The absence of VGLUT3 predisposes to cocaine abuse by increasing dopamine and glutamate signaling in the nucleus accumbens.

Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.

Hemodynamic effects of intravenous nicardipine in severely pre-eclamptic women with a hypertensive crisis.

OBJECTIVE: Nicardipine permits rapid control of blood pressure in women with severe pre-eclampsia (PE) and hypertensive crisis. Our objective was to investigate its maternal and fetal hemodynamic effects. METHODS: Ten severely pre-eclamptic pregnant women who required intravenous nicardipine for severe hypertension were included in this prospective observational trial. Maternal macrocirculation was assessed by transthoracic echocardiography. Maternal microcirculatory perfusion was examined sublingually with the sidestream dark field imaging technique. Fetal hemodynamics were assessed by Doppler examinations of the uteroplacental and fetal circulations. Maternal cardiac output, total vascular resistance, mitral E/A ratio and capillary heterogeneity index, uterine artery pulsatility index and fetal cerebroplacental ratio were considered primary outcomes. Paired measurements, obtained before administration of nicardipine infusion and after stabilization of blood pressure, were compared. RESULTS: Administration of nicardipine significantly reduced the mean arterial blood pressure (median difference, 26 mmHg; P = 0.002) and total vascular resistance (median difference, 791 dynes × s/cm(5) ; P = 0.002) in all included women. This induced a reflex tachycardia with consequent increase in cardiac output of 1.55 L/min (P = 0.004). There were no significant changes in the other determinants of maternal or fetal hemodynamic parameters. CONCLUSIONS: Nicardipine effectively reduces blood pressure through selective afterload reduction that triggers an increase in cardiac output, without affecting maternal diastolic function, or microcirculatory, uteroplacental or fetal perfusion. This hemodynamic response is uniform and predictable. Fetomaternal cardiovascular profiling can be achieved by combining transthoracic echocardiography with obstetric Doppler.

Efficacy of DL-methionine hydroxy analogue-free acid in comparison to DL-methionine in growing male white Pekin ducks.

The present study was performed to assess the bioefficacy of DL-methionine hydroxy analogue-free acid (MHA) in comparison to DL-methionine (DLM) as sources of methionine for growing male white Pekin ducks in the first 3 wk of life. For this aim, 580 1-day-old male ducks were allocated into 12 treatment groups and received a basal diet that contained 0.29% of methionine, 0.34% of cysteine and 0.63% of total sulphur containing amino acids or the same diet supplemented with either DLM or MHA in amounts to supply 0.05, 0.10, 0.15, 0.20, and 0.25% of methionine equivalents. Ducks fed the control diet without methionine supplement had the lowest final body weights, daily body weight gains and feed intake among all groups. Supplementation of methionine improved final body weights and daily body weight gains in a dose dependent-manner. There was, however, no significant effect of the source of methionine on all of the performance responses. Evaluation of the data of daily body weight gains with an exponential model of regression revealed a nearly identical efficacy (slope of the curves) of both compounds for growth (DLM = 100%, MHA = 101%). According to the exponential model of regression, 95% of the maximum values of daily body weight gain were reached at methionine supplementary levels of 0.080% and 0.079% for DLM and MHA, respectively. Overall, the present study indicates that MHA and DLM have a similar efficacy as sources of methionine for growing ducks. It is moreover shown that dietary methionine concentrations of 0.37% are required to reach 95% of the maximum of daily body weight gains in ducks during the first 3 wk of life.

The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation.

During the periparturient phase, cows are typically in an inflammation-like condition, and it has been suggested that inflammation associated with the development of stress of the endoplasmic reticulum (ER) in the liver contributes to the development of fatty liver syndrome and ketosis. In the present study, we investigated the hypothesis that feeding grape seed and grape marc meal extract (GSGME) as a plant extract rich in flavonoids attenuates inflammation and ER stress in the liver of dairy cows. Two groups of cows received either a total mixed ration as a control diet or the same total mixed ration supplemented with 1% of GSGME over the period from wk 3 prepartum to wk 9 postpartum. Dry matter intake during wk 3 to 9 postpartum was not different between the 2 groups. However, the cows fed the diet supplemented with GSGME had an increased milk yield and an increased daily milk protein yield. Cows supplemented with GSGME moreover had a significantly reduced mRNA abundancy of fibroblast growth factor (FGF) 21, a stress hormone induced by various stress conditions, in the liver in wk 1 and 3 postpartum. In contrast, mRNA abundances of a total of 3 genes involved in inflammation and 14 genes involved in ER stress response, as well as concentrations of triacylglycerols and cholesterol, in liver samples of wk 1 and 3 postpartum did not differ between the 2 groups. Overall, this study shows that supplementation of GSGME did not influence inflammation or ER stress in the liver but increased milk yield, an effect that could be due to effects on ruminal metabolism.

Assessment of the ability of the Privigen® purification process to deplete thrombogenic factor XIa from plasma.

BACKGROUND AND OBJECTIVES: Activated clotting factor FXI (FXIa) has been postulated to play a significant role in thromboembolic events potentially associated with the administration of intravenous immunoglobulin. The purpose of this study was to demonstrate that thrombogenic agents, in particular FXIa and FXI, are depleted or inactivated in Privigen(®) . MATERIALS AND METHODS: The ability of the purification process to deplete FXIa from plasma was studied. All steps of the Privigen(®) production were investigated for potential activation of FXI to FXIa with spiking experiments. RESULTS: Privigen(®) contains no procoagulant activity as determined by FXIa chromogenic assay, non-activated partial thromboplastin time (NaPTT) and thrombin generation assays (TGA, FXIa-like activity). The coagulation times were >200 s in the NaPTT test. FXIa was below the detection limit of 0·14 ng/ml (chromogenic assay) and below the quantification limit of 0·2 ng/ml (TGA). FXIa spiking experiments showed that the analytical methods used can detect traces of procoagulant activity in immunoglobulin samples. FXIa spiking and kinetic experiments during the octanoic acid fractionation step showed that a substantial reduction in FXIa specific activity (by ≥99·9% within 40 min of octanoic acid incubation) was reached already at an early stage of the manufacturing process. These results were confirmed in vivo: in a modified Wessler test, no thrombus was reported. CONCLUSION: The Privigen(®) manufacturing process has the capability to remove thrombogenic factors: octanoic acid precipitation, designed to remove a variety of contaminants during immunoglobulin purification, also removes almost all FXIa from plasma and further purification steps do not activate FXI.

Microcirculation in women with severe pre-eclampsia and HELLP syndrome: a case-control study.

OBJECTIVE: To compare microcirculatory perfusion in women with severe pre-eclampsia against that in healthy pregnant women, and secondly in women with severe pre-eclampsia with or without HELLP syndrome (haemolysis, elevated liver enzymes, and low platelets). DESIGN: Case-control study. SETTING: University Hospital Rotterdam, the Netherlands. POPULATION: Twenty-three women with severe pre-eclampsia and 23 healthy pregnant controls, matched for maternal and gestational age. Out of the 23 women with severe pre-eclampsia, ten presented with HELLP syndrome. METHODS: Microcirculation was analysed sublingually by a non-invasive sidestream dark-field imaging device (SDF). MAIN OUTCOME MEASURES: Perfused vessel density (PVD), microcirculatory flow index (MFI), and heterogeneity index (HI) were calculated for both small vessels (∅ < 20 µm; capillaries) and non-small vessels (∅ > 20 µm; venules and arterioles). RESULTS: There were no significant differences between women with severe pre-eclampsia and healthy controls. Women with pre-eclampsia and HELLP syndrome showed a reduced PVD (P = 0.045), MFI (P = 0.008), and increased HI (P = 0.002) for small vessels, as compared with women with pre-eclampsia but without HELLP syndrome. CONCLUSIONS: Sidestream dark-field is a novel, promising technique in obstetrics that permits the non-invasive evaluation of microcirculation. We did not observe major differences in sublingual microcirculatory perfusion between women with severe pre-eclampsia and healthy pregnant controls. In women with severe pre-eclampsia, the presence of HELLP syndrome is characterised by impaired capillary perfusion.

Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice.

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

The association of the Friesinger score and estimated glomerular filtration rate in an urban South Asian patient population.

AIM: The correlation between kidney function and coronary artery disease (CAD) severity as assessed by an angiographic score has not yet been studied in the South Asian population. We sought to estimate the association by performing a single-center, cross-sectional study. PATIENTS AND METHODS: The estimated glomerular filtration rate (eGFR) calculated by the CKD-EPI equation and the Friesinger score to quantify the severity of CAD were the primary endpoints in patients undergoing coronary angiograms. RESULTS: The mean eGFR was significantly lower in participants with a Friesinger score of > 5 compared to participants with a score of < 5 (73 vs. 86 ml/min/1.73 m(2) by MDRD). In univariate analysis, an eGFR of < 55 ml/min/1.73 m(2) was associated with a 9.5-fold increased odds of a higher Friesinger score compared to an eGFR >= 55 ml/min/1.73 m2 (p = 0.043), which was unchanged in multivariate analysis. In multivariate analysis, a 10 ml/min/1.73 m(2) decrease in eGFR was associated with a 1.63-fold increased odds of a higher score (95% CI 1·10 - 2.37, p = 0.042). Traditional risk factors such as a history of previous CAD, hypertension, and dyslipidemia remained predictors of a higher Friesinger score. CONCLUSION: Our study demonstrates that kidney function as assessed by eGFR is a significant independent predictor of severity of CAD as determined by the Friesinger score.

Differences in DNA methylation of insulin-like growth factor 2 and cadherin 13 in patients with preeclampsia.

OBJECTIVES: In a previous mass spectrometry study of our research group, 25 proteins were found to be differentially expressed in cerebrospinal fluid of patients with preeclampsia compared to controls. The objective of the current study was to investigate DNA methylation of the genes encoding for the former mentioned proteins in an independent dataset. STUDY DESIGN: In a nested case-control study of the Rotterdam Periconceptional Cohort, placental tissue, umbilical cord white blood cells and human umbilical vein endothelial cells (HUVEC) were obtained of 13 patients with early-onset preeclampsia, 16 patients with late-onset preeclampsia and 83 normotensive controls (27 patients with fetal growth restriction, 20 patients with spontaneous preterm birth and 36 uncomplicated pregnancies). DNA methylation of 783 CpGs in regions of 25 genes was measured. MAIN OUTCOME MEASURES: DNA methylation of selected candidate genes in early- and late-onset preeclampsia compared to fetal growth restriction, spontaneous preterm birth and uncomplicated controls. RESULTS: From the 783 CpGs of the 25 selected genes, 15 CpGs were differentially methylated between early-onset preeclampsia and spontaneous preterm birth (3.80 E-5 ≤ p ≤ 0.036). Four CpGs were differentially methylated between early-onset preeclampsia and fetal growth restriction (0.0002 ≤ p ≤ 0.037) and 13 CpGs were differentially methylated between early onset preeclampsia and uncomplicated controls (0.0001 ≤ p ≤ 0.04). CONCLUSION: Differences in DNA methylation were found in placental tissue, umbilical cord white blood cells and HUVEC of patients with early onset preeclampsia compared to (un)complicated controls, but not in patients with late-onset preeclampsia. The genes showing the largest differential methylation encode insulin-like growth factor 2 binding protein and receptor and cadherin 13.

Single walled carbon nanotubes induce indirect cytotoxicity by medium depletion in A549 lung cells.

The ability of two types of single walled carbon nanotubes (SWCNT), namely Arc Discharge (AD) and HiPco single walled carbon nanotubes, to induce an indirect cytotoxicity in A549 lung cells by means of medium depletion was investigated. The nanotubes were dispersed in a commercial cell culture medium and subsequently removed by centrifugation and filtration. Spectroscopic analysis confirmed the removal of the nanotubes and showed differing degrees of alteration of the composition of the medium upon the removal of the nanotubes. The ability to induce an indirect cytotoxic effect by altering the medium was evaluated using two endpoints, namely the Alamar Blue (AB) and the Clonogenic assay. Exposure of the A549 cells to the depleted medium which had previously contained carbonaceous nanoparticles, revealed significant cytotoxicity for both endpoints employed. The results presented demonstrate that single walled carbon nanotubes can induce an indirect cytotoxicity by alteration of cell culture medium (in which they have previously been dispersed) which potentially results in a false positive toxic effect being observed in cytotoxicity studies.

Clock controls circadian period in isolated suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals, and one molecular regulator of circadian rhythms is the Clock gene. Here we studied the discharge patterns of SCN neurons isolated from Clock mutant mice. Long-term, multielectrode recordings showed that heterozygous Clock mutant neurons have lengthened periods and that homozygous Clock neurons are arrhythmic, paralleling the effects on locomotor activity in the animal. In addition, cells in dispersals expressed a wider range of periods and phase relationships than cells in explants. These results suggest that the Clock gene is required for circadian rhythmicity in individual SCN cells and that a mechanism within the SCN synchronizes neurons and restricts the range of expressed circadian periods.

Early- and late-onset preeclampsia and the DNA methylation of circadian clock and clock-controlled genes in placental and newborn tissues.

The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case-control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09E-5≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78E-5≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36E-4≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52E-6≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL of early-onset PE compared with spontaneous preterm birth. Implications of the tissue-specific variations in epigenetic programming for circadian performance and long-term health need further investigation.

The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons.

Before their exocytotic release during stimulation of nerve terminals, nonpeptide neurotransmitters are loaded into synaptic vesicles by specific transporters. Recently, a protein initially identified as brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) has been shown to represent a vesicular glutamate transporter (VGLUT1). In this study, we investigated whether a highly homologous "differentiation-associated Na(+)-dependent inorganic phosphate transporter" (DNPI) is involved in glutamatergic transmission. Vesicles isolated from BON cells expressing recombinant DNPI accumulated l-glutamate with bioenergetical and pharmacological characteristics identical to those displayed by VGLUT1 and by brain synaptic vesicles. Moreover, DNPI localized to synaptic vesicles, at synapses exhibiting classical excitatory features. DNPI thus represents a novel vesicular glutamate transporter (VGLUT2). The distributions of each VGLUT transcript in brain were highly complementary, with only a partial regional and cellular overlap. At the protein level, we could only detect either VGLUT1- or VGLUT2-expressing presynaptic boutons. The existence of two VGLUTs thus defines distinct subsets of glutamatergic neurons.

Interaction of FliI, a component of the flagellar export apparatus, with flagellin and hook protein.

FliI is a key component of the flagellar export apparatus in Salmonella typhimurium. It catalyzes the hydrolysis of ATP which is necessary for flagellar assembly. Affinity blotting experiments showed that purified flagellin and hook protein, two flagellar axial proteins, interact specifically with FliI. The interaction of either of the two proteins with FliI, increases the intrinsic ATPase activity. The presence of either flagellin or hook protein stimulates ATPase activity in a specific and reversible manner. A Vmax of 0.12 nmol Pi min-1 microgram-1 and a Km for MgATP of 0.35 mM was determined for the unstimulated FliI; the presence of flagellin increased the Vmax to 0.35 nmol Pi min-1 microgram-1 and the Km for MgATP to 1.1 mM. The stimulation induced by the axial proteins was fully reversible suggesting a direct link between the catalytic activity of FliI and the export process.

Four-factor prothrombin complex concentrate reverses apixaban-associated bleeding in a rabbit model of acute hemorrhage.

BACKGROUND: Apixaban is a direct factor Xa inhibitor approved for the treatment and prevention of thromboembolic disease. There is a lack of data regarding its reversal in cases of acute bleeding or prior to emergency surgery that needs addressing. OBJECTIVES: This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex(®) /Kcentra(®) , CSL Behring) can effectively reverse apixaban-associated bleeding in an in vivo rabbit model and evaluated the correlations between in vivo hemostasis and in vitro coagulation parameters. METHODS: For dose-finding purposes, anesthetized rabbits were treated with a single intravenous dose of apixaban (800-1600 µg kg(-1) ) and, following a standardized kidney incision, volume of blood loss and time to hemostasis were measured. In a subsequent study phase, anesthetized rabbits were treated with apixaban 1200 µg kg(-1) followed by 4F-PCC (6.25-100 IU kg(-1) ), and the effects on the same bleeding parameters were assessed. In parallel, coagulation parameters were monitored. RESULTS: Dose-dependent increases in time to hemostasis and total blood loss were observed post apixaban administration. Preincision treatment with 4F-PCC resulted in a statistically significant reversal in bleeding time (all doses) and volume (doses ≥ 12.5 IU kg(-1) ). Of the coagulation parameters measured, thrombin generation initiated using the RD reagent (phospholipids only) was the most sensitive to in vivo measures of 4F-PCC's hemostatic efficacy, although some correlations were also observed for prothrombin time and whole blood clotting time. CONCLUSIONS: In this rabbit model of acute hemorrhage, 4F-PCC showed potential for reversing the bleeding effects of apixaban. Clinical data in apixaban-treated patients are needed to confirm these results.

Glial and light-dependent glutamate metabolism in the suprachiasmatic nuclei.

The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system.

The role of Clock in the developmental expression of neuropeptides in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the dominant circadian pacemaker in mammals. To understand better the ontogeny of mouse SCN and the role of the pacemaker in peptide expression, the authors examined the distribution of cells that were immunoreactive for vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) in wild type and Clock mutant mice at two developmental stages. Clock homozygous mice failed to show the dramatic increase in the number of VIP-immunoreactive (VIP-ir) neurons from postnatal day 6 (P6) to P30 that was found in the SCN of wild type mice. The number of AVP-ir neurons was relatively constant in the postnatal SCN but was significantly reduced in Clock/Clock mice. The effects of the Clock mutation varied with position in the SCN for both peptides. Densitometry of immunolabeled brains indicated that the Clock mutation reduced AVP expression specifically in the SCN and not in other brain areas. The SCN did not significantly change shape or size with age or Clock genotype. Taken together, these results indicate that the neonatal mouse SCN has its full complement of cells, some of which are not yet mature in their neuropeptide content. Furthermore, the observation that the Clock mutation appears to act on a subset of AVP and VIP cells suggests heterogeneity within these cell classes in the SCN.

Retinal anatomy of Chorocaris chacei, a deep-sea hydrothermal vent shrimp from the Mid-Atlantic Ridge.

Exploration of deep-sea hydrothermal vents over the past quarter century has revealed that they support unique and diverse biota. Despite the harsh nature of the environment, vents along the Mid-Atlantic Ridge are dominated by large masses of highly motile Bresiliid shrimp. Until 1989, when it was discovered that the vent shrimp Rimicaris exoculata possesses a hypertrophied dorsal eye, many believed that animals populating hydrothermal vents were blind. Chorocaris chacei (originally designated Rimicaris chacei) is a Bresiliid shrimp found at hydrothermal vent fields along the Mid-Atlantic Ridge. Like R. exoculata, C. chacei has a hypertrophied retina that appears to be specialized to detect the very small amount of light emitted from the orifices of black smoker hydrothermal vent chimneys. C. chacei lacks the sophisticated compound eyes common to other decapod crustaceans. Instead, it has a smooth cornea, with no dioptric apparatus, apposed by a tightly packed, massive array of photosensitive membrane. Photoreceptors in the C. chacei retina are segmented into a hypertrophied region that contains the photosensitive membrane and an atrophied cell body that is roughly ten times smaller in volume than the photosensitive segment. The microvillar photosensitive membrane is consistent in structure and ultrastructure with the rhabdoms of decapod and other invertebrate retinas. However, the volume density of photosensitive membrane (> or =60%) exceeds that typically observed in invertebrate retinas. The reflecting pigment cells commonly found in decapod retinas are represented in the form of a matrix of white diffusing cells that exhibit Tyndall scattering and form an axial sheath around the photoreceptors. All photoreceptor screening pigment granules and screening pigment cells are restricted to the region below the photoreceptor nuclei and are thereby removed from the path of incident light. No ultrastructural evidence of rhythmic cycling of photosensitive membrane was observed. The morphological adaptations observed in the C. chacei retina suggest that it is a high-sensitivity photodetector that is of functional significance to the animal.

Circadian rhythms in firing rate of individual suprachiasmatic nucleus neurons from adult and middle-aged mice.

The suprachiasmatic nucleus contains a biological clock that drives circadian rhythms in vivo and in vitro. It has been suggested that the suprachiasmatic nucleus is a primary target of the aging process, because age-related changes in behavioral rhythms are mirrored in alterations in circadian pacemaker function. Using long-term, single-cell recording, we assessed the effect of age on firing-rate patterns of individual suprachiasmatic nucleus neurons of young adult (2-4 months) and middle-aged (9-11 months) C3H mice. Individual suprachiasmatic nucleus neurons from adult mice maintained in culture for at least one week exhibited robust circadian rhythms in spontaneous activity that were similar in the free-running period (23.7+/-0.3 h mean+/-S.E.M.) to recordings from neurons dispersed from neonatal tissue, and showed evidence of entrainment to prior light cycles by exhibiting peak activity, in vitro, approximately 4.0+/-0.3 h (mean+/-S.E.M.) after the time of expected light onset. Aging led to a decreased amplitude of impulse activity in dispersed suprachiasmatic nucleus neurons and increased variability in the circadian waveform. From these results we suggest that age-related deterioration in circadian clock function occurs at the level of individual cells, which may account for some of the age-related deficits observed in the expression of behavioral rhythmicity.

Localization of VGLUT3, the vesicular glutamate transporter type 3, in the rat brain.

We have recently identified a third subtype of glutamate vesicular transporter (VGLUT) named VGLUT3. In the present study, we provide a detailed account of the regional and cellular distributions of VGLUT3 in the rat brain, using specific nucleotide probes and antisera. The distribution of VGLUT3 protein was compared with that of the other vesicular transporters (VGLUT1 and VGLUT2). All the areas expressing VGLUT3 also contain high levels of VGLUT1 and -2 proteins, but, at a finer level of analysis, the distribution of the three subtypes differs. Unlike VGLUT1 and -2, VGLUT3 expression is limited to discrete cell populations. Neurons containing VGLUT3 transcript are essentially observed in the caudate-putamen, the olfactory tubercle, the nucleus accumbens, the hippocampus, the interpeduncular nucleus and the dorsal and medial raphe nuclei. More scattered populations of VGLUT3 expressing neurons are found in the cerebral cortex. The distribution of the VGLUT3 protein, as determined with specific antisera, overlaps with that of the transcript in the caudate-putamen, olfactory tubercles, hippocampus, cortex, interpeduncular nucleus, and raphe nuclei, suggesting that VGLUT3 is essentially present in local projection neurons in these regions. Microscopic examination reveals staining of terminals and perikarya. Furthermore, co-localization studies indicate that VGLUT3 is present in GABAergic interneurons in the hippocampus, as well as in the interpeduncular nucleus. However, other regions, such as the substantia nigra (pars compacta), the ventral tegmental area, and the parabigeminal nucleus, receive a dense VGLUT3 terminal labeling although they do not contain VGLUT3 expressing neurons. In these regions, VGLUT3 immunoreactivity may be present in terminals of long projecting neurons. This subclass of glutamatergic afferents differs from other "classical" excitatory terminals that express VGLUT1 or VGLUT2. The distribution of VGLUT3 in the rat brain suggests an unsuspected function of vesicular glutamate transport in subsets of interneurons and in neuromodulatory neurons.