomotor activity in response to an additional AMPH challenge was examined. Sixty-four female Sprague–Dawley rats (Charles River Laboratories, Montreal, QC, Canada) weighing 220–250 g were pair-housed and were the original N of this study. Cages were located in a 21 °C room with a 12-h reverse light–dark cycle (lights off at 09.00 h), with ad libitum access to food and water. Bedding consisted of a 50 : 50 mixture of corncob and beta-chip. All testing and surgical procedures were performed during the dark phase of the diurnal cycle.