Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

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.

Pineal serotonin N-acetyltransferase activity: abrupt decrease in adenosine 3',5'-monophosphate may be signal for "turnoff".

Dispersed pinealocytes have been used to study the role of adenosine 3',5'-monophosphate (cyclic AMP) in the "turnoff" of N-acetyltransferace activity. Activity was first stimulated 100-fold by treating cells with 1-norepinephrine. 1-Propranolol acted stereospecifically to rapidly reverse this, resulting in a 70 percent loss of enzyme activity within 15 minutes. An even more rapid 1-propranolol-induced decreased in cyclic AMP also occurred. This together with the observation that the inhibitory effect of 1-propranolol on N-acetyltransferase was blocked by dibutyryl cyclic AMP and phosphodiesterase inhibitors indicate that an abrupt decrease in cyclic AMP may be the signal for the rapid decrease in pineal N-acetyltransferase activity.

Replacement, Reduction, Refinement - Animal welfare progress in European Pharmacopoeia monographs: activities of the European Pharmacopoeia Commission from 2007 to 2017.

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.

Elaborating European Pharmacopoeia monographs for biotherapeutic proteins using substances from a single source.

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.

Causes of death in DSM-III schizophrenics and other psychotics (atypical group). A comparison with the general population.

Causes of death were determined in a cohort of DSM-III schizophrenics and in a group of "other" psychotics (with schizoaffective disorder, schizophreniform disorder, and atypical psychoses), and comparisons were made with the general population. Five causes of death were considered in this analysis: (1) infections, (2) neoplasms, (3) cardiovascular, (4) unnatural, and (5) other. Suicide occurred in excess among other psychotics compared with schizophrenics and with the general population.

A suspension culture of pinealocytes: regulation of N-acetyltransferase activity.

A method to prepare suspension cultures of highly viable pinealocytes is described. Pineal glands of neonatal or adult rats can be used; the best yield is obtained from neonatal glands. Morphological examination of the cells indicates that they have a normal, healthy appearance and retain some structures typical of neonatal pinealocytes. During the first 24 h of culture, the cells aggregate into small clusters; after several days, larger aggregates can be seen. Biochemical studies indicate that adrenergic stimulation of these cells causes a 30- to 100-fold increase in serotonin N-acetyltransferase activity, a response which is unique to the pineal gland. The relative order of potency of several adrenergic agonists is l-isoproterenol greater than l-norepinephrine greater than or equal to l-epinephrine greater than phenylephrine. Serotonin, tyramine, histamine, carbachol, gamma-aminobutyric acid, cocaine, and desmethylimipramine are inactive. Studies using adrenergic and metabolic inhibitors indicate that the regulation of N-acetyltransferase in these cells has the same characteristics of that seen in adult tissue. In addition, these cells synthesize protein and RNA from radioactive precursors, convert tryptophan to serotonin, and N-acetylate endogenous serotonin.

Catecholamine Metabolism in Locus Coeruleus Neurons: A Study of its Activation by Sciatic Nerve Stimulation in the Rat.

Sciatic nerve stimulation, which strongly activates noradrenergic locus coeruleus (NA-LC) neurons, was used in anaesthetized rats as a model to study the transneuronal control of catechol metabolism in this nucleus. We show, using in vivo electrochemistry and biochemical post-mortem assays, that a prolonged (20 min) unilateral sciatic nerve electrical stimulation led to a reversible enhancement (80 - 130%) of both endogenous and in vivo extracellular levels of 3,4-dihydroxyphenylacetic acid (DOPAC) within the contralateral LC region. An elevation in DOPAC levels was also observed in the ipsilateral nucleus but was always significantly lower. The response was abolished by a pretreatment with kynurenic acid, a non-selective excitatory amino acid (EAA) antagonist known to block footshock-induced excitations of NA-LC neurons: in antagonist-treated rats, the stimulation induced a non-significant effect (+ 30%) on endogenous DOPAC levels, which contrasted with the highly significant effect (+ 113%) observed in vehicle-treated animals. As the major source of EAA afferents to the LC originates in the nucleus paragigantocellularis, we made an attempt to suppress activation by a section of these fibres. An incision performed obliquely (45 degrees ) between LC and PGi greatly and significantly attenuated, but did not totally suppress, the increase in DOPAC endogenous content due to the stimulation. These experiments indicate that a peripheral stimulus provokes an activation of catecholamine metabolism within the soma - dendritic region of the NA-LC cells. They suggest that this effect may be mediated, at least in part, by afferent pathways originating from the medulla which utilize an EAA as transmitter.

Regulation of dopamine release by impulse flow and by autoreceptors as studied by in vivo voltammetry in the rat striatum.

Extracellular dopamine concentration has been monitored in the striatum of pargyline treated, anaesthetized rats using differential normal pulse voltammetry. The catechol oxidation current recorded with electrochemically treated carbon fiber electrodes disappeared when the dopaminergic terminals were selectively destroyed by 6-hydroxydopamine. Calibration of the basal oxidation current revealed that the extracellular dopamine concentration was 26 nM. Brief and moderate electrical stimulation of the nigrostriatal pathway at the level of the medial forebrain bundle induced a large increase in the dopamine current. The observed elevation in the dopamine signal lasted as long as the stimulation. It varied with the frequency (0-25 Hz) of the pulses in an exponential manner. Stimulation pulses distributed in a bursted pattern were twice as potent as an equivalent number of pulses regularly spaced. High frequency stimulations (50 Hz) were also investigated in anaesthetized rats (without pargyline) with untreated carbon fiber electrodes; they induced a very large increase in the dopamine extracellular concentration (up to 8-15 microM). Interruption of the dopaminergic impulse flow either by an electrolytic lesion or by a low dose of apomorphine (0.05 mg/kg) caused an immediate decrease of the dopamine current. The time courses and amplitudes (-70%) of these effects were identical. Subsequent injection of haloperidol (0.5 mg/kg) reversed the apomorphine effect up to +360% of the control basal value. Administration of dopaminergic antagonists such as haloperidol (0.05 and 0.5 mg/kg) or metoclopramide (2 mg/kg) significantly increased the dopamine current up to 317, 340 and 215% of the respective control values. Nomifensine (4 mg/kg) produced a big increase (+417%) of the extracellular dopamine levels. The effect of electrical stimulation of the dopaminergic pathway was potentiated by drugs such as amphetamine (2 mg/kg), nomifensine (4 mg/kg) or haloperidol (0.05 and 0.5 mg/kg) but was not altered by apomorphine (0.05 mg/kg). The study by in vivo voltammetry of the variations in the striatal extracellular dopamine concentrations shows that the release of dopamine is under the influence of both the frequency of impulse flow and of dopaminergic striatal autoreceptors.

In vivo noradrenaline release evoked in the anteroventral thalamic nucleus by locus coeruleus activation: an electrochemical study.

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.

Inhibition of nigral dopamine neurons by systemic and local apomorphine: possible contribution of dendritic autoreceptors.

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.

Effect of haloperidol and sulpiride on dopamine metabolism in nucleus accumbens and olfactory tubercle: a study by in vivo voltammetry.

Differential pulse voltammetry used with electrochemically pretreated carbon fibre microelectrodes enables separation between the two peaks corresponding to the ascorbic acid and catechol oxidation currents. The effects of haloperidol and sulpiride on the 3,4-dihydroxyphenylacetic acid peak recorded in the nucleus accumbens and olfactory tubercle of rats were studied. Chloral hydrate anaesthetized preparations and chronic preparations were used. A microdevice was designed to implant electrodes in freely moving rats. Voltammograms were recorded every minute in each structure in acute preparations and every 2 min in chronic preparations. In acute preparations haloperidol induced a similar dose-dependent increase in the catechol oxidation peak in both structures. Sulpiride at all doses only induced an increase in the olfactory tubercle. In chronic preparations haloperidol and sulpiride had even larger effects on the 3,4-dihydroxyphenylacetic acid peak in both regions. In these preparations sulpiride induced a significant increase in nucleus accumbens. The effects induced by haloperidol in the two regions were greater than those induced by sulpiride. The main conclusions of this study are that the results of voltammetry agree with biochemical results on the effects of haloperidol and sulpiride on dopamine metabolism. An interaction of chloral hydrate with the effects of the two neuroleptics was also observed.

Long-term alteration in tyrosine hydroxylase mRNA levels in rat locus coeruleus after intraventricular injection of 5,6-dihydroxytryptamine.

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.

Lack of glucocorticoids enhances the early activation of the medullary catecholaminergic cell groups triggered by restraint stress.

We have investigated whether the stress-induced activation of the medullary catecholaminergic neurons, that was shown previously to provide the main central activation input to the hypothalamo-pituitary-adrenocortical axis during an immobilization stress, is sensitive to circulating corticosteroids. Experiments were carried out on adrenalectomized rats that were first maintained on corticosterone in the drinking water for 5 days following surgery and then switched to corticosterone-free water 15 h before stress application. Some of the latter animals were injected with dexamethasone. Activation of the brainstem Catecholaminergic neurons was estimated by assaying 3,4-dihydroxyphenylacetic acid (DOPAC), a side metabolite of the noradrenaline and adrenaline biosynthesis pathway that was established previously as a reliable index of the activity of these neurons. In the so-called A(1) C(1) and A(2) C(2) Catecholaminergic groups of the medulla, lack of corticosterone under these experimental conditions did not modify the basal level of DOPAC but led to a further enhancement (+ 35% to 40%) of the approximately 2-fold increase in DOPAC content observed 15 min after the onset of the 5-min immobilization stress. No significant further enhancement of the stress-induced DOPAC increase was observed in the locus coeruleus. In these adrenalectomized rats, dexamethasone pretreatment prevented the enhancement of the stress-induced increase in DOPAC level observed in the medullary cell groups but did not abolish the response to stress. Lack of endogenous corticosteroids led to a 10-fold enhancement of the adrenocorticotropin increase following immobilization stress. Pretreatment with dexamethasone fully abolished the stress-induced increase in adrenocorticotropin plasma level. Our results show that circulating corticosteroids reduce the stress-induced activation of the medullary A(1) and A(2) C(2) groups i.e. those that contribute mainly to the catecholaminergic innervation of the hypothalamo-pituitary-adrenocortical axis within the hypothalamic paraventricular nuclei. However, since the feedback regulation of the central catecholaminergic systems is much less efficient than the feedback actually observed on the adrenocorticotropin secretion, we suggest that it is likely to play a minor physiological role in the overall feedback regulation exerted by circulating corticosteroids on the hypothalamo-pituitary-adrenocortical axis.

Retrograde axonal transport after radioactive serotonin injections into the olfactory bulb: a biochemical analysis of transported radioactive material.

A biochemical analysis of radioactive compounds was performed in the olfactory bulb (OB) and raphe dorsalis (RD) after injection of radioactive [(3)H] or [(14)C]serotonin (5-HT ranging from 10(?2) M to 10(?7) M) into the OB of rats treated or not with a monoamine-oxidase inhibitor (MAOI). In the OB of untreated rats, radioactivity was associated with precipitated protein and soluble perchloric acid (PCA) fractions. High performance liquid chromatography (HPLC) analysis of the PCA-supernatant gave 4 radioactive peaks: one associated with endogenous 5-HT, another with endogenous 5-hydroxyindole acetic acid (5-HIAA) and two without any relationship with endogenous hydroxyindoles: a '5-HT derivative A' and a '5-HT derivative B'. The presence of these '5-HT derivatives' was significantly reduced after treatment with 5,6-dihydroxytryptamine. In the RD, radioactivity was associated with the protein fraction and with '5-HT derivative A'. The kinetic analysis (from 30 min to 46 h) of the '5-HT derivative A' was characterized by a disappearance in the OB and an accumulation in the RD corresponding to a rate of migration in a range of 0.7 to 2 mm/h. This compound was absent or negligible in other non-serotoninergic neurons (such as the Locus Coeruleus, Amygdala and Cortex piriformis). No clear evidence for retrograde transport of radioactive 5-hydroxytryptophan (5-HTP) or 5-HIAA was found. At lower concentration of 5-HT injected into the OB, the half lives and the times of maximal accumulation for 5-HIAA, '5-HT derivative A' and '5-HT derivative B' were increased. The specific activity of 5-HT and 5-HIAA was also increased. The selective radioactive accumulation in the cell bodies of RD neurons after injection of radioactive 5-HT into the OB is discussed as resulting from a selectivity in (a) the uptake by 5-HT nerve terminals; (b) the metabolism of 5-HT into '5-HT derivative A' in the OB; (c) the retrograde axonal transport of '5-HT derivative A'. This '5-HT derivative A' could represent a messenger between nerve terminals and cell bodies and could be involved in homeostatic mechanisms that maintain cellular dynamics. When a MAOI was used, '5-HT-derivative A' and [(3)H]5-HT were found in the OB and also in the RD cell bodies, and to a lesser extent, in the non-serotoninergic cell bodies. These results indicate that MAO inhibition produces a relative non-selectivity in the 'uptake-metabolism and retrograde axonal transport' systems.

Further characterization of the tyrosine hydroxylase induction elicited by reserpine in the rat locus coeruleus and adrenals.

This study shows that reserpine causes a long lasting increase in the amount of protein and activity of tyrosine hydroxylase (TH) in central noradrenergic neurons of the locus coeruleus (LC) and in peripheral cells of the adrenal gland. In these structures, the drug effect appears to be reversible (maximum at day 2-4 after injection) and is entirely dose-dependent. Variations in TH activity were measured both in vitro in optimal enzymatic conditions and in vivo. Reserpine did not modify the ratio between activities measured in vivo and in vitro. But it decreased the specific activity of the enzyme as measured in homogenates of LC. The reversibility of the reserpine effect on all parameters studied appeared faster in adrenal than in central LC neurons. Reserpine did not change the activity and amount of TH in the region of the substantia nigra where dopaminergic neurons are located, except at a high dose (10 mg/kg): a significant elevation (+36%) of the TH protein amount was then observed 2 days after treatment. Moreover, reserpine did not influence the amount of neuron specific enolase in the LC region at a time when maximal effect was observed for TH, thus showing the biochemical specificity of this regulation. Concomitant injection of clonidine at two doses 30 min before (100?g/kg) and 3 h after (50?g/kg) reserpine administration did not affect the induction by reserpine alone. Thus, stimulation of LC noradrenergic cell firing resulting from reserpine administration is probably of negligible importance in the genesis of TH induction by this drug.

Pharmacological effects of GABA on serotonin metabolism in the rat brain.

The pharmacological effects of GABA-related drugs were studied on the serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA) contents of various regions of the rat brain. These effects were examined in the nuclei raphe dorsalis, magnus and centralis and in structures receiving a dense serotonin innervation such as the habenula complex and subcommissural organ. The GABA agonist, muscimol, increased the 5-HT contents and reduced 5-HIAA levels in structures containing serotoninergic terminals suggesting an inhibitory effect of GABA on the firing of serotoninergic neurons with concomitant reduction of 5-HT utilisation. In contrast, the GABA antagonist, bicuculline, probably stimulated 5-HT turnover since its intraperitoneally administration produced significant increase of 5-HT and/or 5-HIAA levels in the same brain regions. These data are in agreement with a transsynaptic inhibitory control of GABA on serotoninergic neurons. Drugs which inhibit the GABA catabolism such as amino-oxyacetic acid or gamma-vinyl-GABA and which should elevate GABA levels in the synaptic gap were capable of increasing or decreasing the 5-HT and the 5-HIAA levels depending on the experimental conditions. These results suggest that several processes are probably involved in the control of serotoninergic neurons by GABA in the rat brain. Among them, an intracellular effect of GABA on 5-HT metabolism might well occur in cells containing both GABA and 5-HT.

Culture of rat locus coeruleus explants: a model to study transneuronal regulation of tyrosine hydroxylase in central noradrenergic neurons.

A culture method is described to maintain explants of rat locus coeruleus in vitro for several days. Both anatomical and biochemical methods were used to control the evolution of the cultures with time. Explants were first examined 7 days after culture by immunohistochemistry with specific antisera directed against enzymes specific for non-neuronal or neuronal cells. Non-neuronal enolase immunoreactive cells were observed throughout the explants except at their edge where outgrowth zones were observed. Neuronal cells which were separately stained by three markers (neuron-specific enolase, tyrosine hydroxylase and dopamine-?-hydroxylase antisera) share common morphological features: they are grouped in clusters; their soma have similar shape and size and the staining was always seen inside the cytoplasm of the cell bodies and their proximal processes. These results show that noradrenergic neurons are still present in the explants 7 days after culture. Biochemical characteristics of the explants were followed throughout culture. Non neuronal enolase activity doubled during the first week in vitro. In contrast neuron specific enolase activity decreased to about half its initial value. Similarly the amount of 1-[(3)H]noradrenaline taken up in the explants decreased abruptly during the two first days in culture to reach a stable value. Tyrosine hydroxylase activity decreased significantly to about a third its initial value. All biochemical markers became stable after 7 days in culture. The cholinergic control of tyrosine hydroxylase in the noradrenergic cells has been investigated using 1-week old cultures. Tyrosine hydroxylase activity in locus coeruleus explants incubated in the presence of oxotremorine (10(?5) M, during 24 h) is significantly increased (+108%) as compared to non treated tissues. This increase was abolished if atropine sulfate (10(?5) M) was present together with oxotremorine. Immunotitrations of tyrosine hydroxylase in homogenates of the explants treated with the cholinergic agonist revealed that the increased enzyme activity is due to a change in its catalytic properties without variation in the number of enzyme molecules (delayed activation). This experiment indicates that an activation of tyrosine hydroxylase can result from the stimulation of cholinergic receptors located in the locus coeruleus region. The present investigation shows that central noradrenergic neurons of the locus coeruleus region can survive in culture conditions. These neurons retain most of their in situ morphological and biochemical characteristics. This in vitro approach appears to be an interesting means to study tyrosine hydroxylase regulation in central noradrenergic neurons.

Subthalamic nucleus modulates burst firing of nigral dopamine neurones via NMDA receptors.

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.

Quantitative evaluation of octadecaneuropeptide-like immunoreactivity in hippocampus, cortex and cerebellum of long-term isolated male Wistar rats.

Quantitative evaluation of octadecaneuropeptide-like immunoreactivity (ODN-Li) was carried out in hippocampus, cortex and cerebellum of group-reared or three month isolated male Wistar rats, whether muricidal or not. ODN-Li was lower in cortex and cerebellum of isolated animals, (mainly in muricidal) than of group-reared ones. A similar trend was apparent in the hippocampus. No significant differences were observed between muricidal and non-muricidal animals. The results are discussed in terms of the involvement of the endogenous ligands of the GABA-benzodiazepine receptor complex in the integrations related to mood and behaviour.

Fast in vivo monitoring of electrically evoked dopamine release by differential pulse amperometry with untreated carbon fibre electrodes.

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.