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J Kehr

Publications and source records attributed to J Kehr.

At least 19 recordsLinked to original sources
PubMed2006

Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection.

The present paper provides an overview on currently developed derivatization chemistries and techniques for determination of monoamine neurotransmitters serotonin (5-HT), norepinephrine (NE) and dopamine (DA) in microdialysis samples by microbore liquid chromatography with fluorescence detection. In mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine (BA), forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity was achieved for reaction with 1,2-diphenylethylenediamine (DPE) rather than with BA, therefore for simultaneous determination of 5-HT, NE and DA in brain microdialysates, a two-step derivatization with BA followed by DPE was developed. The detection limits for 5-HT, NE and DA were 0.2, 0.08 and 0.13 fmol, respectively, in an injection volume of 20 microL, which corresponds to concentrations of 30, 12 and 19.5 pm, respectively in standard solution prior to derivatization. The experimental data presented demonstrate the ability of the technique to simultaneously monitor neuronally releasable pools of monoamine neurotransmitters in the rat and mouse brains at basal conditions and following pharmacological treatments or physiological stimuli. These techniques play an important role in drug discovery and clinical investigation of psychiatric and neurological diseases such as depression, schizophrenia and Parkinson's disease.

Brain↗
PubMed2005

Effect of water stress on lupin stem protein analysed by two-dimensional gel electrophoresis.

Lupinus albus plants can withstand severe drought stress and show signs of recovery 24 h after rewatering (RW). Two-dimensional gel electrophoresis was used to evaluate the effect of water deficit (WD) on the protein composition of the two components of the lupin stem (stele and cortex). This was performed at three distinct stress levels: an early stage, a severe WD, and early recovery. Protein characterisation was performed through mass spectrometric partial sequencing. Modifications in the protein expression were first noticed at 3 days of withholding water, when the plant water status was still unaffected but some decrease in the relative soil water content had already occurred. An increase in serine proteases, possibly associated with WD sensing, was an early alteration induced by WD. When the stress severity increased, a larger number of stem proteins were affected. Immunophilin, serine protease and cysteine protease (well-known components of animal sensing pathways) were some of these proteins. The simultaneous expression of proteases and protease inhibitors that reacted differently to the stress level and to RW was found. Although the level of protease inhibitors was significantly raised, RW did not cause de novo expression of proteins. Many amino acid sequences did not match known sequences of either protein or expressed sequence tag databases. This emphasises the largely unknown nature of stem proteins. Nevertheless, some important clues regarding the way the lupin plant copes with WD were revealed.

Amino Acid Sequence↗
PubMed2004

Effects of isoflurane on prefrontal acetylcholine release and hypothalamic Fos response in young adult and aged rats.

This experiment investigated the influence of age on prefrontal acetylcholine (ACh) release and Fos response in the hypothalamic paraventricular nucleus and the nucleus tractus solitarius (NTS) of rats following isoflurane anesthesia. It is known that isoflurane decreases acetylcholine release in most brain regions. In the present study, we found that the level of prefrontal acetylcholine was significantly lower in 28-month-old rats (14% of baseline) than in 3-month-old rats (38% of baseline) during 2 h of isoflurane anesthesia (P < 0.05). The old rat group showed significantly greater Fos induction in the paraventricular nucleus compared to the young adult rat group (P < 0.05), indicating that the old rats were subjected to stress. No difference in Fos response was noted in the nucleus tractus solitarius. The old rats displayed a significant increase in feeding behavior during the 3-h recovery period (P < 0.05), but there was no difference in overall acetylcholine levels. Taken together, these findings suggest that isoflurane anesthesia influences old rats more profoundly than young adult rats with regard to reductions in acetylcholine release and stress responses. This may have implications for understanding the development of postoperative delirium in aged patients.

Acetylcholine↗
PubMed2004

Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition.

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.

Acetylcholine↗
PubMed2004

Striatal plasticity at the network level. Focus on adenosine A2A and D2 interactions in models of Parkinson's Disease.

Behavioral and microdialysis studies have been performed on antagonistic A(2A)/D(2) interactions in animal models of Parkinson's Disease. The behavioral analysis involved studies on locomotor activity in reserpinized mice, haloperidol-induced catalepsy in rats and rotational behavior in rats with unilateral 6-OHDA lesions of the ascending DA pathways (Ungerstedt model). Dual probe microdialysis studies were indirectly performed on the striatopallidal GABA neurons by studying extracellular glutamate levels in the striatum and globus pallidus of the awake freely moving rat. The striatum was perfused with A(2A) and/or D(2) agonists via reverse microdialysis. The results show that the A(2A) antagonists SCH58261 and KF17837 can increase locomotor activity in reserpinized mice and produce contralateral rotational behavior only after administration of subthreshold doses of l-DOPA or the D(2) like agonist quinpirole. Furthermore, antagonizing the A(2A) receptor (R) reduced haloperidol induced catalepsy. The behavioral results underline the view that A(2A) antagonists act by blocking A(2A) R in A(2A)/D(2) heterodimers where A(2A) R inhibits the D(2) R transduction and D(2) inhibits the adenylate cyclase (AC) activated by A(2A) R. The microdialysis studies show that the A(2A) agonist CGS21680 striatally coperfused with the D(2) agonist quinpirole more potently counteract the D(2) agonist (quinpirole) induced reduction of pallidal glutamate levels in the DA denervated vs the control striatum indicating an enhancement of the inhibitory A(2A)/D(2) interaction. In the DA denervated but not in the control striatum the A(2A) agonist CGS21680 could strongly increase striatal glutamate levels, indicating an increased receptor signaling in the A(2A) R located on the striatal glutamate terminals, where also D(2) like R exist, here probably as D(4). Thus, the signaling of this A(2A) R may be set free by the loss of D(4) tone on the AC activated by A(2A) in this postulated A(2A)/D(4) heteromer on the glutamate terminals. Taken together, the results indicate that the antiparkinsonian actions of A(2A) antagonists probably are produced by blockade of A(2A) R in the A(2A)/D(2) heterodimers mainly located in the striatopallidal GABA neurons.

Adenosine A2 Receptor Antagonists↗
PubMed2004

The role of alpha1- and alpha2-adrenoreceptors on venlafaxine-induced elevation of extracellular serotonin, noradrenaline and dopamine levels in the rat prefrontal cortex and hippocampus.

The role of adrenergic alpha1- and alpha2-adrenoreceptors in augmentation of venlafaxine-induced elevation of extracellular serotonin (5-HT),noradrenaline (NA) and dopamine (DA) levels in the rat prefrontal cortex (PFC) and hippocampus (HIPP) was studied by in vivo microdialysis in anaesthetized rats. The alpha1-adrenoreceptor antagonist prazosin given alone (0.3 mg/kg, s.c.) induced only a moderate reduction of hippocampal 5-HT and NA levels. The alpha2-adrenoreceptor antagonist idazoxan (1.5 mg/kg, s.c.) causes moderate increases in the levels of 5-HT and DA in the PFC. The mixed 5-HT and NA reuptake inhibitor venlafaxine (10 mg/kg, i.p.) increased the efflux of 5-HT, NA and DA almost equally, to approximately 200% of the control levels in the PFC. The levels of 5-HT increased to 310%, an effect approximately twice the effect on NA in the HIPP. Venlafaxine also produced a moderate increase in DA levels in the PFC but had no effect in the HIPP. Pre-treatment with prazosin caused a significant attenuation of the venlafaxine induced 5-HT effect in the PFC, and a moderate increase in DA levels in the HIPP. Prazosin had no significant effect on the venlafaxine-induced increase of the NA levels in PFC or HIPP. A combined treatment of venlafaxine with idazoxan increased the venlafaxine NA and DA effects in PFC by a factor of two and resulted in a very robust five-fold augmentation of NA and DA concentrations in the HIPP. In summary, idazoxan was found to produce a potent enhancement of the venlafaxine effect to increase extracellular NA and DA levels in the PFC and, in particular, in the HIPP. Idazoxan had no effect on venlafaxine-induced elevation of extracellular 5-HT levels in either PFC or HIPP and prazosin induced a decrease of 5-HT in the PFC. The present data suggest that blockade of alpha2-adrenoreceptors may play an important role in augmentation of the effects of mixed monoamine reuptake inhibitors.

Adrenergic alpha-Antagonists↗
PubMed2003

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo.

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2001

Determination of serotonin in microdialysis samples from rat brain by microbore column liquid chromatography with post-column derivatization and fluorescence detection.

The present paper describes a new method for on-line determination of 5-HT in brain microdialysates from awake rats by microbore column liquid chromatography with post-column derivatization and fluorescence detection. The derivatization reagent contained 1 mM benzylamine and 0.5 mM potassium hexacyanoferrate (III), both dissolved in a mixture of acetonitrile and 25 mM borate buffer (pH 11.0) (1:1, v/v). The limit of detection (S/N=3) for 5-HT was 0.5 fmol/20 microl. The samples were injected every 20 min onto a microbore column packed with C18 silica gel. The method exhibits an excellent stability over the periods of at least 12-24 h. The basal levels of 5-HT from 25 awake rats were 7.10+/-1.06 fmol/20 microl in the dorsal hippocampus and 4.64+/-0.91 fmol/20 microl (mean+/-SD) in the striatum. The 5-HT release increased to about 1500% during the perfusion with 100 mM K(+) containing Ringer solution or it was reduced to 60 or 40% during the perfusion with 1 microM tetrodotoxin or calcium free Ringer, respectively. The new method can be used to monitor extracellular 5-HT following acute systemic drug administration.

Animals↗
PubMed2001

Amino acid analysis in five pooled single plant cell samples using capillary electrophoresis coupled to laser-induced fluorescence detection.

In this study 21 amino acid standards, samples of pure phloem sap and samples of pooled mesophyll cells were derivatized with fluorescein isothiocyanate, separated by capillary electrophoresis and detected with laser-induced fluorescence at 488 nm. Two different background electrolytes, a sodium borate buffer containing sodium dodecyl sulfate and a sodium borate buffer containing alpha-cyclodextrin, were used for the separation. Using the sodium dodecyl sulfate buffer, 14 amino acid standards could be separated, spiking identified 12 amino acids in pure phloem sap and 13 amino acids in pooled mesophyll cells. With the alpha-cyclodextrin containing background electrolyte, a resolution of 20 amino acid standards could be attained, 17 amino acids in pure phloem sap and 10 amino acids in mesophyll cells could be assigned. Leucine and isoleucine comigrated in both buffer systems. All separations were performed with a voltage of +20 kV and completed within 30 min. The detection limits obtained were in the fmol range for the sodium dodecyl sulfate and in the pmol range for the alpha-cyclodextrin background electrolyte. Compared to the one published capillary electrophoresis-based method for the determination of amino acids from few plant cells, the procedure described here allows very high sensitivity due to the use of laser-induced fluorescence detection and opens the possibility to dilute and measure pl samples with an fully automated, commercially available CE system.

Amino Acids↗
PubMed2001

Microdialysis in freely moving mice: determination of acetylcholine, serotonin and noradrenaline release in galanin transgenic mice.

In the present study, we describe micro-surgical methods for simultaneous implantation of a microdialysis probe and an intraventricular injection cannula via their respective guide cannulas into the mouse brain. Basal and stimulated release of acetylcholine (ACh), serotonin (5-HT) and noradrenaline (NA) was determined in the ventral hippocampus of freely moving mice. NA and 5-HT were determined in one run by a newly developed HPLC method based on precolumn derivatization with benzylamine and fluorescence detection. The mice with a loss-of-function mutation of the galanin gene (KO) and the mice that over-expressed galanin (OE) were studied. No significant differences in basal, potassium-stimulated or scopolamine-induced extracellular ACh levels were observed in 4-month-old wild-type (WT) and KO mice. In the aged, 10-month-old animals, the basal extracellular ACh levels were significantly reduced in both WT and KO groups. Galanin (1 nmol i.c.v.) caused a significant reduction of basal extracellular NA by about 40% in both WT and galanin OE mice, however, in the latter group the effect was delayed by almost 2 h. A 10-min forced swimming stress caused a higher increase in release of NA and 5-HT in the OE group than in the corresponding WT mice. Finally, venlafaxin (10 mg/kg i.p.) increased extracellular NA to 400% of the control values in the CBA mice, but only to 250% in the C57BL mice. It is concluded that galanin may play an important role in the cholinergic mechanisms underlying cognitive disorders. Furthermore, modulation by galanin and by behavioral activation, of NA and 5-HT neurotransmission in galanin over-expressing mice indicates its possible role in the aetiology of mood disorders.

Acetylcholine↗
PubMed2001

Matrix-assisted laser desorption/ionization time of flight mass spectrometry peptide mass fingerprints and post source decay: a tool for the identification and analysis of phloem proteins from Cucurbita maxima Duch. separated by two-dimensional polyacrylamide gel electrophoresis.

A combination of gel electrophoresis and mass spectrometry was used to analyze the soluble proteins from phloem sap of Cucurbita maxima Duch. Phloem proteins were separated using two-dimensional gel electrophoresis. Coomassie-stained spots were cut out and subjected to tryptic digestion. To identify proteins, peptide mass fingerprints were determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. In addition, MALDI-TOF post source decay measurements were used to obtain partial sequence information for the proteins. Results from both approaches were used for database searches. In this study, 17 proteins in the mass range 5-50 kDa were analyzed. Of these proteins six could be clearly identified, seven showed significant homologies to known plant proteins, and four were not significantly homologous to database entries. The present study suggests that the applied method is feasible for a large-scale analysis and identification of phloem proteins derived from different organs or from plants kept under various physiological conditions.

Amino Acid Sequence↗
PubMed2001

High resolution spatial analysis of plant systems.

Analysis of organisms using techniques that provide high spatial and temporal resolution is of increasing interest in many disciplines of biomedical research. Although the examination of animal tissues has been the main focus to date, the recent development and improvement of methods for the sampling and handling of single cells and for their biochemical analysis now provide tools for investigating plant as well as animal cells.

Biochemistry↗
PubMed2001

Immunological analysis of phloem sap of Bacillus thuringiensis corn and of the nontarget herbivore Rhopalosiphum padi (Homoptera: Aphididae) for the presence of Cry1Ab.

Phloem sap of transgenic Bacillus thuringiensis (Bt) corn expressing a truncated form of the B. thuringiensis delta-endotoxin Cry1Ab, sap sucking aphids feeding on Bt corn and their honeydew were analysed for presence of Cry1Ab using ELISA. Phloem sap of Bt and non-Bt corn was collected using a newly developed technique with a microcapillary being directly inserted into the phloem tubes. Using this technique, no Cry1Ab was detected in the phloem sap. In contrast, measurable concentrations of Cry1Ab in the range of 1 ppb were detected when phloem sap of pooled leaf samples was extracted using EDTA buffer. This was probably because of Cry1Ab toxin released from damaged cells. When analysing apterous adults of Rhopalosiphum padi L. and their honeydew, no Cry1Ab could be detected. In contrast, Cry1Ab was clearly detected in both larvae of the leaf chewing herbivore Spodoptera littoralis (Boisduval) and their faeces, showing that Cry1Ab is detectable after ingestion and excretion by herbivores. These results suggest that R. padi ingests or contains no or only very low concentrations of Cry1Ab in the range of the detection limit. In consequence it is hypothesized that R. padi as an important prey for beneficial insects in corn is unlikely to cause any harm to its antagonists due to mediating Bt toxin.

Animals↗
PubMed2001

An Arabidopsis inositol phospholipid kinase strongly expressed in procambial cells: synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in insect cells by 5-phosphorylation of precursors.

We have cloned a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) cDNA (AtP5K1) from Arabidopsis thaliana. By the application of cell permeabilization and short-term nonequilibrium labelling we show that expression of AtP5K1 in Baculovirus-infected insect (Spodoptera frugiperda) cells directs synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. The same phosphoinositides were produced by isolated whole-cell membrane fractions of AtP5K1-expressing insect cells. Their synthesis was not affected by adding defined precursor lipids, that is PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, or PtdIns(4,5)P2, in excess, indicating that substrates for the plant enzyme were not limiting in vivo. Enzymatic dissection of lipid headgroups revealed that AtP5K1-directed synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 proceeds via 5-phosphorylation of precursors. Analysis of promoter-reporter gene (beta-glucuronidase) fusions in transgenic plants revealed that expression of the AtP5K1 gene is strongest in vascular tissues of leaves, flowers, and roots, namely in cells of the lateral meristem, that is the procambium. Single-cell sampling of sap from flower stem meristem tissue and neighbouring phloem cells, when coupled to reverse transcriptase--polymerase chain reaction, confirmed preferential expression of AtP5K1 in procambial tissue. We hypothesize that AtP5K1, like animal and yeast PIP5K, may be involved in the control of cell proliferation.

Animals↗
PubMed2000

Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons.

The neuropeptide galanin colocalizes with choline acetyltransferase, the synthetic enzyme for acetylcholine, in a subset of cholinergic neurons in the basal forebrain of rodents. Chronic intracerebroventricular infusion of nerve growth factor induces a 3- to 4-fold increase in galanin gene expression in these neurons. Here we report the loss of a third of cholinergic neurons in the medial septum and vertical limb diagonal band of the basal forebrain of adult mice carrying a targeted loss-of-function mutation in the galanin gene. These deficits are associated with a 2-fold increase in the number of apoptotic cells in the forebrain at postnatal day seven. This loss is associated with marked age-dependent deficits in stimulated acetylcholine release, performance in the Morris water maze, and induction of long-term potentiation in the CA1 region of the hippocampus. These data provide unexpected evidence that galanin plays a trophic role to regulate the development and function of a subset of septohippocampal cholinergic neurons.

Animals↗
PubMed2000

Fetal ventral mesencephalic grafts functionally reduce the dopamine D2 receptor supersensitivity in partially dopamine reinnervated host striatum.

Grafting of ventral mesencephalic tissue in Parkinson's disease results in a partial dopaminergic reinnervation of host brain and dopamine agonist-induced rotational behavior is not completely reversed. To study a possible malfunction of the grafts, extracellular recordings with local applications of quinpirole were utilized and the neurophysiological results showed that a normalization of the upregulated dopamine D2 receptor supersensitivity occurred in reinnervated areas of the host striatum as well as in noninnervated areas remote from the graft innervation. Furthermore, the inhibitory effects on striatal nerve cell firing rate by the D1 receptor agonist SKF 81297 were not different in noninnervated or reinnervated areas of the striatum compared to the control side as seen from the dose-response curves. However, spontaneous striatal neuronal firing was significantly upregulated in noninnervated areas, while it was normalized in areas reached by graft-derived nerve fibers. Dual-probe microdialysis studying potassium-evoked glutamate release revealed that there was no difference in extracellular glutamate levels measured within or lateral to graft dopamine reinnervation. Thus, the upregulated spontaneous activity was not due to a difference in extracellular glutamate levels. The remaining rotational behavior seen after grafting was studied and recordings were performed in the striatum following systemic injection of the D1/D2 agonist apomorphine. The results revealed that apomorphine at the dose used to elicit turning behavior (0.05 mg/kg) still affected striatal neurons in noninnervated areas, while no effect was detected in reinnervated areas and in the intact side. However, a lower dose of apomorphine (0.005 mg/kg) showed no effects on striatal firing in graft reinnervated striata but only after dopamine depletion. In conclusion, the D2 supersensitivity is downregulated in graft-reinnervated striatum as well as in striatal areas lateral to the reinnervation when using selective D2 agonists, but the downregulation is not completely normalized when studying combined effects of D1/D2 agonists. Furthermore, the striatal neurons were firing significantly faster in noninnervated areas compared to reinnervated areas of graft-reinnervated striatum, which was most likely not due to changes in the glutamatergic input.

Animals↗