Genome-wide profiles indicate wolf population connectivity within the eastern Carpathian Mountains.

The Carpathian Mountains provide critical wildlife habitat in central Europe, and previous genome-wide studies have found western Carpathian Mountain wolves (Canis lupus) to be a separate population. Whereas differentiation to the north may be explained by a lowland-mountain transition and habitat fragmentation, the eastern Carpathian Mountains extending through Romania appear to offer continuous wildlife habitat southward. Our objective was to assess gene flow patterns and population connectivity among wolves in Romania, western Ukraine, and the Republic of Moldova. We sought to determine if the Carpathian Mountain region is best described by a north-south gradient in genetic profiles, or whether Romanian wolves show population structure with northern individuals clustering with western Ukraine. We genotyped 48 individuals with 170 000 single nucleotide polymorphism markers, and successful profiles from Romania (n = 27) and Moldova (n = 2) were merged with existing data from western Ukraine (n = 10). Expected heterozygosity was 0.234 (SE 0.001) for Romania and 0.229 (SE 0.001) for western Ukraine, whereas observed heterozygosity values were 0.230 (SE 0.001) versus 0.231 (SE 0.001). Population structure analyses with a maximum likelihood method supported K = 1 population, followed by K = 2 where Romania formed one cluster, and western Ukraine and Moldova formed another. Principal component analysis results were broadly consistent with K = 2. Pairwise FST between western Ukraine and Romania was 0.042 (p = 0.001). Our findings indicated weak population differentiation, and future research may clarify whether the spatial distribution of genetic diversity in the region is associated with environmental and ecological factors such as terrain ruggedness and the distribution of prey species.

Electronic and magnetic properties of the [Ni(salophen)]: An experimental and DFT study.

The effect of the coordination of a Ni(II) ion on the electronic and magnetic properties of the ligand salophen were experimentally and theoretically evaluated. The complex [Ni(salophen)] was synthesized and characterized through FTIR and an elemental analysis. Spectral data obtained using DMSO as a solvent showed that the ligand absorption profile was significantly disturbed after the coordination of the metal atom. In addition to a redshift of the salophen ligand absorption bands, mainly composed by π → π∗ electronic transitions, additional bands of around 470 nm were observed, resulting in a partial metal-to-ligand charge transfer. Furthermore, a significant increment of its band intensities was observed, favoring a more intense absorption in a broader range of the visible spectrum, which is a desired characteristic for applications in the field of organic electronics. This finding is related to an increment of the planarity and consequent electron delocalization of the macrocycle in the complex, which was estimated by the calculation of the current strengths at the PBE0/cc-pVTZ (Dyall.v3z for Ni(II)) level.

Late quaternary dynamics in the Madeira River basin, southern Amazonia (Brazil), as revealed by paleomorphological analysis.

Ancient drainage systems are being increasingly documented in the Amazon basin and their characterization is crucial for reconstructing fluvial evolution in this area. Fluvial morphologies, including elongate belts, are well preserved along the Madeira River. Digital Elevation Model from the Shuttle Radar Topography Mission favored the detection of these features even where they are covered by dense rainforest. These paleomorphologies are attributed to the shifting position of past tributaries of the Madeira River through avulsions. These radial paleodrainage networks produced fan-shaped morphologies that resemble distributary megafans. Distinguishing avulsive tributary systems from distributary megafans in the sedimentary record is challenging. Madeira´s paleodrainage reveals the superposition of tributary channels formed by multiple avulsions within a given time period, rather than downstream bifurcation of coexisting channels. Channel avulsion in this Amazonian area during the late Quaternary is related to tectonics due to features as: (i) straight lineaments coincident with fault directions; (ii) northeastward tilting of the terrain with Quaternary strata; and (iii) several drainage anomalies, including frequent orthogonal drainage inflections. These characteristics altogether lead to propose that the radial paleodrainage present at the Madeira River margin results from successive avulsions of tributary channels over time due to tectonics.

Serum and plasma levels of FGF-2 and VEGF in healthy blood donors.

VEGF-A and FGF-2 are two angiogenic growth factors involved in the growth and invasion of solid tumours and haematological malignancies. They are also believed to play an important role in cardiovascular and inflammatory diseases. Several studies dealing with measurement of circulating FGF-2 and VEGF have been published during the last decade. We have studied the levels of FGF-2 and VEGF-A in serum and EDTA plasma from 80 healthy blood donors. The samples were analyzed using the most widely used and commercially available ELISAkits. There was no significant effect of age on any of the assays and no effect of sex on P-FGF-2, S-VEGF-A and P-VEGF-A. Using the 97.5th percentile we obtained the following reference values: P-FGF-2 < 6.4 ng/l; S-VEGF-A < 500 ng/l; P-VEGF-A < 80 ng/l. Separate gender based reference intervals were made for S-FGF-2 as women had significantly higher S-FGF-2 values. Reference values for S-FGF-2 were <4.0 ng/l (men) and <10.8 ng/l (women).

Evaluation of the effect of high-energy proton irradiation treatment on meningiomas by means of 11C-L-methionine PET.

A remnant meningioma of WHO grade I that is located at the base of the skull and is treated with radiotherapy has to be followed up for at least 5-10 years to evaluate the treatment effect and detect recurrence. The tumour has to grow considerably to show detectable volume increase on computed tomography (CT) or magnetic resonance imaging (MRI). Owing to the location at the base of the skull, a small increase in tumour volume may be hazardous. It is thus important to find a method to evaluate treatment effects earlier and potentially detect those tumours that have a tendency to grow. Nineteen patients with intracranial meningiomas were given irradiation with the 180-MeV proton beam at the Svedberg Laboratory, Uppsala, Sweden. The fractionation schedule used was in general a total dose of 24 Gy in four consecutive daily 6-Gy fractions. Serial 11C-Lmethionine PET examinations were used to evaluate the effect of stereotactic proton beam treatment. The radioactivity uptake in the tumour was evaluated as the ratio to the uptake in normal brain tissue. The follow-up period thus far is 36 months. In 15 of the 19 patients, 11C-L-methionine uptake was reduced 36 months after irradiation compared with the pre-treatment uptake of the tracer. In the total patient group the average reduction was 19.4%. Our results reveal that proton beam irradiation of meningiomas had an inhibitory effect on the methionine uptake in the meningiomas, although tumour size remained unchanged. The combination of unchanged tumour morphology and a reduction in methionine uptake after irradiation suggests that 11C-L-methionine PET might enable earlier evaluation of the treatment effect than is possible with CT or MRI.

Effects of intermittent and continuous subchronic administration of raclopride on motor activity, dopamine turnover and receptor occupancy in the rat.

With the purpose of finding means to circumvent the marked pharmacokinetic differences of raclopride between rats and man, the effects of intermittent and continuous administration of raclopride were compared in rats. Intermittent administration of raclopride via subcutaneous injections resulted in a prompt increase of dopamine (DA) turnover and decrease of motor activity but these effects were of short duration, probably due to rapidly decreasing raclopride DA D2 receptor occupancy. In contrast, but similar to schizophrenic patients on raclopride treatment, stable plasma raclopride levels and a steady DA D2 receptor occupancy above 70% were produced in the caudate-putamen and nucleus accumbens/olfactory tubercle, when raclopride was administered continuously via minipumps at daily doses above 2 mg/kg. Tolerance to the acute effects of raclopride on DA turnover and locomotion was found with both routes of administration but it was more marked with continuous administration. At continuous raclopride administration, tolerance to the effects of raclopride on DA turnover and spontaneous motor activity as well as supersensitivity to amphetamine-induced motor activity occurred when 70% or more of DA D2 receptor sites were occupied, i.e. the same degree of receptor occupancy as found in patients given therapeutic doses of raclopride.

Differential effects of neuroleptic agents on hepatic cytochrome P-450 isozymes in the male rat.

We report the effects of various dopamine receptor-blocking drugs on gene and protein expression, as well as the activity of several hepatic cytochrome P-450 (CYP) enzymes in the male Sprague-Dawley rat. At equipotent doses (with respect to receptor blockade and behavioural tests), the dopamine D2-receptor selective sulpiride and remoxipride gave a conspicuous down-regulation of CYP2C11 and its associated androstenedione 16 alpha-hydroxylation activity as well as of the CYP2C11-specific mRNA. The average immunoidentified CYP2C11 levels correlated with the CYP2C11-specific mRNA levels in all treatment groups (r = 0.994), indicating a transcriptional mechanism. The CYP3A protein was also selectively down-regulated. In contrast, androstenedione 5 alpha-reduction was significantly increased. Clozapine, a non-selective neuroleptic, gave the same effects on the steroid metabolism as sulpiride and remoxipride. In contrast, diverging effects were observed for clozapine, compared to sulpiride and remoxipride, on the immunoidentified CYP1A2, CYP2B1, and CYP3A. These proteins were elevated by clozapine, and down-regulated by sulpiride and remoxipride. Our results are of interest for the interpretation of preclinical dose ranging toxicity tests of neuroleptic agents in rats. They may also be relevant in relation to certain interactions and adverse reactions observed in the clinical use of these drugs. The down-regulation of certain CYP enzymes is most likely mediated by an interaction with the growth hormone secretion.

Cellular uptake of intracerebroventricularly administered biotin- or digoxigenin-labeled antisense oligodeoxynucleotides in the rat.

1. Antisense oligodeoxynucleotides (ODNs) internally labeled with biotin or digoxigenin were injected into the lateral ventricle of rats and the distribution of the labeled ODNs was examined at several timepoints following the intracerebroventricular (icv) injections. The stability of these injected antisense ODNs, which had no backbone modifications, was also studied by performing recovery experiments. 2. The most intense labeling was observed near the injection site, in periventricular areas, and in perivascular regions. Many of the labeled cells appeared to be neurons, and both the cytoplasm and the nuclei were stained. The labeled cells were detected 15 min after icv injection, demonstrating that the antisense ODNs were taken up rapidly by cells in the parenchyma. The digoxigeninated antisense ODNs were presented in both the cytoplasmic and the nuclear fractions of rat brain extracts, however, the levels appeared to be much lower in the nuclear fractions. 3. Antisense ODNs injected into the lateral ventricle seemed to follow the bulk flow of cerebrospinal fluid (CSF), i.e., from the injection site in the lateral ventricle, through the ventricular system, to the subarachnoid spaces and the perivascular spaces. From the ventricular and perivascular spaces, the antisense ODNs diffused into the extracellular space and were taken up by cells. The full-length digoxigeninated antisense ODNs were detectable within cells after only 15 min, indicating their rapid uptake. In addition, the antisense ODNs appeared to be relatively stable in the brain since the full-length digoxigeninated ODNs were still detectable after 4 hr.

Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing.

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.

Activation of a bulbospinal opioidergic projection by pain stimuli in the awake rat.

We examined the expression of preproenkephalin mRNA in pain-modulating regions of the rat's brainstem using in situ hybridization histochemistry. We found that neurones in the Kölliker-Fuse nucleus, which receives projections from spinal nociceptive-specific cells, express enkephalin mRNA, that this expression is increased by noxious pinch applied to the skin of awake animals, and that these enkephalinergic neurones are part of a descending system that terminates in the rostroventral medulla and the spinal cord. These findings show that natural noxious stimuli activate opioidergic bulbospinal neurones that could directly modulate spinal nociceptive transmission.

Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions.

The excitatory amino acid, L-glutamate, acting through its N-methyl-D-aspartate (NMDA) receptor, may contribute to neuronal death following cerebral vascular occlusion. In support of this hypothesis, NMDA receptor antagonists reduce the volume of infarction produced by occlusion of the middle cerebral artery in vivo and attenuate Ca2+ influx and neuronal death elicited by L-glutamate or NMDA in vitro. A complementary DNA coding for a major component of the NMDA receptor channel complex, a single protein of M(r) 105.5K (NMDA-R1), has been isolated from rat brain. Here we demonstrate that inhibition of the synthesis of NMDA-R1 by treatment with antisense oligodeoxynucleotides selectively reduces the expression of NMDA receptors, prevents the neurotoxicity elicited by NMDA in vitro and reduces the volume of the focal ischaemic infarction produced by occlusion of the middle cerebral artery in the rat.

Subchronic treatment of rats with remoxipride fails to modify sigma binding sites in the brain.

The effects of 14 days' treatment of rats with haloperidol, remoxipride or both combined on the sigma binding sites in whole brain and liver were determined. The compounds were given subcutaneously via osmotic minipumps at a dose of 0.25 mg/rat/day (corresponding to about 1 mg/kg body weight/day at start) for haloperidol and 2.5 mg/rat/day (10 mg/kg/day) for remoxipride hydrochloride. The sigma recognition sites were determined after a washout period of 2 days with the radioligand [3H](+)-N-propyl-3-(3-hydroxyphenyl)piperidine ([3H](+)-3-PPP). It was found that the haloperidol treatment but not the remoxipride treatment decreased the density of the sigma sites in the brain, without any effect on the affinities of the ligands. Haloperidol had no effect on the binding of [3H](+)-3-PPP to the sigma recognition sites in the liver.

Origin of neuronal inputs to the region of the tuberomammillary nucleus of the rat brain.

The origin of afferent connections of the hypothalamic tuberomammillary nucleus has been examined by using retrograde and anterograde tracing techniques. Retrogradely labeled neurons were found in about 70 cell groups of the forebrain and brainstem after injection of tracer into the ventral subgroup of the tuberomammillary nucleus. The majority of the labeled neurons were seen in the forebrain, with particularly large numbers in the infralimbic cortex, lateral septal nucleus, and preoptic region. The anterograde tracing experiments supported the general results of the retrograde tracing experiments. However, we did not observe any single cell group that selectively projected to the cell-rich core of the nucleus. In general, only a few fibers entered the core, whereas many labeled fibers seemed to terminate immediately adjacent to the cell group. Thus the target for the afferents is not primarily the perikarya of the neurons of the tuberomammillary nucleus, but either dendrites radiating out from the nucleus or neurons not belonging to the tuberomammillary nucleus. The results of the present study demonstrate that the histaminergic tuberomammillary nucleus derives its main input from the limbic forebrain. Through their widespread projections, the histaminergic neurons may transmit information originating from the limbic system to most if not all parts of the brain.

Different laminar distributions of dopamine D1 and D2 receptors in the rat hippocampal region.

The distribution of dopamine (DA) D1 and D2 receptors in the hippocampal region was studied using in vitro receptor autoradiography with 125I-SCH 23982 and 125I-NCQ 298, respectively. The specific binding of both ligands indicated the existence of D1 as well as D2 receptors in the rat hippocampal region. A closer analysis revealed, however, a different laminar distribution of the two receptor subtypes: layers with a high density of one DA receptor subtype had low density of the other. Thus, in the entorhinal cortex, which contained the highest densities of both subtypes, the following pattern was seen. Layers 2, 4, 5 and 6 had high densities of D1 receptors and low densities of D2 receptors while layers 1 and 3 had high densities of D2 receptors and low densities of D1 receptors. The parasubiculum contained D1 receptors but not D2 receptors and the presubiculum had D2 receptors in layer 2 but few D1 receptors. Similar patterns were recorded in Ammon's horn: the part of stratum lacunosum-moleculare which was rich in D1 was poor in D2 receptors. The interdigitating laminar distribution of D1 and D2 receptors suggests that the effects of DA are mediated via D1 and D2 receptors located at different levels along the intrinsic hippocampal circuit.

Dopamine D2 receptors in the rat, monkey and the post-mortem human hippocampus. An autoradiographic study using the novel D2-selective ligand 125I-NCQ 298.

The distribution of dopamine D2 receptors in the hippocampal region of the rat, monkey and the postmortem human brain was studied with in vitro receptor autoradiography using the selective salicylamide ligand 125I-NCQ 298. Specific binding was defined in the presence of the D2-selective compound raclopride. In all 3 species, higher densities of specifically bound 125I-NCQ 298 was found in the retrohippocampal structures than in the hippocampus proper. In the rat, layers 1 and 3 of the entorhinal cortex and layer 2 of the presubiculum were found to be rich in specific binding sites. In the monkey, the highest densities were detected in the deep layers (4 through 6) of the entorhinal cortex (EC) and in layer 2 of the presubiculum. Relatively high density of binding was found in the granule cell layer of area dentata. In the human brain, less specific binding was seen as compared to the other two species; the highest densities occurred in the outer layers of the presubiculum and in the hilus of area dentata. These findings show that D2 receptors are present in the hippocampal region and that the retrohippocampal region, including the entorhinal cortex, is enriched in dopamine D2 receptors.

GABA-like immunoreactivity in the tuberomammillary nucleus: an electron microscopic study in the rat.

The organization of GABAergic elements in the histaminergic tuberomammillary nucleus has been examined by using antibodies against gamma-aminobutyric acid (GABA) and light and electron microscopy. Most neuronal perikarya of the ventral subgroup of the tuberomammillary nucleus were GABA immunoreactive (GABA-i). The morphology of the GABA-i perikarya was similar to the morphology of histaminergic perikarya described by Hayashi et al. ('84: J. Comp. Neurol. 229: 223-241) and Wouterlood et al. ('86: J. Comp. Neurol. 252:227-243). The GABA-i perikarya were contacted by relatively few terminals. The mean bouton covering ratio of GABA-i perikarya was 6.1%, whereas the mean bouton covering ratio for GABA-i dendrites in the tuberomammillary nucleus was 31%. Some of the presynaptic terminals were GABA-i. In addition, GABA-i perikarya and dendrites formed close contacts that never presented synaptic specializations. These results suggest that neurons of the histaminergic tuberomammillary nucleus contain the neurotransmitter GABA. Furthermore, GABA may act as a modulator of cellular processes within the tuberomammillary nucleus.

Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chloride channels.

The endocrine part of the pancreas plays a central role in blood-glucose regulation. It is well established that an elevation of glucose concentration reduces secretion of the hyperglycaemia-associated hormone glucagon from pancreatic alpha 2 cells. The mechanisms involved, however, remain unknown. Electrophysiological studies have demonstrated that alpha 2 cells generate Ca2+-dependent action potentials. The frequency of these action potentials, which increases under conditions that stimulate glucagon release, is not affected by glucose or insulin. The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is present in the endocrine part of the pancreas at concentrations comparable to those encountered in the central nervous system, and co-localizes with insulin in pancreatic beta cells. We now describe a mechanism whereby GABA, co-secreted with insulin from beta cells, may mediate part of the inhibitory action of glucose on glucagon secretion by activating GABAA-receptor Cl- channels in alpha 2 cells. These observations provide a model for feedback regulation of glucagon release, which may be of significance for the understanding of the hypersecretion of glucagon frequently associated with diabetes.

Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation.

Monoaminergic innervation of a histamine-producing cell group, the tuberomammillary nucleus in the posterior hypothalamus, was investigated in the rat by light and electron microscopic immunohistochemical techniques. Immunohistochemical staining of sections of the posterior hypothalamus was demonstrated afferent fibers immunoreactive to tyrosine hydroxylase in ventral and medial subgroups of the tuberomammillary nucleus afferent fibers immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenyletanolamine-N-methyltransferase (PNMT), and serotonin (5-HT). TH- and DBH-immunoreactive fibers were similar and were evenly and densely distributed throughout the tuberomammillary nucleus. Fibers stained with 5-HT antibodies were also present throughout the tuberomammillary nucleus but exhibited the densest labeling in the dendritic layer adjacent to the glia limitans in the ventral subgroup. Innervation by PNMT-immunoreactive axons was sparse. Electron microscopic analysis of TH-, DBH-, and 5-HT-immunoreactive fibers in the tuberomammillary nucleus revealed vesicle-containing terminal boutons, which formed synapses with dendrites of varying size. Synaptic contacts with nerve cell bodies were not found. Retrograde transport of the fluorescent dye Fast Blue injected into the tuberomammillary nucleus, combined with immunofluorescent staining with anti-TH, anti-DBH, anti-PNMT, and anti-5-HT antibodies, showed that monoaminergic input to the tuberomammillary nucleus originated mainly from the adrenergic and noradrenergic cell groups C1-C3 and A1-A2, respectively, and from the serotoninergic cell groups B5-B9 as designated by Dahlström and Fuxe ('65). Few double-labeled neurons were found in the nucleus locus coeruleus and the dopaminergic cell groups of the rostral brain stem. The present findings suggest that the activity of the histamine-producing neurons of the tuberomammillary nucleus is influenced by monoaminergic neurons in the ventrolateral and dorsomedial medulla oblongata and the raphe nuclei of the rostral brainstem.

Neonatal vasopressin antagonist treatment facilitates adult copulatory behavior in female rats and increases hypothalamic vasopressin content.

This study confirms that i.c.v. administration of Arg-vasopressin (AVP, 20 ng) inhibits copulatory behavior in female rats (lordosis response, LR) and demonstrates that this inhibitory effect is blocked by the vasopressin antagonist (3-mercapto-3-methylbutyryl-Tyr-[Me])arginine vasopressin (dPTyr(Me)AVP, 20 ng, i.c.v.). The effects of neonatal AVP antagonist treatment on adult female copulatory behavior, hypothalamic content of AVP and AVP-like immunoreactive (AVP-ir) neurons were examined. dPTyr(Me)AVP was given to female rats 1 microgram/animal/day s.c. from day 1 through to day 7 (day 0 = day of birth). The females were ovariectomized as adults and sexual receptivity activated by submaximal doses of estradiol plus progesterone. The LR was significantly facilitated in the neonatally dPTyr(Me)AVP treated females who also showed a higher content and an increased number of AVP-ir neurons in the suprachiasmatic nucleus compared to saline controls. Functional, biochemical and immunocytochemical evidence is provided that neonatal exposure to an AVP antagonist induces persistent changes in central vasopressinergic neuronal mechanisms.