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1.
Pharmeur Bio Sci Notes ; 2018: 12-36, 2018.
Article in English | MEDLINE | ID: mdl-29845933

ABSTRACT

Since the opening for signature of the European Convention for the Protection of Animals Used for Experimental and Other Scientific Purposes in 1986, the European Pharmacopoeia Commission and its experts have carried out a programme of work committed to Replacing, Reducing and Refining (3Rs) the use of animals for test purposes. While updates on achievements in the field of the 3Rs are regularly provided, this article summarises the activities of the Ph. Eur. Commission in this field within the last decade.


Subject(s)
Animal Testing Alternatives/standards , Animal Welfare/standards , Pharmacopoeias as Topic/standards , Vaccines/standards , Advisory Committees , Animal Testing Alternatives/legislation & jurisprudence , Animals , Europe , Humans , Toxicity Tests/standards
2.
Pharmeur Bio Sci Notes ; 2016: 129-134, 2016.
Article in English | MEDLINE | ID: mdl-28279254

ABSTRACT

For more than twenty years, the European Pharmacopoeia (Ph. Eur.) monographs for biotherapeutic proteins have been elaborated using the multisource approach (Procedure 1), which has led to robust quality standards for many of the first-generation biotherapeutics. In 2008, the Ph. Eur. opened up the way towards an alternative mechanism for the elaboration of monographs (Procedure 4-BIO pilot phase), which is applied to substances still under patent protection, based on a close collaboration with the Innovator company, to ensure a harmonised global standard and strengthen the quality of the upcoming products. This article describes the lessons learned during the P4-BIO pilot phase and addresses the current thinking on monograph elaboration in the field of biotherapeutics. Case studies are described to illustrate the standardisation challenges associated with the complexity of biotherapeutics and of analytical procedures, as well as the approaches that help ensure expectations are met when setting monograph specifications and allow for compatibility with the development of biosimilars. Emphasis is put on monograph flexibility, notably by including tests that measure process-dependent microheterogeneity (e.g. glycosylation) in the Production section of the monograph. The European Pharmacopoeia successfully concluded the pilot phase of the P4-BIO during its 156th session on 22-23 November 2016.


Subject(s)
Biosimilar Pharmaceuticals/analysis , Factor IX/analysis , Factor VIIa/analysis , Pharmacopoeias as Topic/standards , Biological Therapy/methods , Biological Therapy/trends , Biosimilar Pharmaceuticals/therapeutic use , Europe , Factor IX/therapeutic use , Factor VIIa/therapeutic use , Humans , Pilot Projects
3.
Anal Bioanal Chem ; 404(1): 29-42, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22638881

ABSTRACT

The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.


Subject(s)
DNA, Plant/genetics , Genomics/methods , Plants, Genetically Modified/genetics , Plasmids/genetics , Zea mays/genetics , Calibration , Gene Dosage , Genomics/standards
4.
Sleep Med ; 3 Suppl: S3-7, 2002 Nov.
Article in English | MEDLINE | ID: mdl-14592159

ABSTRACT

BACKGROUND: Although there is a relatively high rate of occurrence of sporadic cases of restless legs syndrome (RLS), the systematic study of family history of RLS in populations of RLS patients has been very limited. The objective of the present study was to determine the risk of RLS for first- and second-degree relatives of a population of primary RLS patients not selected for the number of affected relatives in their families and to obtain an estimate of the degree of genetic involvement in RLS. METHODS: Consecutively consenting patients from two different sites who met the criteria for RLS completed a worksheet that asked them to indicate their current age, the date of their earliest RLS symptoms, and the names and RLS status of all of their first- and second-degree relatives. Controls with no clinical history of RLS also completed the worksheet. RESULTS: First- and second-degree relatives of patients with RLS had a significantly greater risk of RLS than the first- (P<0.001) and second-degree relatives (P<0.003) of controls. The risk of RLS was found to be greater for first-degree relatives of early-onset, rather than late-onset, RLS probands (P<0.001). CONCLUSIONS: This study provides a complete systematic examination of the risk of RLS among relatives of RLS probands and controls using the same assessment methodology. Although the results are consistent with a genetic etiology for RLS, they do not support the presence of one simple, Mendelian-inherited major gene in most RLS families.

5.
J Biol Chem ; 273(36): 23093-7, 1998 Sep 04.
Article in English | MEDLINE | ID: mdl-9722536

ABSTRACT

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.


Subject(s)
Adenosine Triphosphate/pharmacology , Cyclic AMP/biosynthesis , Epithelial Cells/drug effects , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/analogs & derivatives , Animals , Arachidonic Acid/pharmacology , Autocrine Communication/drug effects , Cyclooxygenase Inhibitors/pharmacology , Dogs , Indomethacin/pharmacology , Paracrine Communication/drug effects , Prostaglandins/pharmacology , Receptors, Purinergic P2/classification , Receptors, Purinergic P2Y1 , Receptors, Purinergic P2Y2 , Signal Transduction/drug effects , Suramin/pharmacology , Thionucleotides/pharmacology , Uridine Triphosphate/pharmacology
6.
Neuroreport ; 5(10): 1185-8, 1994 Jun 02.
Article in English | MEDLINE | ID: mdl-7919161

ABSTRACT

The role of the subthalamic nucleus in the burst firing of dopamine neurones of the substantia nigra was investigated using extracellular single unit recordings combined with pressure or iontophoretic micro-injections in anaesthetized rats. Inhibition of subthalamic neurones by pressure injection of gamma-aminobutyric acid (GABA) regularized the burst firing pattern in eight out of 17 dopamine neurones. Bicuculline injection near subthalamic neurones increased their firing rate and increased burst discharge in a subpopulation of dopamine neurones tested (34 out of 102). The increase was depressed by iontophoresis of the N-methyl-D-aspartate (NMDA) antagonist (+-)2-amino,5-phosphonopentanoic acid (AP-5), but not of the non-NMDA antagonist, 6-cyano,7-nitroquinoxaline-2,3-dione (CNQX). These data suggest that the subthalamic nucleus promotes burst discharge in a subpopulation of substantia nigra dopamine neurones via NMDA receptors.


Subject(s)
Dopamine/metabolism , Receptors, N-Methyl-D-Aspartate/physiology , Substantia Nigra/physiology , Thalamic Nuclei/physiology , 2-Amino-5-phosphonovalerate/pharmacology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Bicuculline/administration & dosage , Bicuculline/pharmacology , Electrophysiology , Iontophoresis , Male , Microinjections , Neurons/drug effects , Neurons/physiology , Rats , Rats, Sprague-Dawley , Receptors, Amino Acid/antagonists & inhibitors , Receptors, Amino Acid/immunology , Receptors, Amino Acid/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Substantia Nigra/cytology , gamma-Aminobutyric Acid/administration & dosage , gamma-Aminobutyric Acid/pharmacology
7.
Brain Res Mol Brain Res ; 22(1-4): 107-12, 1994 Mar.
Article in English | MEDLINE | ID: mdl-7912398

ABSTRACT

The time course variations in tyrosine hydroxylase (TH) activity and specific mRNA were measured in the rat locus coeruleus (LC) and substantia nigra after an intracerebroventricular (i.c.v.) injection of 5,6-dihydroxytryptamine (5,6-DHT), a neurotoxin known to selectively destroy serotoninergic neurons. In this study, the TH activity and TH mRNA were both analyzed from homogenates of single tissue samples (micropunches). TH mRNA was extracted and quantified by densitometry using a northern blot method and an artificial TH RNA as an external standard. 5,6-DHT injection led to a long-lasting increase in TH activity and TH mRNA in LC but not in substantia nigra. The elevation in LC was progressive and reached its maximum value (+75%) at day 4 and day 8 after 5,6-DHT. This effect on TH activity was accompanied by a parallel change in TH mRNA whose amplitude was +57%, +81% and +45% at day 2, 4, and 8 respectively after the neurotoxin injection. Return to normal values was observed at day 16. Variations in TH activity and TH mRNA in LC were of similar amplitude. These results suggest that serotonin could be a potent modulator of TH gene expression within noradrenergic LC neurons.


Subject(s)
5,6-Dihydroxytryptamine/pharmacology , Locus Coeruleus/drug effects , RNA, Messenger/metabolism , Tyrosine 3-Monooxygenase/genetics , Animals , Injections, Intraventricular , Locus Coeruleus/metabolism , Male , Rats , Rats, Sprague-Dawley , Time Factors
8.
Int J Periodontics Restorative Dent ; 14(1): 34-47, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8005769

ABSTRACT

To obtain esthetic results with composite resin restorations, it is necessary to consider the following elements: the structure of composite resin; tint modifiers; opaquers; generic form; and surface texture. This article discusses these elements and presents clinical examples of techniques for achieving esthetic composite resin restorations.


Subject(s)
Composite Resins/chemistry , Dental Restoration, Permanent/methods , Prosthesis Coloring , Cuspid , Esthetics, Dental , Humans , Incisor
9.
Neurosci Lett ; 167(1-2): 33-6, 1994 Feb 14.
Article in English | MEDLINE | ID: mdl-8177526

ABSTRACT

Maudsley reactive (MR) and Maudsley nonreactive (MNRA) rats were submitted to a single session of acute 5-min immobilization stress and immediately sacrificed by decapitation. Subsequent neurochemical analysis revealed an elevation of 3,4-dihydroxyphenylacetic acid levels in the locus coeruleus and in the ventrolateral medulla, but not in the dorsomedial medulla, of rats of the two strains compared with nonstressed controls. This response was greater in the MR than in the MNRA group, suggesting a strain difference in the reactivity of the central noradrenergic cells to acute stress.


Subject(s)
Brain/physiopathology , Norepinephrine/physiology , Stress, Psychological/physiopathology , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Brain/metabolism , Brain/pathology , Corticosterone/blood , Immobilization , Locus Coeruleus/metabolism , Male , Medulla Oblongata/metabolism , Mice , Mice, Neurologic Mutants , Neurons/metabolism , Rats , Rats, Sprague-Dawley
10.
Brain Res ; 638(1-2): 196-202, 1994 Feb 28.
Article in English | MEDLINE | ID: mdl-8199859

ABSTRACT

It has previously been shown that immobilization and ether stress induce activation of the hypothalamo-pituitary-adrenal (HPA) axis and that this activation occurs subsequent to activation of brain stem catecholaminergic neurones. In the present study we have investigated whether the brain stem catecholaminergic (CA) neurons show habituation to chronic daily intermittent exposure to the same restraint stress comparable to that of the HPA axis. The level of activity of the brainstem CA groups was estimated by measurement in tissue punches of content of 3,4-dihydroxyphenylacetic acid (DOPAC), a side metabolite of noradrenaline and adrenaline biosynthesis which has been shown to be a reliable index of the stress-induced activation of the CA groups. The level of activity of the HPA axis was determined by measurement of plasma corticosterone and adrenocorticotropic hormone (ACTH) levels. The animals were submitted to a 15 min restraint stress daily. They were sacrificed at the end of the stress session on day 3, 5 and 10. The ACTH response to the acute restraint stress whilst unchanged on day 3 was significantly decreased on day 5 (-54%) and day 10 (-70%) compared to the response in naive rats. The approximately twofold increase in DOPAC level induced by acute restraint stress in the so-called CA medullary group A1/C1 of naive rats was reduced in daily restraint rats on day 5 (-22%) and day 10 (-30%) but was unchanged on day 3. A small (-20%) decrease of the stress-induced DOPAC response in the A2/C2 CA group and locus coeruleus was also observed on day 10.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
3,4-Dihydroxyphenylacetic Acid/metabolism , Adrenocorticotropic Hormone/metabolism , Brain Stem/metabolism , Corticosterone/metabolism , Habituation, Psychophysiologic , Hypothalamo-Hypophyseal System/physiology , Pituitary-Adrenal System/physiology , Stress, Psychological , Adrenocorticotropic Hormone/blood , Animals , Blood Glucose/metabolism , Corticosterone/blood , Male , Rats , Rats, Sprague-Dawley , Reference Values , Restraint, Physical , Time Factors
11.
Anesthesiology ; 79(5): 1072-82, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8238984

ABSTRACT

BACKGROUND: alpha 2-Adrenoceptor agonists, known as antihypertensive agents, may be used during general anesthesia for their anesthetic sparing action and to reduce the occurrence of side effects. Previous studies have shown that the brain's noradrenergic nucleus, locus coeruleus, is an important target in mediating the hypnotic action of alpha 2 agonists. The authors studied the effects of recovery from halothane anesthesia on the electrical activity of locus coeruleus neurons to examine cellular substrates underlying the clinical effectiveness of alpha 2 agonists. METHODS: Experiments were performed in locally anesthetized rats, whose circulatory and acid-base stabilities were ensured by mechanical ventilation and volume infusion. Locus coeruleus neurons were recorded continuously while the rats were anesthetized with halothane (1%) and/or after the halothane was discontinued. RESULTS: Under the influence of halothane, locus coeruleus cells exhibited a slow, regular spontaneous discharge (1.95 +/- 0.23 Hz), and contralateral foot or tail pinch elicited a prominent, phasic activation in locus coeruleus neurons. Such phasic activation was blocked by local ejection of kynurenic acid, an excitatory amino acid antagonist, close to recorded neurons, but not by clonidine (up to 64 micrograms.kg-1). Thirty minutes after the halothane was discontinued, the mean firing rate of locus coeruleus neurons was increased by 87 +/- 20%. This excitation resulted from a prominent increase in bursting activity (21 +/- 5% of spikes in bursts vs. 4 +/- 1%) and was reversed by halothane readministration. This activation also was reduced by local ejection of kynurenic acid. Halothane discontinuance revealed the reactivity of locus coeruleus neurons to nonnoxious, sensory stimuli, and considerably reduced the apparent potency of intravenous administration of clonidine to inhibit locus coeruleus activity (effective dose for 50% of maximal effect (ED50), 25.48 +/- 8.26 micrograms.kg-1 vs. 4.81 +/- 0.80 micrograms.kg-1 under halothane). This decrease was caused by the persistence of bursting activity after the administration of clonidine, which was completely suppressed by readministration of halothane or local application of kynurenic acid. CONCLUSION: The data demonstrate: (1) that halothane withdrawal increases locus coeruleus neuronal activity via excitatory amino acid input, and this withdrawal-induced activity is characterized by a prominent burst (phasic) discharge; (2) that sedative doses of clonidine inhibit the tonic component of locus coeruleus activity but not the phasic activation of locus coeruleus neurons; and (3) that readministration of halothane or local ejection of an excitatory amino acid antagonist fully suppresses the bursting activity unaffected by clonidine.


Subject(s)
Anesthesia Recovery Period , Anesthesia, Inhalation , Clonidine/pharmacology , Halothane , Locus Coeruleus/physiology , Animals , Locus Coeruleus/cytology , Male , Rats , Rats, Sprague-Dawley , Time Factors
12.
Eur J Neurosci ; 5(8): 1024-8, 1993 Aug 01.
Article in English | MEDLINE | ID: mdl-7904220

ABSTRACT

Microiontophoretic application of selective agonists for the three major excitatory amino acid receptors, N-methyl-D-aspartate (NMDA), quisqualate and kainate, increased the discharge rate of noradrenergic locus coeruleus (LC) neurons in vivo. NMDA activation was selectively attenuated by iontophoretic application of 2-amino-5-phosphonopentanoate (AP5), an antagonist at NMDA receptors, whereas kainate- and quisqualate-evoked responses were attenuated by both NMDA and non-NMDA antagonists iontophoresis. NMDA- and quisqualate-evoked responses were significantly decreased by co-iontophoresis of serotonin (5-HT). When the NMDA receptor-mediated component of the response to kainate was blocked with AP5 iontophoresis, 5-HT increased the response of LC neurons to kainate. These results revealed that 5-HT differentially modulates the responsiveness of LC neurons to excitatory amino acids, depending on the receptor subtypes responsible for the neuronal activation.


Subject(s)
Locus Coeruleus/physiology , Neurons/physiology , Receptors, Amino Acid/physiology , Serotonin/pharmacology , 2-Amino-5-phosphonovalerate/pharmacology , Amino Acids/physiology , Animals , Electrophysiology , Iontophoresis , Kainic Acid/pharmacology , Locus Coeruleus/cytology , Male , Neurons/drug effects , Quisqualic Acid/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Amino Acid/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors
13.
J Neurosci Methods ; 48(3): 241-50, 1993 Jul.
Article in English | MEDLINE | ID: mdl-8412306

ABSTRACT

The combination of electrochemically treated carbon-fiber electrodes with DPV, DNPV or DPA represents a wide range of possibilities. As shown in this review, the choice of treatment and measurement technique depends on the purpose. As regards in vivo monitoring of 5-HIAA or DOPAC from very small brain nuclei, electrochemically treated carbon-fiber electrodes appear very potent and inexpensive. The main limitation of the established electrochemical techniques, including those discussed here, is that the unequivocal measurement of the basal extracellular neurotransmitter level cannot be achieved unless animals are treated with pargyline. On the other hand, this monitoring is feasible with in vivo dialysis. Therefore, electrochemical techniques, on the one hand, and in vivo dialysis, on the other hand, present different advantages. The former are much more potent than the latter in two respects. First, due to the much smaller size of the sensor, electrochemical techniques are more suitable for studying small brain nuclei. Second, since electrochemical techniques exhibit a better temporal resolution, they are recommended for investigating the relationship between impulse flow and neurotransmitter release. However, when high anatomical or temporal resolution is not required, in vivo dialysis is more suitable for recording the basal monoamine release.


Subject(s)
Brain Chemistry , Catechols/analysis , Indoles/analysis , Animals , Carbon , Electrochemistry , Electrodes , Humans
14.
Gen Comp Endocrinol ; 90(1): 1-13, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8504914

ABSTRACT

The adrenal gland of amphibians is composed of a mixed population of adrenochromaffin and corticosteroid-secreting cells. It has previously been shown that chromaffin cells synthesize several bioactive substances (including biogenic amines and neurotransmitters) which may act locally to regulate corticosteroid secretion. In the present report, we have studied the secretory activity of adrenochromaffin cells in Rana ridibunda. Frozen sections of adrenal gland were immunolabeled with antisera against tyrosine hydroxylase or phenylethanolamine-N-methyltransferase. Comparison of homologous fields on consecutive sections indicated that 77% of catecholaminergic cells produce adrenaline. The concentrations of catecholamines were measured by means of high performance liquid chromatography analysis coupled to electrochemical detection. The concentrations of dopamine, noradrenaline, and adrenaline in fresh adrenal tissue were 24 +/- 4, 763 +/- 68, and 1032 +/- 118 ng/mg wet weight, respectively. After a 12-hr perifusion period, the concentration of adrenaline in the tissue was reduced by 62%, whereas noradrenaline only decreased by 22%. The secretion rates of adrenaline and noradrenaline from perifused adrenal slices significantly diminished during the first 7 hr of the experiment and then remained relatively stable for about 10 hr. Exposure of adrenal tissue to a depolarizing concentration of potassium (55 mM) induced an immediate and substantial rise of adrenaline and noradrenaline release and a delayed increase in corticosterone output. Acetylcholine, which stimulates corticosterone secretion from frog adrenocortical cells, induced a slight but not significant increase of adrenaline and noradrenaline release. Similarly, the selective cholinergic agonists muscarine and nicotine did not significantly affect catecholamine release, while muscarine mimicked the stimulatory action of acetylcholine on corticosterone secretion. This study validates the use of the perifusion model to investigate the mechanism of control of catecholamine release from frog adrenochromaffin tissue. The results presented herein indicate that, in contrast to mammals, the secretion of catecholamines from the amphibian adrenal gland is not regulated by cholinergic inputs.


Subject(s)
Adrenal Cortex Hormones/metabolism , Adrenal Medulla/metabolism , Catecholamines/metabolism , Rana ridibunda/metabolism , Adrenal Medulla/drug effects , Animals , Immunohistochemistry , In Vitro Techniques , Male , Membrane Potentials/physiology , Parasympathomimetics/pharmacology , Perfusion , Potassium/physiology
15.
Eur J Pharmacol ; 235(2-3): 205-10, 1993 Apr 28.
Article in English | MEDLINE | ID: mdl-8099552

ABSTRACT

The effects of desipramine on sensory-evoked (sciatic nerve stimulation) activation of locus coeruleus neurones were investigated in vivo by using treated carbon-fibre electrodes in conjunction with either differential normal pulse voltammetry or differential pulse amperometry. Firstly, the amplitude of the sensory-evoked increase in extracellular noradrenaline, monitored in thalamic locus coeruleus terminals, was not modified by desipramine (10 mg/kg), whereas that evoked by direct activation of locus coeruleus neurones was greatly increased: +143% for dorsal noradrenergic bundle stimulation and +761% for glutamate ejection in the locus coeruleus. Secondly, desipramine administered at the same dose significantly reduced (-48%) the activation of locus coeruleus neurones (monitored at the somato-dendritic level) evoked by prolonged sciatic nerve stimulation. Our results indicate that acute treatment with desipramine does not potentiate overall noradrenergic transmission by locus coeruleus neurones during sensory stimulation.


Subject(s)
Desipramine/pharmacology , Locus Coeruleus/drug effects , Norepinephrine/metabolism , Thalamic Nuclei/drug effects , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Desipramine/administration & dosage , Electric Stimulation , Electrodes , Glutamates/pharmacology , Glutamic Acid , Locus Coeruleus/metabolism , Locus Coeruleus/pathology , Male , Rats , Rats, Sprague-Dawley , Sciatic Nerve , Thalamic Nuclei/physiology
16.
Neuroscience ; 52(4): 961-72, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8095714

ABSTRACT

The anteroventral thalamic nucleus is innervated by noradrenergic terminals exclusively originating in the locus coeruleus, a densely packed cell group located in the dorsotegmental part of the pons. In urethane-anaesthetized rats, electrical stimulations of locus coeruleus axons (dorsal noradrenergic bundle; 14 Hz, 20 s) evoked a rapid increase in the signal (catechol oxidation current) measured within the anteroventral thalamic nucleus by the use of carbon fibre electrodes combined with electrochemistry. This effect was reproducible and immediately reversible. Evoked changes in this current were found to be due to oxidation of noradrenaline released from terminals. The amplitude of the evoked noradrenaline release varied non-linearly with the frequency of stimulation. We investigated the influence of locus coeruleus activation on noradrenaline release measured in the anteroventral thalamic nucleus every second by means of differential pulse amperometry: (i) chemical activation of locus coeruleus by local injection of glutamate (0.2-0.8 nmol) immediately and consistently evoked noradrenaline release in a dose-dependent manner; and (ii) peripheral stimulation of the sciatic nerve (20 s)--known to enhance the firing rate of locus coeruleus neurons-evoked a noradrenaline release similar to that produced by a stimulation of the dorsal noradrenergic bundle at 8-10 Hz. Pharmacological and kinetic characteristics of the noradrenaline release were the same for central or peripheral stimulation of locus coeruleus neurons. Our results indicate that in vivo electrochemistry, because of its sensitivity and its high space and time resolution, is well suited for studies of evoked noradrenaline release from locus coeruleus terminals. This approach allowed us to describe the characteristics of central noradrenaline release evoked by central and peripheral stimulations of short duration. In particular, we observed a very close relationship between impulse flow and evoked noradrenaline release.


Subject(s)
Locus Coeruleus/physiology , Norepinephrine/metabolism , Thalamic Nuclei/physiology , Animals , Axons/physiology , Clonidine/pharmacology , Desipramine/pharmacology , Electric Stimulation , Electrochemistry/methods , Evoked Potentials , Glutamates/metabolism , Glutamic Acid , Male , Oxidation-Reduction , Pargyline/pharmacology , Rats , Rats, Sprague-Dawley , Sciatic Nerve/physiology , Thalamic Nuclei/drug effects , Yohimbine/pharmacology
17.
Eur J Neurosci ; 5(2): 137-44, 1993 Feb 01.
Article in English | MEDLINE | ID: mdl-8261095

ABSTRACT

Midbrain dopamine neurons in vivo discharge in a single-spike firing pattern or in a burst-firing pattern. Such activity in vivo strikingly contrasts with the pacemaker activity of the same dopamine neurons recorded in vitro. We have recently shown that burst activity in vivo of midbrain dopamine neurons is due to the local activation of excitatory amino acid receptors, as microapplication of the broad-spectrum antagonist of excitatory amino acids, kynurenic acid, strongly regularized the spontaneous firing pattern of these dopamine neurons. In the present study, we investigated which subtypes of excitatory amino acid receptors are involved in the burst-firing of midbrain dopamine neurons in chloral hydrate-anaesthetized rats, iontophoretic or pressure microejections of 6-cyano, 7-nitroquinoxaline-2,3-dione (CNQX), a non-N-methyl-D-aspartate (NMDA) receptor antagonist, did not alter the spontaneous burst firing of dopamine neurons (n = 36). In contrast, similar ejections of (+-)2-amino,5-phosphonopentanoic acid (AP-5), a specific antagonist at NMDA receptors, markedly regularized the firing pattern by reducing the occurrence of bursts (n = 52). In addition, iontophoretic ejections of NMDA, but not kainate or quisqualate, elicited a discharge of these dopamine neurons in bursts (n = 20, 12 and 14, respectively). These data suggest that burst-firing of midbrain dopamine neurons in vivo results from the tonic activation of NMDA receptors by endogenous excitatory amino acids. In view of the critical dependency of catecholamine release on the discharge pattern of source neurons, excitatory amino acid inputs to midbrain dopamine neurons may constitute a major physiological substrate in the control of the dopamine level in target areas.


Subject(s)
Dopamine/physiology , Mesencephalon/physiology , Neurons/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Animals , Electrophysiology , Iontophoresis , Kainic Acid/pharmacology , Male , Mesencephalon/cytology , N-Methylaspartate/pharmacology , Neurotoxins/antagonists & inhibitors , Quisqualic Acid/pharmacology , Rats , Rats, Sprague-Dawley
18.
J Neurosci Methods ; 45(3): 183-90, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1363483

ABSTRACT

Differential pulse amperometry has previously been used in combination with electrochemically treated carbon fibre electrodes. In order to improve the time resolution, this technique was combined here with untreated electrodes. Dopamine release was evoked in the nucleus accumbens of rats anaesthetized with urethane by electrical stimulation of the mesolimbic dopaminergic pathway. The differential oxidation current appearing at +200 mV was recorded every 1 s and was proportional to the dopamine concentration from 0.5 to 50 microM. At this voltage these untreated electrodes were not sensitive to the main catechol metabolite (DOPAC) and poorly sensitive to ascorbic acid. The electrically evoked increase in the oxidation current corresponded exclusively to dopamine. It was enhanced by nomifensine, amphetamine, haloperidol and pargyline and reduced by alpha-methyl-p-tyrosine (A-MPT). The results show that the evoked DA release was facilitated by increasing the stimulation frequency from 10 to 40 Hz. The method was sufficiently sensitive to detect dopamine release evoked by electrical stimulation at 10 Hz and its time resolution was 1 s.


Subject(s)
Dopamine/metabolism , 3,4-Dihydroxyphenylacetic Acid/metabolism , Amphetamine/pharmacology , Animals , Dopamine/chemistry , Electric Stimulation , Electrochemistry , Electrodes, Implanted , Haloperidol/pharmacology , Male , Methyltyrosines/pharmacology , Neural Pathways/drug effects , Neural Pathways/metabolism , Neural Pathways/physiology , Nomifensine/pharmacology , Nucleus Accumbens/drug effects , Nucleus Accumbens/metabolism , Nucleus Accumbens/physiology , Oxidation-Reduction , Pargyline/pharmacology , Rats , Rats, Sprague-Dawley , Tyrosine 3-Monooxygenase/antagonists & inhibitors , alpha-Methyltyrosine
19.
Neuroscience ; 49(4): 879-91, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1359456

ABSTRACT

Peripheral administration of low doses of dopamine agonist apomorphine induces a strong and short-latency inhibition of dopamine neurons in the substantia nigra, presumably via the activation of somatodendritic autoreceptors. We studied the site of action of apomorphine in anesthetized rats using volume-controlled pressure microejection combined with single unit recordings. Microapplication of apomorphine in the immediate vicinity of nigral dopamine neurons did not mimic the effect of intravenous administration of apomorphine (50 micrograms/kg), regardless of the concentration or volume used (10(-10)-10(-2) M, 10-100 nl). In contrast, the inhibition produced by systemic apomorphine was mimicked by drug application at a site 300 microns lateral and 600 microns ventral from the recording site in the zona reticulata of the substantia nigra, a region rich in dendrites of dopamine neurons. The inhibition induced by such a distant application of apomorphine could be reversed by systemic injection of D2, but not D1, receptor antagonists. Non-dopaminergic substances such as GABA, bicuculline or lidocaine were more effective when ejected close to rather than distant from the recording site, in a manner opposite to that of apomorphine. Similar to apomorphine, dopamine and D2 receptor agonists were more potent when intranigral applications were made at sites distant from, rather than close to, the recorded dopamine cells. Ejection of D2 antagonists in the substantia nigra zona reticulata attenuated the inhibitory effect of subsequent systemic apomorphine. Our results, together with other previous studies on the location of D2 receptors on dopamine neurons, suggest that peripheral administration of low doses of apomorphine inhibits nigral dopamine neurons by acting at D2 receptors located on the dendrites of these neurons.


Subject(s)
Apomorphine/pharmacology , Dopamine/physiology , Neurons/physiology , Substantia Nigra/physiology , Animals , Apomorphine/administration & dosage , Benzazepines/pharmacology , Bicuculline/pharmacology , Dopamine Agents/pharmacology , Dose-Response Relationship, Drug , Ergolines/pharmacology , Evoked Potentials/drug effects , Haloperidol/pharmacology , Injections, Intravenous , Lidocaine/pharmacology , Male , Microinjections , Neurons/drug effects , Phenethylamines/pharmacology , Quinpirole , Rats , Rats, Wistar , Substantia Nigra/drug effects , gamma-Aminobutyric Acid/pharmacology
20.
Eur J Pharmacol ; 219(1): 169-72, 1992 Aug 14.
Article in English | MEDLINE | ID: mdl-1397045

ABSTRACT

The effects of the phencyclidine derivative, N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine (BTCP), on the electrical activity of noradrenaline (NA) neurons of the locus coeruleus (LC) were studied in halothane-anesthetized rats. Systemic administration of BTCP potently inhibited LC neurons (ID50 of 1.1 +/- 0.1 mg/kg i.v.). This effect was mimicked by local microejection of BTCP into the LC. Both the systemic and local effects of BTCP were blocked by alpha 2-adrenoceptor antagonists and prevented by prior depletion of catecholamines with reserpine. These and other data suggest that BTCP behaves as a potent indirect NA agonist (i.e. via NA re-uptake and/or release systems).


Subject(s)
Locus Coeruleus/drug effects , Phencyclidine/analogs & derivatives , Receptors, Adrenergic/drug effects , Animals , Dopamine/metabolism , Male , Norepinephrine/metabolism , Phencyclidine/pharmacology , Rats , Rats, Inbred Strains
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