A Review of Studies on the System-Wide Implementation of Evidence-Based Psychotherapies for Posttraumatic Stress Disorder in the Veterans Health Administration.

Since 2006, the Veterans Health Administration (VHA) has instituted policy changes and training programs to support system-wide implementation of two evidence-based psychotherapies (EBPs) for posttraumatic stress disorder (PTSD). To assess lessons learned from this unprecedented effort, we used PubMed and the PILOTS databases and networking with researchers to identify 32 reports on contextual influences on implementation or sustainment of EBPs for PTSD in VHA settings. Findings were initially organized using the exploration, planning, implementation, and sustainment framework (EPIS; Aarons et al. in Adm Policy Ment Health Health Serv Res 38:4-23, 2011). Results that could not be adequately captured within the EPIS framework, such as implementation outcomes and adopter beliefs about the innovation, were coded using constructs from the reach, effectiveness, adoption, implementation, maintenance (RE-AIM) framework (Glasgow et al. in Am J Public Health 89:1322-1327, 1999) and Consolidated Framework for Implementation Research (CFIR; Damschroder et al. in Implement Sci 4(1):50, 2009). We highlight key areas of progress in implementation, identify continuing challenges and research questions, and discuss implications for future efforts to promote EBPs in large health care systems.

Diabetes induces GABA receptor plasticity in murine vagal motor neurons.

Autonomic dysregulation accompanies type-1 diabetes, and synaptic regulation of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV) is altered after chronic hyperglycemia/hypoinsulinemia. Tonic gamma-aminobutyric acid A (GABAA) inhibition prominently regulates DMV neuron activity, which contributes to autonomic control of energy homeostasis. This study investigated persistent effects of chronic hyperglycemia/hypoinsulinemia on GABAA receptor-mediated inhibition in the DMV after streptozotocin-induced type-1 diabetes using electrophysiological recordings in vitro, quantitative (q)RT-PCR, and immunohistochemistry. Application of the nonspecific GABAA receptor agonist muscimol evoked an outward current of significantly larger amplitude in DMV neurons from diabetic mice than controls. Results from application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP), a δ-subunit agonist, suggested that GABAA receptors containing δ-subunits contributed to the enhanced inducible tonic GABA current in diabetic mice. Sensitivity to THIP of inhibitory postsynaptic currents in DMV neurons from diabetic mice was also increased. Results from qRT-PCR and immunohistochemical analyses indicated that the altered GABAergic inhibition may be related to increased trafficking of GABAA receptors that contain the δ-subunit, rather than an expression change. Overall these findings suggest increased sensitivity of δ-subunit containing GABAA receptors after several days of hyperglycemia/hypoinsulinemia, which dramatically alters GABAergic inhibition of DMV neurons and could contribute to diabetic autonomic dysregulation.

The effect of the Rim Cutter on cement pressurization and penetration on cemented acetabular fixation in total hip arthroplasty: an in vitro study.

The Rim Cutter (Stryker Orthopedics, Mahwah, New Jersey) is a tool designed to cut a ledge inside the rim of the acetabulum, onto which a precisely trimmed, cemented, flanged cup can be fitted. The aim was to investigate the effect of the Rim Cutter on the intra-acetabular cement mantle pressure and the depth of cement penetration during cup insertion. The study had two parts. In the first part, hemi-pelvis models were fitted with pressure sensors. Pressure in the acetabulum was measured on insertion of a conventional cemented flanged cup with and without the use of a Rim Cutter to prepare the rim of the acetabulum. The second part assessed cement penetration when the same cups were inserted into a foam shell model. The shell was mounted in a jig and had holes drilled in it; the distance that cement penetrated into the holes was measured. A significant increase in cement pressure at the apex (p = 0.04) and the rim (p = 0.004) is seen when the Rim Cutter is used. Cement penetration in the Rim Cutter group was significantly increased at the rim of the acetabulum (p = 0.003). Insertion of a flanged cup after the acetabulum is prepared with the Rim Cutter leads to a significant increase in cement pressure and penetration during cup insertion in vitro when compared with conventional flanged cups.

Dietary methylsulfonylmethane supplementation and oxidative stress in broiler chickens.

Methylsulfonylmethane (MSM) is an organic, sulfur-containing compound widely used as a dietary supplement to improve joint health and treat arthritic pain. An experiment was conducted to study the effects of feeding 0.05% MSM to broilers exposed to diet-induced oxidative stress on tissue MSM distribution, growth performance, oxidative stress biomarkers, and immune responsivity. A total of 528 birds were allocated to 4 dietary treatments (fresh oil-no MSM, fresh oil-MSM, oxidized oil-no MSM, oxidized oil-MSM) as provided ad libitum to 11 replicate cages of 12 birds per treatment. Blood and tissue samples were collected to analyze MSM concentrations, and oxidative stress biomarkers including concentrations of thiobarbituric acid reactive substances (TBARS), total antioxidant capacity (TAC), total glutathione, and glutathione peroxidase (GPx) and reductase (GR) activities. Additionally, blood samples collected at day 25 were used to quantify T-cell (TC) populations using flow cytometry. Overall, MSM was quantified in all tissues and plasma samples of MSM-treated groups at all time points. Oxidized oil reduced (P = 0.006) feed intake over the 21-d feeding period, but MSM did not affect growth equally across time points. No effects (P > 0.2) of MSM or oil type were observed on TC populations. In the presence of oxidized oil, MSM reduced (P = 0.013) plasma TBARS and increased (P = 0.02) liver GPx at day 21, and increased (P = 0.06) liver GR at day 7. Irrespective of dietary oil type, groups supplemented with MSM showed higher plasma TAC at day 7 (P = 0.023), liver GPx activity at day 21 (P = 0.003), and liver GR activity at day 7 (P = 0.004) compared with groups not receiving MSM. In conclusion, 0.05% dietary MSM supplementation partially protected birds from oxidative stress but did not affect immune cell profiles.

Dietary supplementation with anti-IL-10 antibody during a severe Eimeria challenge in broiler chickens.

Attenuation of host IL-10 activity during Eimeria infection may elicit a robust Th1 response to eliminate the parasite from the gut epithelium. An experiment was conducted to study the effects of feeding IL-10 neutralizing antibody delivered via a dried egg product (DEP) on growth performance, immune responsivity, and gut health outcomes during a severe challenge with either Eimeria acervulina (study 1) or Eimeria tenella (study 2) following FDA CVM #217 protocol to test anticoccidial products. A total of 720 male Ross 308 chicks were used in each study, with 15 replicate cages of 12 birds and the following 4 treatments: sham-inoculated (uninfected) control diet (UCON), Eimeria-infected control diet (ICON), and Eimeria-infected control diet supplemented with DEP at 2 levels (165 [I-165] or 287 [I-287] U/tonne in study 1 and 143 [I-143] or 287 [I-287] U/tonne in study 2). Individual birds assigned to infected treatment groups received a single oral dose of either 200,000 E. acervulina (study 1) or 80,000 E. tenella (study 2) oocysts at 12 d of age (i.e., d post inoculation [DPI] 0), whereas uninfected birds were sham-inoculated with tap water. A one-way ANOVA was performed on outcomes including growth performance, hematology, serum chemistry profiles, immunophenotyping profiles, and intestinal lesion scores. In both studies, DPI 0 to 7 weight gain, feed intake, and feed conversion ratio were worse (P < 0.05) in all infected groups compared with the UCON group. Compared with ICON, DEP supplementation elicited no differences on overall growth performance. Histopathology and lesion scores revealed severe damage to the gut epithelium owing to the Eimeria challenge, yet DEP supplementation did not improve these outcomes or oocyst shedding, hematological measurements, or serum chemistry. However, DEP supplementation improved (P < 0.05) the percentage of circulating CD3+ cells at 6 DPI in study 2. These results indicate that DEP does not appear to elicit a coccidiostatic effect during a severe infection with E. acervulina or E. tenella.

Four novel SPG3A/atlastin mutations identified in autosomal dominant hereditary spastic paraplegia kindreds with intra-familial variability in age of onset and complex phenotype.

Mutation of the atlastin gene (SPG3A) is responsible for approximately 10% of autosomal dominant hereditary spastic paraplegia (AD-HSP) cases. The goal of this study was to identify novel disease causing atlastin mutations. Atlastin nucleotide variations were detected by direct sequencing of all 14 exons in 70 autosomal dominant (AD), 16 single sibship and 14 sporadic spastic paraplegia patients. Six mis-sense mutations (four of which were novel) were identified in six unrelated AD-HSP kindreds in exons 4, 7 and 8 of the atlastin gene. One kindred with a novel mutation showed variability in clinical phenotype and age of onset. Mutations are predicted to decrease GTPase activity, cause morphological abnormalities of the endoplasmic reticulum and prevent maturation of the Golgi complex resulting in impaired vesicle trafficking. Our study significantly adds to the spectrum of mutations and clinical phenotype of SPG3A. We advocate that all spastin mutation negative AD-HSP kindreds should be screened for pathogenic atlastin mutations regardless of age of onset or phenotypic complexity.

Effects of Yucca schidigera-derived saponin supplementation during a mixed Eimeria challenge in broilers.

An experiment was conducted to determine if dietary Yucca-derived saponin supplementation could ameliorate the immune and growth responses of broilers during a mixed coccidian challenge. A total of 576 two-day-old male Ross 308 broiler chicks were housed in galvanized starter batteries and randomly assigned to 1 of 4 dietary treatment groups (12 replicate cages of 12 birds). Dietary treatments were corn-soybean meal-based and included 1) control diet + sham-inoculated (Ucon), 2) control diet + Eimeria oocyst challenge (Icon), 3) control diet with 250 mg/kg Yucca-derived saponin product + Eimeria oocyst challenge (ISap250), and 4) control diet with 500 mg/kg of Yucca-derived saponin product + Eimeria oocyst challenge (ISap500). On study day 14, birds were orally inoculated with 1.5 mL of tap water containing Eimeria acervulina, E. maxima, and E. tenella (100,000, 40,000, and 30,000 oocysts/dose, respectively), or sham-inoculated with 1.5 mL of tap water. Eimeria-challenged birds exhibited a reduction in growth compared with uninfected birds (P < 0.001); however, there were no detectable differences due to dietary treatment among Eimeria-challenged groups. Mucosal thickness in the jejunum was increased (P < 0.042) in all infected groups and there were no differences among infected groups; however, saponin supplementation included at 250 mg/kg was not significantly different from the uninfected birds. Lymphocytes as a percentage of total white blood cells were increased (P < 0.014) in all Eimeria-challenged groups at 7 D post-inoculation compared with uninfected birds, but birds supplemented at 250 mg/kg were not different from uninfected birds. Cecal and duodenal IFN-γ expression increased with infection when compared with sham-inoculated birds. Cecal and duodenal IL-1ß expression increased (P < 0.008 and P < 0.039) due to infection, and ISap250 and ISap500 treatments ameliorated IL-1ß expression to levels not different from sham-inoculated birds. These results suggest that saponin supplementation may provide some immunomodulatory effects during a mixed coccidian challenge as evidenced by lymphocyte responses, changes in intestinal structure, and alterations in cecal and duodenal inflammatory cytokine mRNA expression.

Toxicity and tissue distribution of methylsulfonylmethane following oral gavage in broilers.

An experiment was conducted to investigate the toxicity and tissue distribution of methylsulfonylmethane (MSM) following oral gavage in broilers. A total of four hundred and thirty-two 15-day-old Ross 308 male broilers were allotted to 6 treatments with 6 replicates of 12 birds per replicate and administered a single oral dose of MSM at 0, 50, 100, 300, 1,000, or 2,000 mg/kg BW (Study 1). Another one hundred and sixty-eight 3-day-old chicks were allotted to either control or test group (Study 2) and administered a daily oral gavage of either 0 or 1, 500 mg/kg BW of MSM for 21 D consecutively. Blood and tissue samples were collected over a 48 h (Study 1) or 21 D (Study 2) period and analyzed for MSM concentrations. Toxicity was assessed through changes in hematology and clinical blood chemistry. In Study 1, plasma MSM concentrations were below 167 µg/mL at all time-points in birds receiving up to 300 mg/kg BW, and were significantly higher (P < 0.05) in birds receiving 1,000 or 2,000 mg/kg BW. Similarly, only the highest 2 MSM dosages elicited increased lymphocyte and decreased heterophil counts at 8 h (P < 0.003) and decreased hematocrit at 48 h (P = 0.015). Growth performance variables were unaffected by MSM in Study 2, and plasma and tissue MSM concentrations were highest on study day 21, with MSM-dosed birds always exhibiting higher (P < 0.03) concentrations compared with the control. Birds in Study 2 that were dosed with MSM had decreased liver enzyme concentrations at day 7 and 21 and decreased glucose and phosphorus at day 7. Importantly, MSM was detected in plasma and all tissues of control groups, confirming that MSM is synthesized de novo in chickens. In conclusion, oral MSM at either acute (single dose at 1,000 to 2,000 mg/kg BW) or sub-chronic (1,500 mg/kg BW daily for 21 D) concentrations did not cause any adverse effects on growth or clinical outcomes and appeared to be absorbed and distributed throughout the body.

Rapid inhibition of neurons in the dorsal motor nucleus of the vagus by leptin.

The peptide leptin conveys the availability of adipose energy stores to the brain. Increasing evidence implicates a significant role for extrahypothalamic sites of leptin action, including the dorsal vagal complex, a region critical for regulating visceral parasympathetic function. The hypothesis that leptin suppresses cellular activity in the dorsal motor nucleus of the vagus nerve (DMV) was tested using whole-cell patch-clamp recordings in brainstem slices. Leptin caused a rapid membrane hyperpolarization in 50% of rat DMV neurons. Leptin also hyperpolarized a subset of gastric-related neurons (62%), identified after gastric inoculation with a transneuronal retrograde viral tracer. The hyperpolarization was associated with a decrease in input resistance and cellular responsiveness and displayed characteristics consistent with an increased K+ conductance. Perfusion of tolbutamide (200 microM) reversed the leptin-induced hyperpolarization, and tolbutamide or wortmannin (10-100 nM) prevented the hyperpolarization, indicating that leptin activated an ATP-sensitive K+ channel via a phosphoinositide-3-kinase-dependent mechanism. Leptin reduced the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs), whereas inhibitory postsynaptic currents (IPSCs) were largely unaffected. Electrical stimulation of the nucleus tractus solitarii (NTS) resulted in constant-latency EPSCs, which were decreased in amplitude by leptin. The paired-pulse ratio was increased, suggesting leptin effects involved activation of receptors presynaptic to the recorded neuron. A leptin-induced suppression of EPSCs, but not IPSCs, evoked by focal photolytic uncaging of glutamate within the NTS was also observed, supportive of leptin effects on the glutamatergic NTS projection to the DMV. Therefore, leptin directly hyperpolarized and indirectly suppressed excitatory synaptic activity to DMV neurons involved in visceral regulation, including gastric-related neurons.

Nano measurements with micro-devices: mechanical properties of hydrated collagen fibrils.

The mechanical response of a biological material to applied forces reflects deformation mechanisms occurring within a hierarchical architecture extending over several distinct length scales. Characterizing and in turn predicting the behaviour of such a material requires an understanding of the mechanical properties of the substructures within the hierarchy, the interaction between the substructures, and the relative influence of each substructure on the overall behaviour. While significant progress has been made in mechanical testing of micrometre to millimetre sized biological specimens, quantitative reproducible experimental techniques for making mechanical measurements on specimens with characteristic dimensions in the smaller range of 10-1000 nm are lacking. Filling this void in experimentation is a necessary step towards the development of realistic multiscale computational models useful to predict and mitigate the risk of bone fracture, design improved synthetic replacements for bones, tendons and ligaments, and engineer bioinspired efficient and environmentally friendly structures. Here, we describe a microelectromechanical systems device for directly measuring the tensile strength, stiffness and fatigue behaviour of nanoscale fibres. We used the device to obtain the first stress-strain curve of an isolated collagen fibril producing the modulus and some fatigue properties of this soft nanofibril.

5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain, and serotonin (5-HT) can modify the response of the SCN circadian oscillator to light. 5-HT1B receptor-mediated presynaptic inhibition has been proposed as one mechanism by which 5-HT modifies retinal input to the SCN (Pickard et al., 1996). This hypothesis was tested by examining the subcellular localization of 5-HT1B receptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1B receptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1B receptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1B receptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1A or 5-HT7 receptor antagonists did not block this effect. Moreover, in cells demonstrating an evoked EPSC in response to optic nerve stimulation, TFMPP had no effect on the amplitude of inward currents generated by local application of glutamate. The effect of TFMPP on light-induced phase shifts was also examined using 5-HT1B receptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1B receptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN.

The prevalence of carbon-13 in respiratory carbon dioxide as an indicator of the types of endogenous substrate. The change from lipid to carbohydrate during the respiratory rise in potato slices.

Isotope discrimination is a common feature of biosynthesis in nature, with the result that different classes of carbon compounds frequently display different (13)C/(12)C ratios. The (13)C/(12)C ratio of lipid in potato tuber tissue is considerably lower than that for starch or protein. We have collected respiratory CO(2) from potato discs in successive periods through 24 hr from the time of cutting-an interval in which the respiration rate rises 3-5-fold. The (13)C/(12)C ratio of the evolved CO(2) was determined for each period, and compared with the (13)C/(12)C ratios of the major tissue metabolites. In the first hours the carbon isotope ratio of the CO(2) matches that of lipid. With time, the ratio approaches that typical of starch or protein. An estimation has been made of the contribution of lipid and carbohydrate to the total respiration at each juncture. In connection with additional observations, it was deduced that the basal, or initial, respiration represents lipid metabolism-possibly the alpha-oxidation of long chain fatty acids-while the developed repiration represents conventional tricarboxylic acid cycle oxidation of the products of carbohydrate glycolysis. The true isotopic composition of the respiratory CO(2) may be obscured by fractionation attending the refixation of CO(2) during respiration, and by CO(2) arising from dissolved CO(2) and bicarbonate preexisting in the tuber. Means are described for coping with both pitfalls.

Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT1B and 5-HT7 receptors.

Serotonin (5-HT) and 5-HT receptor agonists can modify the response of the mammalian suprachiasmatic nucleus (SCN) to light. It remains uncertain which 5-HT receptor subtypes mediate these effects. The effects of 5-HT receptor activation on optic nerve-mediated input to SCN neurons were examined using whole-cell patch-clamp recordings in horizontal slices of ventral hypothalamus from the male mouse. The hypothesis that 5-HT reduces the effect of retinohypothalamic tract (RHT) input to the SCN by acting at 5-HT1B receptors was tested first. As previously described in the hamster, a mixed 5-HT(1A/1B) receptor agonist, 1-[3-(trifluoromethyl)phenyl]-piperazine hydrochloride (TFMPP), reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by selectively stimulating the optic nerve of wild-type mice. The agonist was negligibly effective in a 5-HT1B receptor knockout mouse, suggesting minimal contribution of 5-HT1A receptors to the TFMPP-induced reduction in the amplitude of the optic nerve-evoked EPSC. We next tested the hypothesis that 5-HT also reduces RHT input to the SCN via activation of 5-HT7 receptors. The mixed 5-HT(1A/7) receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), reduced the evoked EPSC amplitude in both wild-type and 5-HT1B receptor knockout mice. This effect of 8-OH-DPAT was minimally attenuated by the selective 5-HT1A receptor antagonist WAY 100635 but was reversibly and significantly reduced in the presence of ritanserin, a mixed 5-HT(2/7) receptor antagonist. Taken together with the authors' previous ultrastructural studies of 5-HT1B receptors in the mouse SCN, these results indicate that in the mouse, 5-HT reduces RHT input to the SCN by acting at 5-HT1B receptors located on RHT terminals. Moreover, activation of 5-HT7 receptors in the mouse SCN, but not 5-HT1A receptors, also results in a reduction in the amplitude of the optic nerve-evoked EPSC. The findings indicate that 5-HT may modulate RHT glutamatergic input to the SCN through 2 or more 5-HT receptors. The likely mechanism of altered RHT glutamatergic input to SCN neurons is an alteration of photic effects on the SCN circadian oscillator.

Presynaptic ionotropic glutamate receptors modulate GABA release in the mouse dorsal motor nucleus of the vagus.

Regulation of GABA release in the dorsal motor nucleus of the vagus (DMV) potently influences vagal output to the viscera. The presence of functional ionotropic glutamate receptors (iGluRs) on GABAergic terminals that rapidly alter GABA release onto DMV motor neurons has been suggested previously, but the receptor subtypes contributing to the response are unknown. We examined the effect of selective activation and inhibition of iGluRs on tetrodotoxin-insensitive, miniature inhibitory postsynaptic currents (mIPSCs) in DMV neurons using patch-clamp recordings in brainstem slices from mice. Capsaicin, which activates transient receptor potential vanilloid type 1 (TRPV1) receptors and increases mIPSC frequency in the DMV via an iGluR-mediated, heterosynaptic mechanism, was also applied to assess GABA release subsequent to capsaicin-stimulated glutamate release. Application of glutamate, N-methyl-d-aspartate (NMDA), or kainic acid (KA), but not AMPA, resulted in increased mIPSC frequency in most neurons. Inhibition of AMPA/KA receptors reduced mIPSC frequency, but selective antagonism of AMPA receptors did not alter GABA release, implicating the presence of presynaptic KA receptors on GABAergic terminals. Whereas NMDA application increased mIPSC frequency, blocking NMDA receptors was without effect, indicating that presynaptic NMDA receptors were present, but not activated by ambient glutamate levels in the slice. The effect of NMDA was prevented by AMPA/KA receptor blockade, suggesting indirect involvement of NMDA receptors. The stimulatory effect of capsaicin on GABA release was prevented when AMPA/KA or NMDA, but not AMPA receptors were blocked. Results of these studies indicate that presynaptic NMDAR and KA receptors regulate GABA release in the DMV, representing a heterosynaptic arrangement for rapidly modulating parasympathetic output, especially when synaptic excitation is elevated.

Molecular and functional changes in glucokinase expression in the brainstem dorsal vagal complex in a murine model of type 1 diabetes.

Glucose concentration changes in the nucleus tractus solitarius (NTS) affect visceral function and metabolism by influencing central vagal circuits, especially inhibitory, GABAergic NTS neurons. Acutely elevated glucose can alter NTS neuron activity, and prolonged hyperglycemia and hypoinsulemia in animal models of type 1 diabetes results in plasticity of neural responses in the NTS. NTS neurons contributing to metabolic regulation therefore act as central glucose sensors and are functionally altered in type 1 diabetes. Glucokinase (GCK) mediates cellular utilization of glucose, linking increased glucose concentration to excitability changes mediated by ATP-sensitive K(+) channels (KATP). Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and in vitro electrophysiology, we tested the hypothesis that changes in GCK expression in the NTS accompany the development of diabetes symptoms in the streptozotocin (STZ)-treated mouse model of type 1 diabetes. After several days of hyperglycemia in STZ-treated mice, RNA expression of GCK, but not Kir6.2 or SUR1, was decreased versus controls in the dorsal vagal complex. Electrophysiological recordings in vitro indicated that neural responses to acute hyperglycemia, and synaptic responsiveness to blockade of GCK with glucosamine, were attenuated in GABAergic NTS neurons from STZ-treated mice, consistent with reduced molecular and functional expression of GCK in the vagal complex of hyperglycemic, STZ-treated mice. Altered autonomic responses to glucose in type 1 diabetes may therefore involve reduced functional GCK expression in the dorsal vagal complex.

Whole-cell recordings from preoptic/hypothalamic slices reveal burst firing in gonadotropin-releasing hormone neurons identified with green fluorescent protein in transgenic mice.

Central control of reproduction is governed by a neuronal pulse generator that underlies the activity of hypothalamic neuroendocrine cells that secrete GnRH. Bursts and prolonged episodes of repetitive action potentials have been associated with hormone secretion in this and other neuroendocrine systems. To begin to investigate the cellular mechanisms responsible for the GnRH pulse generator, we used transgenic mice in which green fluorescent protein was genetically targeted to GnRH neurons. Whole-cell recordings were obtained from 21 GnRH neurons, visually identified in 200-microm preoptic/hypothalamic slices, to determine whether they exhibit high frequency bursts of action potentials and are electrically coupled at or near the somata. All GnRH neurons fired spontaneous action potentials, and in 15 of 21 GnRH neurons, the action potentials occurred in single bursts or episodes of repetitive bursts of high frequency spikes (9.77 +/- 0.87 Hz) lasting 3-120 sec. Extended periods of quiescence of up to 30 min preceded and followed these periods of repetitive firing. Examination of 92 GnRH neurons (including 32 neurons that were located near another green fluorescent protein-positive neuron) revealed evidence for coupling in only 1 pair of GnRH neurons. The evidence for minimal coupling between these neuroendocrine cells suggests that direct soma to soma transfer of information, through either cytoplasmic bridges or gap junctions, has a minor role in synchronization of GnRH neurons. The pattern of electrical activity observed in single GnRH neurons within slices is temporally consistent with observations of GnRH release and multiple unit electrophysiological correlates of LH release. Episodes of burst firing of individual GnRH neurons may represent a component of the GnRH pulse generator.

Alpha-tocopherol dietary supplement decreases titers of antibody against 5-hydroxymethyl-2'-deoxyuridine (HMdU).

This study evaluated the effects of vitamin E (alpha-tocopherol) on oxidative DNA damage in a randomized double-blind Phase II chemoprevention trial. Oxidative DNA damage was measured by the level of auto-antibody (Ab) against 5-hydroxymethyl-2'-deoxyuridine (HMdU) in plasma. After the baseline screening, eligible subjects (n = 31; plasma samples from 28 subjects were available for this study) were randomized to receive 15, 60, or 200 mg of alpha-tocopherol per day for 28 days. Biomarkers were measured twice at baseline--on day 1 (visit 1) and day 3 (visit 2)--and twice after intervention--on day 17 (visit 3) and day 31 (visit 4). At baseline, there was a highly significant inverse correlation between anti-HMdU Ab titer and plasma vitamin E level (r = -0.53; P = 0.004; n = 28). Smoking did not affect baseline anti-HMdU Ab titer; however, anti-HMdU Ab titer levels at baseline were significantly lower in subjects with above-median (0.75 ounce/day) alcohol consumption (P = 0.008). No significant change in anti-HMdU Ab level occurred at either visit 3 or visit 4 for subjects on the lowest dose, 15 mg alpha-tocopherol per day. Subjects receiving 60 mg of alpha-tocopherol per day had a significant decrease in anti-HMdU Ab level at visits 3 and 4 compared with baseline (P = 0.049 and P = 0.02, respectively). However, subjects receiving the highest dose, 200 mg/day, had less consistent results: a significant decrease in anti-HMdU Ab level was seen at visit 4 (P = 0.04) but not at visit 3. Our results demonstrate an inverse relationship between alpha-tocopherol and anti-HMdU Abs in plasma; oxidative DNA damage can be modulated by short-term dietary supplementation of alpha-tocopherol in some subjects.

Tuberal supraoptic neurons--II. Electrotonic properties.

The electrotonic properties of tuberal supraoptic neurons were studied from conventional intracellular recordings made in the hypothalamo-neurohypophysial explant in vitro. The cable parameters electrotonic dendritic length, and the dendritic to somatic conductance ratio, were estimated using the slopes and intercepts of the first two peeled exponentials of the voltage transients generated by current steps. The estimations were made assuming an equivalent cylinder model consisting of a soma and an attached, lumped dendrite of finite length. An equalizing time constant was resolved in 12 of 17 neurons, allowing calculation of both cable parameters. In only one of these 12 was it necessary to assume a somatic shunt to account for the data. The average value of the dendritic electrotonic length was 1.02, and that of the dendritic to somatic conductance ratio, 4.11. In the remaining five neurons, an equalizing time constant could not be peeled and consequently the dendritic cable parameters could not be estimated. The average input resistance of these 12 neurons was 162 M omega and the average membrane time constant was 11.86 ms. Principal Components Analysis revealed that the variance of input resistance and time constant was largely explained by one factor, while that of dendritic electrotonic length and the dendritic to somatic conductance ratio was explained by a separate, independent factor, suggesting a separation of electrical and morphological parameters, respectively. In addition, the variability of the data indicates that considerable differences in the morphology and specific membrane resistivity exist across supraoptic neurons. An analysis of spontaneously occurring postsynaptic potentials revealed that the shapes of these potentials could not be explained simply by assuming that they were determined by their passive decay from some point along the equivalent cable to the soma. In conclusion, dendrites make a significant and previously unappreciated contribution to the electrotonic behavior of supraoptic neurons. These electrotonic properties are similar to those of many other, morphologically diverse, central nervous system neurons.

Histamine enhances the depolarizing afterpotential of immunohistochemically identified vasopressin neurons in the rat supraoptic nucleus via H1-receptor activation.

Previous studies have demonstrated that histamine primarily excites unidentified neurons in the rat supraoptic nucleus. We investigated the neuromodulatory effects of histamine on immunohistochemically identified vasopressin neurons in the rat supraoptic nucleus using intracellular recording techniques from the hypothalamo-neurohypophysial explant. Exogenous application of histamine (0.1-100 microM) to vasopressinergic neurons produced a small membrane depolarization accompanied by an increase of up to 100% in the amplitude of the depolarizing afterpotential that follows current-evoked trains of action potentials. The enhancement of the depolarizing afterpotential by histamine did not depend upon the depolarization. Further, histamine enhanced the amplitude of the depolarizing afterpotential when blocking the afterhyperpolarizing potential with d-tubocurarine or apamin, and in the presence of tetrodotoxin and d-tubocurarine or apamin, indicating a postsynaptic action of histamine on the depolarizing afterpotential that is not simply a reflection of a decrease in the afterhyperpolarizing potential. These toxins also had no effect on the histamine-induced depolarization. The enhancement of the depolarizing afterpotential by histamine was mimicked by the histamine H1-receptor agonist 2-thiazolylethylamine and was reduced or blocked by the H1-receptor antagonist promethazine, but was not blocked or reduced in the presence of the histamine H2-receptor antagonist, cimetidine. In summary, these results show that the excitatory effect of histamine on immunohistochemically identified vasopressin neurons in the supraoptic nucleus is due in part to the H1-receptor-mediated enhancement of the depolarizing afterpotential independent of any change in the afterhyperpolarizing potential or membrane potential.

Tuberal supraoptic neurons--I. Morphological and electrophysiological characteristics observed with intracellular recording and biocytin filling in vitro.

Previous studies of the tuberal, or retrochiasmatic, portion of the supraoptic nucleus suggest its functional similarity to the more densely populated anterior supraoptic nucleus, but the basic electrophysiological and morphological features of tuberal supraoptic nucleus neurons have not been described. Using the hypothalamo-neurohypophysial explant preparation in the rat, intracellular recordings and biocytin injections were made in tuberal supraoptic nucleus neurons and the results indicate that the two parts of the nucleus are similar. The generally oval-shaped somata of tuberal supraoptic nucleus neurons exhibited short, irregularly shaped appendages, and possessed 2-5 varicose, sparsely branching dendrites oriented in the horizontal plane. Many tuberal supraoptic nucleus neurons could be antidromically stimulated (mean latency = 6.4 ms). Filled neurons had varicose axons which were traced to the median eminence and even as far as the neural stalk, but which did not bifurcate. Both axons and dendrites were sparsely invested with short, hair-like appendages. The input resistance of the recorded neurons (mean = 177.7 M omega) was positively correlated with the membrane time constant (mean = 13.1 ms; r = 0.83). Tuberal supraoptic nucleus neurons displayed a prominent afterhyperpolarization following individual spikes or bursts of spikes, as well as firing frequency adaptation in response to positive current pulses. Although numbering far fewer than those of the anterior supraoptic nucleus, tuberal supraoptic nucleus neurons have axons which are more often intact in this preparation, and a dendritic tree which radiates within the plane of the explant. Thus these neurons should provide a useful model for further study of the electrophysiological and morphological characteristics of mammalian neurosecretory neurons.