Short communication: Chronology of different sexual behaviors and motion activity during estrus in dairy cows.

In studying the efficiency of a variety of methods for estrus detection in a large dairy herd, we suspected a definite sequence of estrus signs. Consequently, we observed a subset of animals continuously between 0400 and 2400 h, making a note of the precise timing and frequency of each sexual behavior. Sixteen Holstein-Friesian cows, >20 d postpartum, were equipped with motion activity-sensing neck collars and had milk progesterone profiles monitored simultaneously. The duration between the first and last observed estrus behavior was (mean ± SE) 14.0 ± 1.9 h, with a range 8.5 to 28.75 h. The duration of standing to be mounted (STBM) was 4.68 ± 1.49 h, with a range of 0.25 to 18.25 h. Sniffing the vulva of another cow occurred on average 5.5 ± 1.3 h (range = 0.25-18.25 h) before the first STBM. By ranking the first appearance of each behavior, we established that sniffing was followed by the active behaviors of mounting another cow and not accepting a mount, as well as the passive behaviors of being sniffed and STBM by another cow. Chin resting occurred before not accepting a mount and STBM. All these behaviors were observed in the reverse order after the last STBM. The mean profile of motion activity revealed an increase in motion activity with the onset of exploratory behaviors, and highest values occurred within the period of STBM. Such distinct behavioral sequences may be controlled by changes in peripheral progesterone and estradiol concentrations, as well as by subtle independent mechanisms via pheromones in differing concentrations or divergent composition.

KNDy neurone activation prior to the LH surge of the ewe is disrupted by LPS.

In the ewe, steroid hormones act on the hypothalamic arcuate nucleus (ARC) to initiate the GnRH/LH surge. Within the ARC, steroid signal transduction may be mediated by estrogen receptive dopamine-, ß-endorphin- or neuropeptide Y (NPY)-expressing cells, as well as those co-localising kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy). We investigated the time during the follicular phase when these cells become activated (i.e., co-localise c-Fos) relative to the timing of the LH surge onset and may therefore be involved in the surge generating mechanism. Furthermore, we aimed to elucidate whether these activation patterns are altered after lipopolysaccharide (LPS) administration, which is known to inhibit the LH surge. Follicular phases of ewes were synchronised by progesterone withdrawal and blood samples were collected every 2 h. Hypothalamic tissue was retrieved at various times during the follicular phase with or without the administration of LPS (100 ng/kg). The percentage of activated dopamine cells decreased before the onset of sexual behaviour, whereas activation of ß-endorphin decreased and NPY activation tended to increase during the LH surge. These patterns were not disturbed by LPS administration. Maximal co-expression of c-Fos in dynorphin immunoreactive neurons was observed earlier during the follicular phase, compared to kisspeptin and NKB, which were maximally activated during the surge. This indicates a distinct role for ARC dynorphin in the LH surge generation mechanism. Acute LPS decreased the percentage of activated dynorphin and kisspeptin immunoreactive cells. Thus, in the ovary-intact ewe, KNDy neurones are activated prior to the LH surge onset and this pattern is inhibited by the administration of LPS.

Kisspeptin, c-Fos and CRFR type 2 co-expression in the hypothalamus after insulin-induced hypoglycaemia.

Normal reproductive function is dependent upon availability of glucose and insulin-induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c-Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin-treated ewes had an increased expression of c-Fos. Furthermore, the percentage of kisspeptin cells co-expressing c-Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre-optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.

The effect of a chronic stressor, lameness, on detailed sexual behaviour and hormonal profiles in milk and plasma of dairy cattle.

The objectives of the present study were to quantify the effects of a biological chronic stressor (lameness) on the duration and frequency of different oestrous behaviours in parallel with milk hormone profiles. Dairy cows 51.8 +/- 1.4 days postpartum (n = 59), including 18 non-lame control cows, were scored for lameness and closely observed for signs of oestrus having had their follicular phases synchronized by administration of gonadotrophin-releasing-hormone (GnRH) followed by prostaglandin F(2alpha) (PG) 7 days later. Lameness shortened the period when herd-mates attempted to mount the lame cows (1.83 +/- 0.69 h vs 5.20 +/- 1.53 h; p = 0.042) but did not affect the overall duration of total behaviours (lame 12.3 +/- 1.3 h vs non-lame 15.2 +/- 1.3 h). Lameness also lowered the intensity of oestrus [1417 +/- 206 points (n = 18) vs 2260 +/- 307 points (n = 15); p = 0.029]. Throughout the synchronized oestrous period, lame cows mounted the rear of herd-mates less frequently (p = 0.020) and tended to chin rest less (p = 0.075). Around the period of maximum oestrous intensity, lameness also diminished the proportion of cows mounting the rear of another cow and chin resting (p = 0.048, p = 0.037, respectively). Furthermore, lame cows had lower progesterone values during the 6 days before oestrous (p < or = 0.05). Fewer lame cows were observed in oestrus following PG (non-lame 83%, lame 53%; p = 0.030); however, if prior progesterone concentrations were elevated, lame cows were just as likely to be observed in oestrus. In conclusion, following endogenous progesterone exposure, lameness shortens the period when herd-mates attempt to mount lame cows but does not affect the incidence of oestrous. However, lame cows are mounted less frequently and express oestrus of lower intensity. This is associated with lower progesterone prior to oestrus but not with abnormal oestradiol or cortisol profiles in daily milk samples.

Influence of somatic cell count, body condition and lameness on follicular growth and ovulation in dairy cows.

The objective of this study was to investigate the effect of somatic cell count (SCC), body condition score (BCS) or lameness score on ovarian follicular growth and ovulation in dairy cows. Seventy four animals 30-80 days post-partum were monitored for all three conditions before synchronization of ovarian follicular phases by administration of gonadotrophin releasing hormone (GnRH) followed seven days later with prostaglandin F2alpha (PG). Ultrasonography of both ovaries twice daily throughout the follicular phase revealed that fewer animals with combined high SCC and lameness (4/9) ovulated compared to healthy animals (19/21; P=0.006) or animals with only high SCC (11/11; P=0.004) or only lameness (21/27; P=0.06). Overall, regardless of the presence of other concurrent conditions, fewer lame cows ovulated than Non Lame animals (30/42 and 30/32; P=0.015). Mean follicular growth and maximum follicular diameter were unaffected by any of the three conditions. However, dominant follicle growth and maximum diameter were greater in the 60 animals that ovulated compared to the 14 that did not; 1.83+/-0.16 versus 0.96+/-0.26mm/day (P=0.014) and 19.4+/-0.4 versus 16.4+/-1.2mm (P=0.003), respectively. In conclusion, lameness reduced the proportion of cows that ovulated and the synergistic effect of high SCC and lameness reduced that proportion further. However, follicular growth and maximum follicular diameter were unaffected by high SCC, low BCS or lameness.

The mGlu2 but not the mGlu3 receptor mediates the actions of the mGluR2/3 agonist, LY379268, in mouse models predictive of antipsychotic activity.

RATIONALE: Group II metabotropic glutamate receptors (mGluRs) comprise the mGluR2 and mGluR3 subtypes, the activation and modulation of which has been suggested to be beneficial for treating schizophrenia. Genetic association studies suggest limited association between mGluR2 and schizophrenia but some association between mGluR3 and schizophrenia. Conversely, pre-clinical studies suggest that mGluR2 may be responsible for mediating the antipsychotic activity of mGluR2/3 agonists, although to date, the role of mGluR3 has not been specifically assessed. OBJECTIVES: The aim of this study is to use recently generated mGluR3 and mGluR2 knockout mice to investigate which of the group II mGluRs mediates the actions of the mGluR2/3 agonist, LY379268, in two mouse models predictive of antipsychotic activity. MATERIALS AND METHODS: LY379268 (0.3-10 mg/kg SC), phencyclidine (PCP; 1-5 mg/kg IP), and amphetamine 1-10 mg/kg IP) were assessed on locomotor activity and behaviour in C57Bl/6J and transgenic mice. LY379268 was then assessed on PCP (5 mg/kg IP)- and amphetamine (2.5 mg/kg IP)-induced hyperactivity and behaviour in C57Bl/6J and transgenic mice. RESULTS: PCP (5 mg/kg)-evoked hyperactivity and behavioural alterations, i.e. circling, falling, stereotypy and ataxia, as well as amphetamine (2.5 mg/kg)-evoked hyperactivity, were dose-dependently attenuated by LY379268 (0.3-3 mg/kg) in C57Bl/6J mice. One milligram per kilogram of LY379268 reversed PCP-evoked hyperactivity and behavioural alterations in wild-type (WT) and mGluR3 knockout mice but not in mice lacking mGluR2. Similarly, 3 mg/kg LY379268 reversed amphetamine-evoked hyperactivity in WT and mGluR3 knockout mice but not in mice lacking mGluR2. CONCLUSION: The mGlu2 but not the mGlu3 receptor subtype mediates the actions of the mGluR2/3 agonist, LY379268, in mouse models predictive of antipsychotic activity.

Differential expression of IEG mRNA in rat brain following acute treatment with clozapine or haloperidol: a semi-quantitative RT-PCR study.

Antipsychotic drugs have been shown to modulate immediate early gene (IEG) expression in rat brain regions that are associated with schizophrenia, which may be directly linked to their immediate therapeutic benefit. In this study, we analysed the expression profile of a series of IEGs (c-fos, c-jun, fra-1, Krox-20, Krox-24, arc, sgk-1, BDNF and NARP) in six rat brain regions (prefrontal cortex, hippocampus, striatum, nucleus accumbens, thalamus and cerebellum). Rats (n=5) were administered either clozapine (20 mg/kg i.p.), haloperidol (1 mg/kg i.p.) or the appropriate vehicle with pre-treatment times of 1, 6 and 24 h. IEG expression was analysed in these regions by Taqman RT-PCR. The spatial and temporal profile of IEG induction following antipsychotic drug treatment correlates with regions associated with the efficacy and side effect profile of each drug. In particular, sgk-1 expression levels after antipsychotic drug treatment may have predictive value when investigating the profile of a novel antipsychotic drug.

Lameness, activity time-budgets, and estrus expression in dairy cattle.

The aim of the present study was to identify specific behavioral patterns that contribute to diminished estrus expression in lame cows. Behavioral scan and focal sampling were used to examine the effect of lameness on daily activity budgets, sexual behavior, feeding activities, and body condition score. A total of 59 milking cows (51.8 +/- 1.4 d postpartum) were monitored on a commercial dairy farm for 5 d following estrus synchronization. Overall, lame cows (n = 39) spent proportionately less time elevated on their feet and more time lying down compared with nonlame cows (n = 20). This included lame cows spending less time walking or standing. Overall, the total proportion of scans in which an estrous behavior was observed was very small but tended to be smaller for lame compared with nonlame cows. Throughout a day, lame cows displayed a lower proportion of estrous behaviors in the early morning. Lameness did not affect durations of drinking, grazing, or ruminating, or how these behavioral states fluctuated throughout the day. Similarly, rumination chewing rates were the same for lame and nonlame cows, and there was no association between lameness and dominance/displacement while feeding at a feed-fence. Lame cows did, however, have a slower bite rate at pasture and had a lower body condition score. Lame cows were also nearer the rear of the herd, both as they left the field and when entering the milking parlor. In conclusion, lame cows have longer lying times and spend less time standing, walking, and expressing an estrous behavior. Lame cows also have a lower bite rate at pasture and are more likely to be of lower body condition score.

GABA control of GnRH release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study examines the involvement of GABA(A or B) receptors in gonadotrophin-releasing hormone (GnRH) release in vitro and determines whether oestradiol modulates gamma-aminobutyric acid (GABA)-GnRH interaction. Within 10 min after ewe killing, hypothalamic slices were dissected and placed in oxygenated Minimum Essential Media (MEM)-alpha at 4 degrees C; within 2 h, slices were singly perifused at 37 degrees C with oxygenated MEM-alpha (0.15 ml/min), with or without oestradiol (24 pg/ml). After 4 h equilibration, fractions were collected for 4 h interposed with a 10 min exposure to specific GABA(A or B) receptor ligands (0.1-10 mM). The GABA(A or B) agonists (muscimol or baclofen) did not greatly influence GnRH release. However, GnRH increased (p < 0.05) after exposure to 10 mM GABA(A or B) antagonists (bicuculline or CGP52432, respectively). The GABA(A) antagonist stimulated greater sustained GnRH release (p < 0.05) in the absence of oestradiol than in its presence. The bioactivity of the released GnRH was studied using a hypothalamus-pituitary sequential double-chamber perifusion. Only after exposure of hypothalamic slices to the GABA(A) antagonist, did the hypothalamic eluate stimulate luteinizing hormone release from pituitary fragments (p < 0.05) confirming that the GABA(A) antagonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is predominantly under GABA(A) receptor inhibitory control and this is attenuated in the presence of oestradiol.

Noradrenergic control of GnRH release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study investigates the influence of alpha(1)-adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates alpha(1)-adrenoreceptor-GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media-alpha (MEM-alpha) at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to specific alpha(1)-adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1-10 mm). The alpha(1)-adrenoreceptor agonist (10 mm) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, alpha(1)-adrenoreceptor agonist (10 mm)-induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus-pituitary sequential double-chamber perifusion. Only after exposure of hypothalamic slices to alpha(1)-adrenoreceptor agonist (10 mm), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 +/- 12.3-36.2 +/- 21.6 ng/ml) confirming that the alpha(1)-adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.

Association of production diseases with motor activity-sensing devices and milk progesterone concentrations in dairy cows.

We have previously established that the efficiency of identifying oestrus with activity-sensing devices can be compromised by common production diseases; the present study was undertaken to determine how these diseases may affect device readings. A total of 67 Holstein-Friesian cows, >20 days postpartum, were equipped with activity-sensing neck collars and pedometers, and simultaneous milk progesterone profiles were also monitored twice a week. The influences of common production stressors on maximum activity and progesterone values were analysed. Approximately 30% potential oestrus events (low progesterone value between two high values) remained unrecognised by both activity methods, and progesterone values in these animals were higher on the potential day of oestrus when both activity methods did not detect an event (0.043 ±â€¯0.004 versus 0.029 ±â€¯0.004 ng/mL; P = 0.03). Data from a subset of 45 cows (two events each) were subjected to mixed models and multiple regression modelling to investigate associations with production diseases. Cow motor activity was lower in lame cows. Maximum progesterone concentrations prior to oestrus increased as time postpartum and body condition score (BCS) increased. There were also fewer days of low progesterone prior to oestrus associated with increases in BCS and maximum progesterone concentrations prior to oestrus. In conclusion, lameness was associated with lower activity values, but this suppression was insufficient to account for lowered oestrus detection efficiency of either device. However, associations were identified between production diseases and progesterone profiles.

Medial prefrontal cortex volume loss in rats with isolation rearing-induced deficits in prepulse inhibition of acoustic startle.

Rearing rats in isolation produces perturbations in behavior and brain neurochemistry suggested to resemble those of schizophrenia. In particular, isolation-reared rats display deficits in prepulse inhibition of acoustic startle that in humans are associated with disorders including schizophrenia and are interpreted as abnormalities in sensorimotor gating. The prefrontal cortex is considered important in the regulation of prepulse inhibition of acoustic startle and postmortem studies suggest that neuropil and total volume, but not total number of neurons, are decreased in this region of the brains of schizophrenic patients. In this study we used design-based stereological techniques to examine the brains of Lister Hooded rats, reared in isolation and which displayed prepulse inhibition of acoustic startle deficits, for changes in morphology compared with the brains of their socially-reared littermates. Pooled data from three batches of animals revealed a significant 7% volume loss of the medial prefrontal cortex of isolation-reared rats whereas neuron number in this region was unchanged. In contrast, volume and total neuron number were unaffected in the rostral caudate putamen. The robust reduction in prefrontal cortical volume observed in isolation-reared rats, in the absence of reductions in total neuron number, suggest that there is a loss of volume of the neuropil. These changes parallel those reported in schizophrenia patients and therefore support the construct validity of this model.

The effects of ziprasidone on regional c-Fos expression in the rat forebrain.

RATIONALE: Typical and atypical antipsychotic drugs produce characteristic patterns of immediate early gene expression in rat forebrain that are considered to reflect their effects in schizophrenia subjects. OBJECTIVE: To use c-Fos immunohistochemistry to investigate the functional neuroanatomical profile of the newly introduced atypical agent ziprasidone. MATERIALS AND METHODS: c-Fos immunohistochemistry was performed on paraformaldehyde-fixed cryosections of rat brains obtained, initially, from animals 2, 4, or 6 h after oral administration of 10 mg/kg ziprasidone or vehicle and, subsequently, from animals 2 h after oral administration of 1, 3, or 10 mg/kg ziprasidone or vehicle. The density of immunoreactive nuclei was assessed in pre-determined forebrain regions. RESULTS: Ziprasidone induced a time-dependent increase in the density of c-Fos-positive nuclei that was maximal at 2 h. At the 2 h time-point, c-Fos expression was significantly (p<0.05) elevated in the shell and core of the nucleus accumbens, lateral and medial caudate putamen, and lateral septum. At 4 h post-dose, c-Fos expression was also significantly increased in the cingulate gyrus. Ziprasidone-induced c-Fos expression was dose-dependent with significant (p<0.05) c-Fos expression observed in the nucleus accumbens (shell and core) and caudate putamen (lateral and medial) at 3 and 10 mg/kg and in the lateral septum at 10 mg/kg. CONCLUSIONS: Increased c-Fos expression in the nucleus accumbens and lateral septum is considered to be predictive of activity against positive symptoms, in the caudate putamen of motor side effect liability, and in the cingulate gyrus of efficacy against negative symptoms. Thus, the observed pattern of c-Fos expression induced in rat brain by ziprasidone is consistent with its reported clinical effects, namely, efficacy against positive symptoms with a therapeutic window over motor side effects and with some activity against negative symptoms.

The joint diabetic-renal clinic in clinical practice: 10 years of data from a District General Hospital.

BACKGROUND: Diabetic nephropathy is the leading cause of end-stage renal failure. Untreated, it causes continuous decline in glomerular function, worsening hypertension and a marked increase in cardiovascular risk. Joint diabetic-renal clinics were established to address these factors and prepare patients for renal replacement therapy. AIM: To determine whether our joint diabetic-renal clinic influenced progression of renal disease, and whether we were able to achieve targets from clinical trials and guidelines in routine practice. DESIGN: Retrospective review. METHODS: We collected data using clinical notes and electronic records for 130 patients attending the clinic over 10 years. RESULTS: Our patients had 62% type 2 and 38% type 1 diabetes. Mean duration of diabetes was 24 years for type 1 and 11 years for type 2 diabetes. At referral, 56% had evidence of vascular disease and 45%, proliferative retinopathy. Baseline median creatinine was 124 micromol/l. Significant improvements were made in systolic BP, diastolic BP and cholesterol (p < 0.001), compared to measurements at presentation. We analysed progression of renal disease by linear regression on 45 patients who had follow-up data for 3 years. Rate of decline of GFR was significantly reduced from 1.09 ml/min/month in the first year to 0.39 ml/min/month in the third year, (p < 0.004). DISCUSSION: Our findings suggest that the rate of deterioration of renal function can be reduced by aggressive management of risk factors. Joint diabetic-renal clinics appear to be useful in achieving targets in routine clinical practice.

High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases.

BACKGROUND: Transcriptional initiation and elongation provide control points in gene expression. Eukaryotic RNA polymerase II subunit 9 (RPB9) regulates start-site selection and elongational arrest. RPB9 contains Cys4 Zn(2+)-binding motifs which are conserved in archaea and homologous to those of the general transcription factors TFIIB and TFIIS. RESULTS: The structure of an RPB9 domain from the hyperthermophilic archaeon Thermococcus celer was determined at high resolution by NMR spectroscopy. The structure consists of an apical tetrahedral Zn(2+)-binding site, central beta sheet and disordered loop. Although the structure lacks a globular hydrophobic core, the two surfaces of the beta sheet each contain well ordered aromatic rings engaged in serial edge-to-face interactions. Basic sidechains are clustered near the Zn(2+)-binding site. The disordered loop contains sidechains conserved in TFIIS, including acidic residues essential for the stimulation of transcriptional elongation. CONCLUSIONS: The planar architecture of the RPB9 zinc ribbon-distinct from that of a conventional globular domain-can accommodate significant differences in the alignment of polar, non-polar and charged sidechains. Such divergence is associated with local and non-local changes in structure. The RPB9 structure is distinguished by a fourth beta strand (extending the central beta sheet) in a well ordered N-terminal segment and also differs from TFIIS (but not TFIIB) in the orientation of its apical Zn(2+)-binding site. Cys4 Zn(2+)-binding sites with distinct patterns of polar, non-polar and charged residues are conserved among unrelated RNAP subunits and predicted to form variant zinc ribbons.

The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster.

BACKGROUND: The HMG-box is a conserved DNA-binding motif that has been identified in many high mobility group (HMG) proteins. HMG-D is a non-histone chromosomal protein from Drosophila melanogaster that is closely related to the mammalian HMG-box proteins HMG-1 and HMG-2. Previous structures determined for an HMG-box domain from rat and hamster exhibit the same global topology, but differ significantly in detail. It has been suggested that these differences may arise from hinge motions which allow the protein to adapt to the shape of its target DNA. RESULTS: We present the solution structure of HMG-D determined by NMR spectroscopy to an overall precision of 0.85 A root mean squared deviation (rmsd) for the backbone atoms. The protein consists of an extended amino-terminal region and three alpha-helices that fold into a characteristic 'L' shape. The central core region of the molecule is highly stable and maintains an angle of approximately 80 degrees between the axes of helices 2 and 3. The backbone dynamics determined from 15N NMR relaxation measurements show a high correlation with the mean residue rmsd determined from the calculated structures. CONCLUSIONS: The structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

Long-term evaluation of isolation-rearing induced prepulse inhibition deficits in rats: an update.

RATIONALE: Rats reared in social isolation from weaning show prepulse inhibition (PPI) deficits which are thought to model the sensorimotor gating deficits seen in schizophrenia and other psychiatric disorders. We have previously reported that ten cohorts of Lister Hooded rats reared in isolation showed robust and reliable PPI deficits. OBJECTIVE: Our methodology differed from those used by others (Weiss and Feldon in Psychopharmacology 156(2-3):305-326, 2001), most notably in the weaning of pups at postnatal day (PND) 28 compared with PND20-22. Since our initial report, we have studied 18 more cohorts weaned at PND28 and one cohort weaned at PND21. METHOD: At weaning, male Lister Hooded pups were singly (isolates) or group (n=5) housed (grouped). Eight weeks later, startle and PPI responses of isolates and grouped rats were investigated using conditions of fixed inter-stimulus interval (ISI) (pulse=110 dB/50 ms; prepulse (PP)=75-80 dB/30 ms; ISI=100 ms). RESULTS: Isolates from 14 of the subsequent 18 cohorts demonstrated PPI deficits, giving an overall success rate of 86% for all 28 cohorts. %PPI ranged from 12 to 26% in the isolates and from 26 to 47% in the grouped for the successful cohorts, compared to 16-30% (isolates) and 19-35% (grouped) for those that failed. Only five out of the 19 subsequent cohorts demonstrated startle hyperreactivity, which was unrelated to PPI response. The isolates from the cohort weaned at PND21 did not show a significant deficit in PPI, suggesting, in our hands at least, a requirement for weaning at PND28. CONCLUSION: The data presented here reinforce our original findings that isolation-rearing of Lister Hooded rats provides a viable, non-pharmacological model of impaired PPI.

Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen-deuterium exchange.

The extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterized by comparing the differences in the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 15N-labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [15N]HMQC spectra of a sample prepared in the same manner exchanged into D2O. The largest changes in either chemical shift or hydrogen-deuterium exchange rate are observed for residues located in the active-site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.

Assignment of the backbone 1H and 15N NMR resonances and secondary structure characterization of barstar.

Barstar, a polypeptide inhibitor of ribonucleases, has been studied by 2D and 3D NMR techniques using uniformly 15N-labeled protein. Backbone (15NH-C alpha H-C beta H) resonances were assigned for all but 5 of the 89 residues. Dihedral angle and deuterium exchange studies were used in conjunction with medium range inter-proton NOEs to characterize the secondary structure of barstar. The protein is composed of four alpha-helices and three short stretches of extended strand. By further analysis of the NOE data three of the helices were found to be parallel to each other with the single disulphide bond linking the second and fourth helices at their C-terminal ends.

Practical and theoretical issues in gene-targeting studies and their application to behaviour.

Some of the major practical and theoretical issues that are associated with gene-targeting studies in mice are discussed. The availability of sufficient space to house the extensive breeding colonies associated with studies in gene-manipulated mice is an important logistical consideration that requires consideration at an early stage. A practical example is discussed which illustrates some of these issues. Problems associated with disease control and methods of maintaining the health status of valuable colonies are also outlined. Differences in the behavioural phenotype of inbred mouse strains pose important issues for study design and selection of host mouse lines. The results from studies exploring variations in the behavioural phenotype of six common inbred strains are briefly outlined. The impact of phenotypic variation on behavioural studies is considered and the implications for experimental design are discussed.