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Dopamine is thought to directly influence the neurophysiological mechanisms of both performance monitoring and cognitive control-two processes that are critically linked in the production of adapted behaviour. Changing dopamine levels are also thought to induce cognitive changes in several neurological and psychiatric conditions. But the working model of this system as a whole remains untested. Specifically, although many researchers assume that changing dopamine levels modify neurophysiological mechanisms and their markers in frontal cortex, and that this in turn leads to cognitive changes, this causal chain needs to be verified. Using longitudinal recordings of frontal neurophysiological markers over many months during progressive dopaminergic lesion in non-human primates, we provide data that fail to support a simple interaction between dopamine, frontal function, and cognition. Feedback potentials, which are performance-monitoring signals sometimes thought to drive successful control, ceased to differentiate feedback valence at the end of the lesion, just before clinical motor threshold. In contrast, cognitive control performance and beta oscillatory markers of cognitive control were unimpaired by the lesion. The differing dynamics of these measures throughout a dopamine lesion suggests they are not all driven by dopamine in the same way. These dynamics also demonstrate that a complex non-linear set of mechanisms is engaged in the brain in response to a progressive dopamine lesion. These results question the direct causal chain from dopamine to frontal physiology and on to cognition. They imply that biomarkers of cognitive functions are not directly predictive of dopamine loss., Competing Interests: The authors have declared that no competing interests exist.

Disturbances of the daily sleep/wake cycle are common non-motor symptoms of Parkinson's disease (PD). However, the impact of dopamine (DA) depletion on circadian rhythms in PD patients or non-human primate (NHP) models of the disorder have not been investigated. We evaluated alterations of circadian rhythms in NHP following MPTP lesion of the dopaminergic nigro-striatal system. DA degeneration was assessed by in vivo PET ([(11)C]-PE2I) and post-mortem TH and DAT quantification. In a light∶dark cycle, control and MPTP-treated NHP both exhibit rest-wake locomotor rhythms, although DA-depleted NHP show reduced amplitude, decreased stability and increased fragmentation. In all animals, 6-sulphatoxymelatonin peaks at night and cortisol in early morning. When the circadian system is challenged by exposure to constant light, controls retain locomotor rest-wake and hormonal rhythms that free-run with stable phase relationships whereas in the DA-depleted NHP, locomotor rhythms are severely disturbed or completely abolished. The amplitude and phase relations of hormonal rhythms nevertheless remain unaltered. Use of a light-dark masking paradigm shows that expression of daily rest-wake activity in MPTP monkeys requires the stimulatory and inhibitory effects of light and darkness. These results suggest that following DA lesion, the central clock in the SCN remains intact but, in the absence of environmental timing cues, is unable to drive downstream rhythmic processes of striatal clock gene and dopaminergic functions that control locomotor output. These findings suggest that the circadian component of the sleep-wake disturbances in PD is more profoundly affected than previously assumed.

The retinal circadian clock is crucial for optimal regulation of retinal physiology and function, yet its cellular location in mammals is still controversial. We used laser microdissection to investigate the circadian profiles and phase relations of clock gene expression and Period gene induction by light in the isolated outer (rods/cones) and inner (inner nuclear and ganglion cell layers) regions in wild-type and melanopsin-knockout (Opn 4 (-/-) ) mouse retinas. In the wild-type mouse, all clock genes are rhythmically expressed in the photoreceptor layer but not in the inner retina. For clock genes that are rhythmic in both retinal compartments, the circadian profiles are out of phase. These results are consistent with the view that photoreceptors are a potential site of circadian rhythm generation. In mice lacking melanopsin, we found an unexpected loss of clock gene rhythms and of the photic induction of Per1-Per2 mRNAs only in the outer retina. Since melanopsin ganglion cells are known to provide a feed-back signalling pathway for photic information to dopaminergic cells, we further examined dopamine (DA) synthesis in Opn 4 (-/-) mice. The lack of melanopsin prevented the light-dependent increase of tyrosine hydroxylase (TH) mRNA and of DA and, in constant darkness, led to comparatively high levels of both components. These results suggest that melanopsin is required for molecular clock function and DA regulation in the retina, and that Period gene induction by light is mediated by a melanopsin-dependent, DA-driven signal acting on retinal photoreceptors.

Spontaneous and/or stimulated neural activity of the nigrostriatal dopamine (DA) pathway makes amines run out from the neurons. This DA dynamic follows a rather complex path, running in or out the terminals, and flushing or diffusing into the extracellular space. The location of this leakage is not limited to the axon terminals; it also occurs from the cell bodies and dendrites. This molecular release mechanism was, for a long time, considered as being produced, in part, by the exocytosis of previously stored vesicles. The DA carrier protein (DAT, DA transporter) embedded in the DA cell membrane is known to clear previously released amines through an inward DA influx. The DAT also appears to be an active vector of amine release. Particular local conditions and the presence of numerous psychostimulant substances are able to trigger an outward efflux of DA through the DAT. This process, delivering slowly large amounts of amine could play a major regulatory role in extracellular DA homeostasis., (© 2011 The Author. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Background: It is increasingly recognized that non-motor symptoms are a prominent feature of Parkinson's disease and in the case of cognitive deficits can precede onset of the characteristic motor symptoms. Here, we examine in 4 monkeys chronically treated with low doses of the neurotoxin MPTP the early and long-term alterations of rest-activity rhythms in relationship to the appearance of motor and cognitive symptoms., Methodology/principal Findings: Behavioral activity recordings as well as motor and cognitive assessments were carried out continuously and in parallel before, during and for several months following MPTP-treatment (12-56 weeks). Cognitive abilities were assessed using a task that is dependent on the functional integrity of the fronto-striatal axis. Rest-activity cycles were monitored continuously using infrared movement detectors of locomotor activity. Motor impairment was evaluated using standardized scales for primates. Results show that MPTP treatment led to an immediate alteration (within one week) of rest-activity cycles and cognitive deficits. Parkinsonian motor deficits only became apparent 3 to 5 weeks after initiating chronic MPTP administration. In three of the four animals studied, clinical scores returned to control levels 5-7 weeks following cessation of MPTP treatment. In contrast, both cognitive deficits and chronobiological alterations persisted for many months. Levodopa treatment led to an improvement of cognitive performance but did not affect rest-activity rhythms in the two cases tested., Conclusions/significance: Present results show that i) changes in the rest activity cycles constituted early detectable consequences of MPTP treatment and, along with cognitive alterations, characterize the presymptomatic stage; ii) following motor recovery there is a long-term persistence of non-motor symptoms that could reflect differential underlying compensatory mechanisms in these domains; iii) the progressive MPTP-monkey model of presymptomatic ongoing parkinsonism offers possibilities for in-depth studies of early non-motor symptoms including sleep alterations and cognitive deficits.

The present experiments aimed at understanding the functional link between dopamine (DA) and glutamate (GLU) during the compensatory processes taking place after partial DA denervation. Lesion of the lateral part of substantia nigra in rats using 6-hydroxydopamine resulted in DA denervation of the lateral region of the ipsilateral caudate/putamen complex (CPc) whereas the medial CPc was spared. In vivo voltammetry revealed a large increase of extracellular dopamine (DA(ext)) in the medial CPc both ipsilateral and contralateral to the lesion. In addition, in vivo microdialysis and HPLC-ED revealed a concomitant increase of extracellular glutamate (GLU(ext)) in the ipsilateral medial CPc. Post-lesion chronic treatment with the putative neuroprotectors amantadine, memantine, and riluzole counteracted the tonic increases of DA(ext) and GLU(ext), revealing a possible role of GLU neurotransmission in the DA over-expression. Finally, acute low doses of GBR12909 had no effect on the DA(ext) in sham- operated animals, but dramatically increased DA(ext) in lesioned animals. The data suggest that a partial unilateral nigral lesion induces a bilateral increase of DA turn-over in the non-denervated striata through GLU afferences to the DA terminals.

Embryonic stem cells (ESC) have the ability of indefinite self-renewal and multilineage differentiation, and they carry great potential in cell-based therapies. The rhesus macaque is the most relevant preclinical model for assessing the benefit, safety, and efficacy of ESC-based transplantations in the treatment of neurodegenerative diseases. In the case of neural cell grafting, tracing both the neurons and their axonal projections in vivo is essential for studying the integration of the grafted cells in the host brain. Tau-Green fluorescent protein (tau-GFP) is a powerful viable lineage tracer, allowing visualization of cell bodies, dendrites, and axons in exquisite detail. Here, we report the first rhesus monkey ESC line that ubiquitously and stably expresses tau-GFP. First, we derived a new line of rhesus monkey ESC (LYON-ES1) that show marker expression and cell cycle characteristics typical of primate ESCs. LYON-ES1 cells are pluripotent, giving rise to derivatives of the three germ layers in vitro and in vivo through teratoma formation. They retain all their undifferentiated characteristics and a normal karyotype after prolonged culture. Using lentiviral infection, we then generated a monkey ESC line stably expressing tau-GFP that retains all the characteristics of the parental wild-type line and is clonogenic. We show that neural precursors derived from the tau-GFP ESC line are multipotent and that their fate can be precisely mapped in vivo after grafting in the adult rat brain. Disclosure of potential conflicts of interest is found at the end of this article.

Sensory stimulations of the forelimb in cats are known to increase dopamine release in the ipsilateral striatum and to decrease it in the homologous contralateral structure. Using positron emission tomography in both humans and cats, the present study shows that such sensory stimulations greatly reduce [(18)F]FDOPA accumulation ipsilateral to the stimulation (by 40.4% and 26.4% in the human caudate and putamen, respectively, and by 33.3% in the cat striatum). This decrease in striatal [(18)F]FDOPA uptake suggests a reduced DA storage resulting from the increased amine release. No change was observed in the contralateral striatum in neither human or cat suggesting, in contrast, that [(18)F]FDOPA accumulation is not facilitated by decreased DA release. These results support the hypothesis that sensory stimulations activate a non-synaptic mode of dopamine release.

Background: Sensory stimulation of the forelimb extremities constitutes a well-established experimental model that has consistently shown to activate dopamine (DA) neurotransmission in the mammals' forebrain., Objectives: To visualize in vivo this modification of striatal DA release in healthy human volunteers using Positron Emission Tomography (PET) and [(11)C]raclopride. Experiments in humans were paralleled by experiments in anesthetized cats. Changes in endogenous DA release were assessed through its competition with [(11)C]raclopride binding (BP(raclo)), a radioligand probing DA D2-receptors., Results: In humans no significant difference of BP(raclo) in caudate (with sensory stimulation: 2.0 +/- 0.3 versus without sensory stimulation: 2.2 +/- 0.3; P = 0.3) or putamen (2.6 +/- 0.3 versus 2.6 +/- 0.2; P = 0.9) ipsilateral to the stimulus was disclosed as a result of sensory stimulation. Similarly, no change of BP(raclo) was observed contralaterally to the stimulation in the caudate nucleus (with sensory stimulation: 2.0 +/- 0.4 versus without sensory stimulation: 2.1 +/- 0.2; P = 0.5) and the putamen (2.5 +/- 0.4 versus 2.6 +/- 0.2; P = 0.4). In cats the same results were obtained in the ipsilateral to stimulation striatum (with sensory stimulation: 2.5 +/- 0.03 versus without sensory stimulation: 2.4 +/- 0.05; P = 0.7). No change was also observed contralaterally to the stimulation (2.4 +/- 0.04 versus 2.5 +/- 0.06; P = 0.6). The [(11)C]raclopride binding remained unchanged by sensory stimuli in both humans and cats., Conclusion: This suggests that the DA release induced by sensory stimulus is mostly extrasynaptic whereas the synaptic DA release is probably small, which fits well with the absence of [(11)C]raclopride displacement. The mechanism of this extrasynaptic DA release could be related to a local action of glutamate on dopaminergic terminals via a thalamo-cortico-striatal loop. Present results also underline homology between cat and human responses to sensory stimuli and validate the use of cat brain to find physiological concepts in humans.

The effects of the electrically evoked serotonin release were evaluated on the binding of (18)F-MPPF in the hippocampus of anesthetized rats. The specific binding of (18)F-MPPF was measured by an implanted beta-microprobe and the serotonin (5-HT) extracellular concentration was measured by microdialysis under the same conditions. Our results showed that the 10-, 20-, or 30-min electrical stimulation of the raphe nucleus elicited a significant increase in extracellular 5-HT, only detectable in the presence of a 5-HT reuptake inhibitor in the perfusate (5 microM clomipramine). Interestingly, the raphe stimulations were associated with a 27-76% reversible decrease of the (18)F-MPPF specific binding in the hippocampus, but an unchanged extracellular (18)F-MPPF collected in dialysates. Considered together, these observations suggest that (18)F-MPPF binding is sensitive to 5-HT released at a neuronal level. This compartment, explored by the beta-microprobe, is probably distinct from the extracellular compartment, explored by microdialysis., (Copyright 2003 Wiley-Liss, Inc.)

Astrocytes release glutamate (Glu) by the mobilisation of intracellular concentrations of Ca++. The rationale of the present work was to test whether Glu and its agonists, known to affect intracellular Ca++ content via the activation of metabotropic and ionotropic receptors, could modulate the astrocytic release of excitatory aminoacids. NMR experiments showed that Glu released uniformly labelled [13C] Glu in the incubation medium of rat astrocytes in primary cultures. Further experiments confirmed this finding and showed that the incubation of these cells with agonists and antagonists of Glu ionotropic and metabotropic receptors, produced a different modulation of Glu and aspartate release. The observed activations of the various receptors suggest a complex modulation of the release of the excitatory aminoacids. Such a release of is interpreted in terms of metabolic microzonation.

Cats were trained to stay in a containment box, without developing any signs of behavioural stress, while their head was maintained in a position that allowed positron emission tomography (PET) experiments to be performed. The binding potential for [(11)C]raclopride (BP(raclo)), a radioligand with good specificity for dopamine (DA) receptors of the D(2) type, was measured in the striatum and in three experimental situations: awake, anaesthetised with ketamine (50 mg kg(-1) h(-1); i.m.) and anaesthetised with halothane (1.5%). Non-specific binding was evaluated in the cerebellum. In the striatum of both sides, the BP(raclo) was unmodified by ketamine anaesthesia when compared with awake animals. In contrast, a large increase in BP(raclo) was observed under halothane anaesthesia. The non-specific binding of [(11)C]raclopride, evaluated in the cerebellum, was also unchanged under ketamine anaesthesia but greatly increased under halothane anaesthesia. To evaluate whether changes in the cerebral blood flow (CBF) resulting from the different experimental situations could be at the root of these discrepancies, injections of [(15)O]H(2)O were performed; measurements revealed a drastically increased CBF under halothane anaesthesia and a slight enhancement under ketamine anaesthesia, when compared with the waking state. These results are the first to be obtained on this topic in awake cats, and show that the BP(raclo) is greatly dependent on alterations in the CBF.

The effects of halothane and ketamine anesthesia on [11C]raclopride binding were assessed in the cat striatum at basal conditions and after drug- or depolarization-induced dopamine (DA) release using Positron Emission Tomography. At baseline, Scatchard analyses revealed that the higher [11C]raclopride binding found under halothane anesthesia was mainly attributable to a higher radioligand apparent affinity. Decreased [11C]raclopride binding was demonstrated following amphetamine under ketamine but not under halothane anesthesia. Under ketamine anesthesia transient DA overflows induced by direct stimulations of DA neurons through an intracerebral electrode induced transient changes in [11C]raclopride binding with a remarkable spatiotemporal accuracy. No effect was observed under halothane anesthesia. The failure to detect competition between DA and [11C]raclopride for binding on D(2)-receptors under halothane anesthesia might reflect, as already reported for other brain receptor systems, a halothane-promoted conversion of D(2)-receptors to a state of lower affinity for DA. It is suggested that the affinity state of receptors is a factor to be considered in in vivo ligand-activation studies.

Unlabelled: Our aim was to show the ability of a recently developed beta(+)-range-sensitive intracerebral probe (SIC) to measure, in vivo, the binding of radioligands in small animals., Methods: The potential of the device for pharmacokinetic studies was evaluated by measurement of the dynamic striatal binding of (11)C-raclopride, a well-documented D(2) dopaminergic receptor ligand, in rat brain after intravenous injection of the labeled compound. The effects of preinjection of the unlabeled ligand (raclopride, 2 mg/kg intravenously) and of increasing the synaptic dopamine level (amphetamine treatment, 1 mg/kg intravenously) or of depleting synaptic dopamine (reserpine pretreatment, 5 mg/kg intraperitoneally) on in vivo (11)C-raclopride binding were monitored by SIC., Results: The radioactivity curves measured as a function of time were reproducible and consistent with previous studies using PET imaging (ratio of striatum to cerebellum, 2.6 +/- 0.3 after 20 min). Further studies showed significant displacement of (11)C-raclopride by its stable analog. Finally, the device proved its capacity to accurately detect changes in (11)C-raclopride binding after a sudden (amphetamine) or a gradual (reserpine) modulation of endogenous dopamine levels., Conclusion: These results show that the new device can monitor binding of PET ligands in anesthetized rodents in vivo, with high temporal resolution.

After injection of 6-hydroxydopamine into the lateral part of the rat substantia nigra, tissue dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were reduced in the corresponding lateral part of the ipsilateral caudate/putamen (CP) complex (13, 40 and 56% of controls, respectively). In this region, tyrosine hydroxylase (TH, the rate limiting enzyme of the DA synthesis) immunoautoradiography decreased by more than 80% as was the case for the binding of tritiated GBR12935 (a specific marker of the DA-carrier protein). In the medial region of the CP, only very moderate reductions of DA, DOPAC and HVA (77, 76 and 84% of controls, respectively) were observed. In this region, TH immunoautoradiography and GBR12935 binding were only reduced by about 20% reflecting weak DA denervation. However, using in vivo voltammetry, extracellular basal DA levels were found to be particularly high in the medial region of CP complex when compared to unoperated animals (up to 235%). In the medial region, TH activity was also significantly increased (161%) but the electrical stimulation of DA fibers produced the same DA overflow in control and lesioned animals. From these results, it may be concluded that elevated basal DA levels in this region cannot be attributed to the reduced DA uptake and/or to an increased ability of DA neurons to release DA in response to impulse flow.

It is well established that midbrain dopamine neurons innervating the striatum, release their neurotransmitter through an exocytotic process triggered by the neural firing and involving a transient calcium entry in the terminals. Long ago, it had been proposed, however, that another mechanism of release could co-exist with classical exocytosis, involving the reverse-transport of the cytosolic amine by the carrier, ordinarily responsible for uptake function. This atypical mode of release could be evoked directly at the preterminal level by multiple environmental endogenous factors involving transient alterations of the sodium gradient. It cannot be excluded that this mode of release participates in the firing-induced release. In contrast with the classical exocytosis of a preformed DA pool, the reverse-transport of DA requires simultaneous alterations of intraterminal amine metabolism including synthesis and displacement from storage compartment. The concept of a reverse-transport of dopamine is coming from the observations that releasing substances, such as amphetamine-related molecules, actually induce this type of transport. A large set of arguments advocates that reverse-transport plays a role in the maintenance of basal extracellular DA concentration in striatum. It was also often evoked in physiopathological situations including ischemia, neurodegenerative processes, etc. The most recent studies suggest that this release could occur mainly outside the synapses, and thus could constitute a major feature in the paracrine transmission, sometimes evoked for DA.

The in vivo and ex vivo distributions and the pharmacological profile of the fluorinated phenylpiperazine derivative p-[(18)F]MPPF (4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-fluorobenzamido]-et hyl piperazine) were evaluated in the cat brain as a potential selective antagonist for 5-HT(1A) receptors using PET. After intravenous injection of p-[(18)F]MPPF in cats, there was a rapid accumulation of radioactivity in the brain, with 4% of the total radioactivity injected present in the brain at 4 minutes postinjection. The highest uptakes of radioactivity were observed in the hippocampus and cingulate cortex, regions known to be rich in 5-HT(1A) receptors, whereas lower levels of radioactivity were observed in the cerebellum. The mean ratio of radioactivity in the hippocampus to the cerebellum was 4.29 (SD = 0.21; n = 5) from 40 to 90 minutes postinjection of p-[(18)F]MPPF. The corresponding ratio for the cingulate cortex was 3.01 (SD = 0.16; n = 5). Specific binding in the hippocampus and the cingulate cortex was markedly reduced following injection of unlabeled WAY-100635 and pindolol but was unaffected by treatment with alpha1, 5-HT(2), or reuptake inhibitor agents indicating reversibility and selectivity of p-[(18)F]MPPF binding to 5-HT(1A) receptors. Ex vivo autoradiographic study with p-[(18)F]MPPF in cat brain sections showed labeling of areas rich in 5-HT(1A) receptors with a regional brain distribution that closely matched that observed using PET. These results indicate that p-[(18)F]MPPF may be a useful candidate for noninvasive PET imaging of 5-HT(1A) receptors in the living human brain., (Copyright 2000 Wiley-Liss, Inc.)

To investigate the contribution of the dopamine (DA) synthesis to both the calcium-dependent and the carrier-mediated, mechanisms of DA release in the striatum, anaesthetized rats were locally superfused in the striatum with a push pull cannula supplied with an artificial CSF containing tritiated tyrosine. DA, dihydroxyphenylacetic acid (DOPAC) and their respective specific activity were measured in effluent and used to evaluate changes in the DA synthesizing rate. Excluding calcium ions from the CSF only partially reduced spontaneous DA release (70%) still leaving a possible carrier-mediated DA release. This effect was not additive with a local superfusion with 0.1 mM a-methyl-p-tyrosine, a blocker of DA synthesis, suggesting that synthesis could already be reduced by calcium-free superfusion. Local superfusion with 100 microM cadmium in the presence or not of calcium ions, increased the DA release (220 and 350%, respectively), simultaneously reducing DA synthesis. Local application of 1 microM calcium ionophore (A23187) was without effect on the basal release of DA but enhanced DA synthesis and increased the amphetamine-evoked and carrier-mediated amine release. We conclude that DA synthesis can be a modulatory process of the firing-independent and carrier-mediated amine release while it weakly affects the classical calcium-dependent release.

Polyamine contents were determined in human temporal lobe epilepsy. In the seven patients studied, stereoelectroencephalography (SEEG) located the epileptogenic focus in Ammon's horn and neuropathological findings were limited to hippocampal gliosis and sclerosis. Each polyamine exhibited a specific regional distribution. The most important variations were observed for spermidine and spermine while putrescine levels varied less. The regional variation was predominant in middle > posterior > anterior parts of the temporal lobe. Spermine contents and the spermidine/spermine (SPD/SPM) index varied especially in the middle and posterior parts of the hippocampus. Metabolic SPD/SPM index and spermidine levels were found to be drastically increased in almost all limbic parts when compared to neocortical regions. The opposite was observed for spermine. The heterogeneous distribution of polyamines was compared to abnormal electrical activities recorded by SEEG: SPD/SPM index and spermidine levels were sharply increased in seizure onset areas and high levels of spermine were detected in temporal cortex propagation areas. The presently reported heterogeneity of polyamine contents might contribute to modulate differentially the local control of excitability in human temporal epilepsy.

The effects of moderate changes in extracellular dopamine concentrations on the in vivo binding of specific dopaminergic D2 radioligands with different affinities and kinetics were investigated in rats. Either [125I]NCQ298 (Kd = 19 pM), or [25I]iodolisuride (Kd = 0.27 nM) or [3H]raclopride (Kd = 1.5 nM) were administered intravenously (IV) to animals 1 h after the intraperitoneal (IP) injection of either alpha-methyl-p-tyrosine (AMPT) (250 mg/kg) or nomifensine (15 mg/kg), or saline. The kinetics of radioactivity concentration in the striatum, cerebellum, and plasma were measured for up to 4 h after [125I]NCQ298 or [125I]iodolisuride injection and up to 1.5 h after [3H]raclopride injection. For each tracer, the striatum-to-cerebellum radioactivity concentration ratios (S/C) and the binding potential (BP), calculated as the association to dissociation binding rate constant ratios (k3/k4), were assessed and related to the changes in extracellular dopamine concentration induced by drug treatments. Results show that S/C and BP of [3H]raclopride were significantly diminished by pretreatment with nomifensine, a drug that increases extracellular dopamine concentration. Nomifensine pretreatment induced no changes in the in vivo binding indexes of the high affinity [125I]NCQ298 and a slight but not significant decrease of the binding indexes of 125I]iodolisuride. Treatment with AMPT, which induced a 40% reduction in dopamine concentration, did not change [125I]NCQ298 binding indexes but slightly increased those of [3H]raclopride and [125I]iodolisuride. In conclusion, the change of dopamine concentration induces modification of radiotracer kinetics. Thus, the combined use of tracers with high and low affinities could allow us to obtain information both on receptor density and neurotransmitter release in vivo. However, as indicated by the [3H]raclopride study with AMPT, small changes in the concentration of intrasynaptic dopamine cannot be easily detected.

Colchicine, an axonal transport blocking agent, was unilaterally injected in the medial forebrain bundle of rats. As early as 18 h after the injection a rapid decrease in TH-mRNA level was observed in the substantia nigra and the ventral tegmental area (SN/VTA) on the injected side. In contrast, TH protein levels remained stable for 48 h, and decreased later in both cells bodies and terminals (caudate/putamen). The number of TH-immunopositive cells in SN/VTA increased after colchicine equally in both sides, excluding a neurotoxic effect. These results suggest that TH gene expression is controlled by a retrogradely transported activating factor rather than by feedback inhibition by the end product, i.e. TH protein.

No-carrier-added 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1 piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) was synthesized by nucleophilic substitution of the corresponding nitro compound in the presence of Kryptofix 222 and K2CO3 by microwave heating (3 min, 500 W) using a remotely controlled radiosynthesis. Baseline separation of p-[18F]MPPF from the nitro derivative was performed on a semipreparative HPLC C18 column. After Sep-Pak formulation, the radiopharmaceutical was obtained with a radiochemical yield of 25% (EOS) in about 70 min. Specific radioactivity averaged between 1-5 Ci/micromol EOS. Labelling of the ortho and meta derivatives was also attempted. Brain uptake of p-[18F]MPPF was studied with PET on fluothane-anesthetized cats. Following intravenous injection of p-[18F]MPPF, high accumulation of radioactivity was observed in the hippocampus and cerebral cortex. Low levels of radioactivity were observed in cerebellum. At 30 min, the mean hippocampus/cerebellum and cortex/cerebellum ratios were 5 and 3.8, respectively. The accumulation of the tracer was blocked by prior administration of reference WAY-100635, demonstrating the specificity of the ligand.

The expression of enkephalin in neurons of the rat forebrain was studied by in situ hybridization and immunohistochemistry after unilateral injections of ibotenic acid into the bed nucleus of the stria terminalis. Initially, we observed that the destruction of nerve cell bodies in this nucleus resulted in a prominent bilateral increase in the number of neuronal perikarya immunoreactive for [Met]enkephalin in the lateral/basolateral amygdaloid complex-especially in the anterior division of the latter nucleus-as compared with NaCl-injected rats. In a separate set of experiments, this effect was associated with a significant (two times) enhancement of the number of nerve cell bodies containing preproenkephalin A messenger RNAs in the same amygdaloid nucleus ipsilateral to the injection, as compared with controls. In the hypothalamus of both experimental and control rats, the nerve cell bodies immunoreactive for [Met]enkephalin were few since the animals were not pretreated with colchicine, and the effects of the lesion were difficult to appreciate. However, using in situ hybridization, numerous nerve cell bodies containing preproenkephalin A messenger RNAs were detected bilaterally in the perifornical area, the paraventricular (parvocellular division) and the ventromedial nuclei of the hypothalamus. In the latter nucleus, the lesion of the bed nucleus of the stria terminalis resulted in a strong decrease (about two times) in the number of labelled cell bodies as compared with the controls, whereas no significant changes were found bilaterally in the paraventricular nucleus. In agreement with some data of the literature, our results indicate that the bed nucleus of the stria terminalis plays an important role in the regulation of neuropeptide genes expression in certain regions of the limbic system. Such a role is often exerted by nerve fibres afferents to the nerve cell bodies considered. However, from numerous neuroanatomical data of the literature, it appears more probable that the induction or inhibition of the expression of enkephalin in presynaptic neurons is due to the disappearance of their postsynaptic target in the bed nucleus of the stria terminalis.

A push-pull cannula supplied with artificial CSF was implanted in the striatum of anaesthetized rats, and the basal extracellular DA and DOPAC was assayed in the superfusates using HPLC and electrochemical detection. Simultaneously, a carbon fibre electrode was implanted in close proximity of the cannula and the evoked DA release was detected by differential pulse amperometry during stimulation of the DA axons. Local treatments with cadmium (100 microM) blocked the evoked DA release (-90%), but substantially increased the basal extracellular DA (+125%). The effects of glutamate agonists NMDA (1 mM) and kainate (0.1 mM), known to increase basal extracellular DA were confirmed (+150% and +60% respectively). It was, however, simultaneously observed that the evoked DA release was inhibited (-80% and -50%, respectively). Amphetamine (1 microM) released DA (+150%) and produced also an increase (+100%) of the evoked DA release. These results, apparently conflicting, show that the two mechanisms releasing dopamine (firing-dependent and not) can be directly and simultaneously observed. These two releasing processes appear to be not strictly antagonist. They are also differently and independently modulated by calcium and by local influences such those conveyed by glutamate.

The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17 alpha or 17 beta E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose-dependent increase in striatal TH activity was observed within minutes after 17 beta (but not 17 alpha) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push-pull cannula supplied with an artificial CSF containing [3H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at 20-min intervals. Addition of 10(-9) M 17 beta (but not 17 alpha) E2 to the superfusing fluid immediately evoked an approximately 50% increase in [3H]DA and [3H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2-induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (Ki(DA)) was calculated. Incubation of striatal slices in the presence of 10(-9) M 17 beta (but not 17 alpha) E2 indeed evoked an approximate twofold increase in the Ki(DA) of one form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty weeks after ibotenic acid lesions of the striatum, the amount of tyrosine hydroxylase (TH) in this structure was markedly increased. This was accompanied by a 3-fold increase in TH mRNA levels in the ipsilateral subtantia nigra (SN). Striatal levels of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) were markedly reduced. In the nucleus accumbens, spared by the lesion, DA neurotransmission was also altered, as evidenced by a reduction of DA and DOPAC, but no increase in TH could be detected. TH mRNA levels were moderately enhanced in the ventral tegmental area (VTA). Thus, lesioning in the striatum induces TH gene activation in both SN and VTA neurones, not strictly related to DA function at the terminal level.

The effects of a chronic imipramine treatment on the mesoamygdaloid pathway of rats were examined. Using semiquantitative immunocytochemical techniques, it was observed that the level of TH mRNA was decreased in the ventral tegmental area (VTA). In contrast, the TH protein was increased in both the VTA and amygdala. The TH activity was decreased in the amygdala when assessed under normal conditions but increased after a preincubation to phosphorylate the enzyme, suggesting a lowering of the protein-specific activity in the terminals. These results show that TH protein turnover in the mesoamygdaloid neurons can be reduced by chronic imipramine treatments, thereby producing an accumulation of inactive TH protein in the neurons while also decreasing TH gene activity in the cell bodies.

The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a Ki(DA) value of 29.92 +/- 0.49 microM, the other being approximately 15-fold more sensitive to DA inhibition with a Ki(DA) of 1.96 +/- 0.09 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low Ki(DA) remained unaffected, whereas the Ki(DA) of the purported active form of TH increased to 62.6 +/- 0.8 microM, suggesting an increase in the enzyme phosphorylation. This increase in the Ki(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12-O-tetradecanoylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.

In the present study, we describe the neurochemical effects of intranigral injections of colchicine in the rat forebrain using immunohistochemistry and in situ hybridization. The observations on the injected side are compared to the contralateral one and to the sham-operated rats. We demonstrate that such injections are able to strongly enhance the immunoreactivity for Met-enkephalin (ME), substance P (SP) and neuropeptide Y (NPY) in numerous nerve cell bodies of the limbic system (injected side), whereas the levels of the corresponding mRNAs are differently modified according to the region examined. A clear correlation between the enhancement of the immunostaining for ME and SP and that of the preproenkephalin (PPA) and preprotachychinin gene transcripts was observed in neuronal perikarya of the medial amygdaloid nucleus (SP), of the dorsolateral hypothalamus (ME) and of the ventromedial hypothalamic nucleus (SP). These observations are interpreted as an induction--or increased expression--of neuropeptide genes in neuronal perikarya postsynaptic to nerve fibers originating in the midbrain and brain stem. In this case, colchicine is thought to block the electrophysiological activity of ascending nerve fibers (anterograde and postsynaptic effect). In the case where the enhancement of the immunoreactivity for the studied neuropeptides was associated with no change or a decreased expression of the corresponding genes in the same brain areas, colchicine may have blocked the axoplasmic transport of peptides in nerve fibers projecting to the midbrain and/or brain stem (6). This may result in a retrograde accumulation of peptides in the nerve cell bodies of origin and, eventually, in a negative feedback regulation of the corresponding encoding genes in these perikarya (retrograde and presynaptic effect of colchicine). The drastic behavioral effects of bilateral intranigral injections of colchicine, on ingestive behavior in particular, have been studied in a following paper.

In the present study, we demonstrate the existence of numerous peptidergic afferents to the bed nucleus of the stria terminalis (BNST) using the retrograde transport of gold-labeled wheat germ agglutinin-apo-peroxidase (G-WGA-HRP) combined with the indirect immunoperoxidase method after intraparenchymatous injections of colchicine. At first, we show that local injections of colchicine alone into the BNST are able to induce the retrograde accumulation of peptides until the nerve cell bodies of origin, probably because of the blockade of axonal transport in nerve terminal arborizations innervating this nucleus. The actual existence of putative peptidergic afferents to the BNST indicated by the local injections of colchicine was established using: a) the retrograde transport of G-WGA-HRP from the BNST combined with immunocytochemistry after administration of colchicine at the same place, b) the anterograde "transport" of the fluorescent tracer DiI from selected nuclei of the forebrain. We demonstrate that the neurons immunoreactive for enkephalins, neurotensin, or substance P that innervate the BNST are localized mainly in the central amygdaloid nucleus, the paraventricular thalamic nucleus, and the ventromedial hypothalamic nucleus ipsilateral to the injection, as well as bilaterally in the magnocellular paraventricular and perifornical regions of the hypothalamus. From these results it may be concluded that intracerebral injections of colchicine constitute a powerful tool to search for multiple peptidergic afferents to a given brain nucleus using only immunohistochemistry. The existence of these pathways, however, must be verified by other neuroanatomical methods because of the problem of nerve fibers of passage.

The short-term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L-3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30-40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a Ki(DA) of 26.4 +/- 2 microM and the other being approximately 10-fold more sensitive to DA inhibition, with a Ki(DA) of 2.56 +/- 0.17 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and poorly active form, respectively. Conversely, after estradiol treatment all TH molecules exhibited the same Ki(DA) of 2.5 +/- 0.3 microM. This effect was stereospecific, because 17 alpha-estradiol could not promote it, whereas with 17 beta-estradiol, it could be observed at only 10(-11) M and after a short delay (30 min). Finally, this decrease in the Ki(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.

Polyamine (tissue) concentrations have been studied in hippocampus and temporal neocortex from patients with temporal lobe epilepsy. Depth electrode recordings demonstrated hippocampal origin of the seizures, the temporal neocortex being involved during the discharge propagation. Neuropathological examination of excised tissues showed glial proliferation or glioma in Ammon's horn (CA), whereas the temporal neocortex did not exhibit any histological abnormality. Polyamine (putrescine or PUT, spermidine or SPD, spermine or SPM) concentrations were determined on surgical samples from the hippocampus and various areas of temporal neocortex. Human post-mortem tissue from temporal lobe regions was used for controls. In post-mortem controls and temporal neocortex specimens from epileptic patients, polyamine levels were similar (in nmol/g wet weight: PUT = 40-100; SPD = 200-350; SPM = 100-200). In CA, polyamine levels exhibited striking changes: SPD content was significantly increased (350-700 nmol/g) while SPM was lowered (50-100). PUT was only increased in CA invaded by the tumoral process (100-180). Accordingly, a very high SPD/SPM molar ratio in the abnormal CA region was observed, indicating an acceleration of polyamine neosynthesis which is usually related to ornithine decarboxylase induction. Metabolic changes in polyamines appear to be selective of human epileptic hippocampus. A relationship between glial proliferation (gliosis or neoplasia), epileptic firing and polyamines is discussed.

A possible interference of the GnRH-associated peptide (GAP) with the metabolism of dopamine in the rat striatum was investigated. The presence of the precursor of the peptide in this central region of dopaminergic terminals was first evidenced using specific RIA. The action of GAP on dopamine release was investigated in the caudate nucleus using the local superfusion with a push-pull cannula supplied with an artificial CSF containing the tritiated precursor of dopamine [( 3H]tyrosine). Addition of GAP (1 microM) to the superfusing fluid resulted in an increase of the release of the newly synthesized dopamine without a significant modification of the total amine release. In situ neutralization of GAP by addition in the CSF of a rabbit serum containing antibodies directed against the GAP produced opposite effects evidencing a tonic function for this peptide. In addition to the increased specific activity of the dopamine released during GAP treatment, the alterations observed in the efflux (and the specific activity) of dihydroxyphenyl acetic acid and the activation of dopamine synthesis obtained in vitro in striatal slices in the presence of GAP led us to conclude that the GAP system could be considered as a positive control of dopamine synthesis.

Rats with unilateral or bilateral partial section of the fornix were impaired on an eight arm radial maze task. Neurochemical analysis of hippocampal tissue four weeks after the lesions revealed a 50% reduction of choline acetyltransferase (ChAT) activity. The cholinergic marker was correlated negatively with the number of errors in the maze; the lower the ChAT activity, the higher the error score. The fornix lesion also induced a 50% reduction in norepinephrine (NE), but no change in the noradrenergic metabolite methylhydroxyphenylglycol (MHPG), suggesting a net increase in turnover of NE in these animals. Additional lesion of the noradrenergic system with the neurotoxin DSP4 reduced both MHPG and NE levels by more than 90%, compared to nonlesioned controls, and reversed the behavioral deficit. This treatment had no further effect on cholinergic markers. There was a significant negative correlation between ChAT activity and the index of NE turnover, suggesting that hyperactivity in the noradrenergic system after fornix section inhibits the spared cholinergic function and thus exacerbates the cognitive deficit. The pattern of neurochemical results bear a striking resemblance to those seen in some Alzheimer's patients and suggest that an equilibrium among neurotransmitters is important to cognitive function.

The afferent connections of the rat substantia nigra pars lateralis have been studied using the retrograde axonal transport of fluorescent latex microspheres. The most numerous groups of retrogradely labelled nerve cell bodies were observed bilaterally in the parabrachial complex and several hypothalamic nuclei, whereas the parietal neocortex, the fundus striati, the central nucleus of the amygdala and the bed nucleus of the stria terminalis were labelled on the injected side only. The neuronal projections from the central amygdaloid nucleus to the substantia nigra pars lateralis and lateral part of the rostral pars compacta have additionally been confirmed by anterograde tracing using wheat-germ agglutinin coupled to horseradish peroxidase. The presence of some peptides in this pathway was studied by combining the use of the same retrograde tracer with immunofluorescence after intra-amygdaloid injections of colchicine. With this method, we have demonstrated that Met-enkephalin, dynorphin and neurotensin are probably utilized as neurotransmitters or co-transmitters in the neurons of the amygdalo-nigral pathway.

Abstract The effects of acute estradiol (E(2)) treatment on both the activity of tyrosine hydroxylase (TH) in the median eminence and the serum level of prolactin (PRL) were investigated. Twelve-day-ovariectomized rats were injected with 17beta-E(2) (25mug sc) at 1100 h and sacrificed hourly from 1200 to 2300 h. TH activity was quantified by measuring the amount of exogenous tyrosine converted to L-DOPA in vitro by aliquots of median eminence homogenates. Serum PRL levels were evaluated by radioimmunoassay. A biphasic response of TH activity to treatment was observed: an immediate decrease occurred-preceding and accompanying a rise in serum PRL-followed by an increase beyond control levels 2 h after the maximal release of PRL. The increase in TH activity could be prevented by the pretreatment of rats with a specific rat PRL antiserum, suggesting it was not due to E(2) per se but rather mediated by the E(2)-induced PRL elevation. To pin-point the process underlying the E(2)-induced decrease in TH activity, we evaluated the kinetic parameters of TH in the median eminence as well as its quantity (by Western blot analysis) in the median eminence and arcuate nucleus. Finally, we used a sensitive dot-blot assay to quantify specific TH messenger ribonucleic acid in the arcuate nucleus. The decrease in TH activity after E(2) treatment paralleled an immediate decrease in the affinity of TH for its pterin cofactor (6-MPH4), while V(max) remained unchanged. A decrease in the amount of TH protein in the arcuate nucleus and median eminence as well as in the TH messenger ribonucleic acid level in the arcuate nucleus was also observed, but the latency of these effects precluded a major involvement in the immediate decline of TH activity. Therefore, when observed separately from those of PRL, E(2) effects on TH in tuberoinfundibular dopaminergic neurons are clearly inhibitory consisting of a 'deactivation' of the enzyme together with a reduction of its synthesis.

Forskolin (FSK) was locally injected into the substantia nigra (SN) of anesthetised rats. The day after injection (24 and 36 hr), tyrosine hydroxylase (TH) activity increased locally in this structure but remained unmodified in the ipsilateral caudate nucleus (CN). The amount of messenger RNA for TH (TH-mRNA) was also increased in the SN 24 hr after the injection. However, TH protein content was modified neither locally in the SN nor in the ipsilateral CN. In addition, the decrease of the ratio between dopamine and its first metabolite in the CN and the SN suggested a decreased activity of the dopaminergic nigral cells. The absence of increase of the protein synthesis in spite of the fact that TH-gene transcription was initiated could be the consequence of the inhibition of dopaminergic cells by the drug. These results confirm that, in vivo, TH induction is cAMP-dependent and demonstrate that the TH-gene activity is not strictly coupled to the activity of dopaminergic cells in the SN.

The effects of chronic treatment with desimipramine (a tricyclic antidepressant), fluoxetine [a specific 5-hydroxytryptamine (5-HT) uptake inhibitor], clorgyline (a specific monoamine oxydase inhibitor of A type), ipsapirone (a specific 5-HT1A receptor agonist) as well as electroconvulsive shock treatment were investigated on rat hippocampal 5-HT1A receptors negatively coupled to adenylyl cyclase. Drugs were injected intraperitoneally in rats for 2 or 3 weeks, and biochemical determinations were made 4 to 72 hr after the final dose. Chronic treatments with desimipramine, ipsapirone and fluoxetine did not induce any change in the 5-HT1A-induced inhibition of the adenylyl cyclase activity. In contrast, chronic treatment with clorgyline and electroconvulsive shock treatment induced a slight but significant reduction of 5-HT's ability to inhibit hippocampal adenylyl cyclase. This indicates that, at least in hippocampal neurons, the 5-HT1A receptor coupled to adenylyl cyclase is not easily desensitized. This was verified in vitro on murine hippocampal neurons in culture, by measuring the effects of intense stimulation (1 and 2 hours), with 5-HT, ipsapirone and 8-hydroxy-2-(di-n-propylamino)tetralin. Indeed, such stimulations did not significantly affect the 5-HT1A receptor-induced inhibition of cAMP production in these hippocampal neurons in culture. Our results indicate that it is not the post-synaptic 5-HT1A receptor of hippocampus that is modified during antidepressant treatments, at least at the level of its coupling to adenylyl cyclase.

The retrograde transport of WGA-HRP adsorbed to colloidal gold was combined with the indirect immunoperoxidase technique to study the origin of enkephalin-containing afferents to the medial and ventral regions of the globus pallidus (GP). On the injected side, the nerve cell bodies labeled retrogradely or double labeled were numerous in the central nucleus of the amygdala (ACe), scattered in the bed nucleus stria terminalis (BNST) and few in the fundus striati. In the ACe, approximately 40% and 20% of the retrogradely labeled perikarya were found immunoreactive for Met-enkephalin and Leu-enkephalin, respectively, whereas they were only 30% and 15% in the BNST. It is concluded that the enkephalinergic afferents of the ventro-medial region of GP, which contains the basal nucleus of Meynert in the rat, are largely of limbic origin.

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.

A push-pull cannula supplied with an artificial cerebrospinal fluid containing the tritiated precursor of dopamine, [3H]tyrosine, was implanted in the caudate nucleus of rats anesthetized with halothane. The extracellular dopamine and dihydroxyphenylacetic acid were measured in successive 20 min fractions (both in their tritiated and unlabeled form) and the ratio between the two forms calculated. Glutamate was added to the superfusing cerebrospinal fluid to investigate its role in the process of dopamine release. The release of dopamine and the efflux of dihydroxyphenylacetic acid were activated by a low concentration (10(-8) M) of glutamate. In contrast, a higher concentration (10(-4) M) of the amino acid reduced the release of dopamine. These results first confirmed the presence of a dual mechanism of control, by glutamate, of the dopamine release in the striatum depending on the extracellular concentration. Secondly, these treatments affected the dihydroxyphenylacetic acid amount and predominantly the tritiated form of dopamine, suggesting that the glutamate induces an important increase of the amine synthesis, in spite of a moderate effect on the release. The reversal of the inhibition by applications of tetrodotoxin (5 x 10(-7) M) and bicuculline (10(-4) M) confirmed that it was mediated by an indirect mechanism involving a GABAergic neurotransmission. In addition, the increase of the spontaneous dopamine release during bicuculline application suggested the existence of a tonic mechanism of inhibition of dopamine release in the striatum. This was confirmed by the fact that local xylocaine-induced anesthesia of the sensory motor cortex increased the spontaneous release of dopamine in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

The release of [3H]DA synthesized from [3H]tyrosine was estimated in the two caudate nuclei (CN) during the unilateral nigral application of glycine and GABA-related compounds in 'encéphale isolé' cats using push-pull cannulae. Glycine (10(-5) M) reduced the release of [3H]DA in both CN and these effects were antagonized by strychnine (10(-5) M). A decrease in [3H]DA release was also seen in both CN during the unilateral nigral application of diazepam (10(-5) M). In contrast, muscimol (10(-6) M) and GABA (10(-5) M) stimulated [3H]DA release on both sides. The effect of GABA was blocked by picrotoxin (10(-5) M). Picrotoxin alone stimulated the release of [3H]DA in the ipsilateral CN and was without effect in the contralateral side. Bicuculline (10(-5) M) stimulated [3H]DA release only in the contralateral CN. A symmetric increase in [3H]DA release in both CN was also observed during the unilateral nigral application of potassium (30 mM). A model involving a facilitatory polysynaptic pathway originating from the substantia nigra (SN) and acting presynaptically on ther terminals of the contralateral DA neurons is proposed to explain the changes in [3H]DA release induced in the contralateral CN in these various situations. The results are discussed taking into account previous data on the reciprocal control of the two dopaminergic pathways induced by the unilateral nigral application of dopaminergic drugs.

In order to study the mechanisms regulating the dopaminergic nigroamygdaloid cells, the release of dopamine was observed in the central nucleus of the amygdaloid complex. Halothane anesthetized rats were implanted, according to the experiment, with one or two push-pull cannulae in the central nuclei of the amygdala (ACE), the substantia nigra (SN) and/or the caudate nucleus (CN). Canulae were supplied with artificial cerebrospinal fluid (CSF) containing tritiated tyrosine, and labeled dopamine [3H]DA was evaluated in successive superfusate fractions. Electrical stimulation of the medial forebrain bundle with an implanted bipolar electrode induced an increase of the [3H]DA release in the ipsi- and contralateral ACE. Electrical stimulation of the SN produced only a very delayed effect in the ipsilateral ACE but an immediate and large increase of [3H]DA release in the contralateral structure. Superfusion of unlabeled DA and alpha-methyl-p-tyrosine in the SN remained ineffective on the [3H]DA release in the ipsilateral ACE. In this structure the release of [3H]DA was, however, decreased by nigral superfusion with gamma-amino-butyric acid (GABA). D-(+)-Amphetamine (1 microM), when superfused in the CN, induced a large enhancement of the [3H]DA release in the ipsilateral ACE simultaneously with the local increase of [3H]DA release. The results presented here are in agreement with the previous studies concerning the anatomical organization of the dopaminergic nigroamygdaloid pathway. The DA cell bodies located in the SN appear insensitive to a local action of DA, perhaps due to a lack of autoreceptors. They are, however, powerfully inhibited by GABA and the relation observed between the [3H]DA release in the CN and ACE support the hypothesis that the SN can act as a relay between the extrapyramidal and limbic systems.

Rats were anesthetized with fluothane and implanted in the caudate nucleus with a push-pull cannula supplied with artificial CSF containing the tritiated precursor of dopamine (DA), [3H]tyrosine. Total DA and dihydroxyphenylacetic acid (DOPAC) were measured in successive 20 min fractions using high performance liquid chromatography and electrochemical detection. Radioisotopic counting of the peaks permitted the calculation of the specific activity of both DA and DOPAC released into the extracellular space. Local applications of potassium (8, 16 and 32 mM) induced a dose-dependent increase of the release of DA with a decrease of its specific activity as evidence of the involvement of a stored DA pool. Base release of DOPAC was increased by repeating potassium applications with a temporary decrease during the applications. Superfusion with alpha-methyl-p-tyrosine produced a decrease of both the [3H]DA and total DA with a simultaneous decrease of its specific activity. This decrease was considered to be an indicator of the involvement by synthesis inhibition of the stored amine, but the simultaneous decrease of the specific activity of DOPAC suggests that this release was intraterminal. These results constitute the first direct observation that DA terminals act with two separate pools (stored and releasable) and suggest that the stored amine is preferentially released intraterminally. Systemic injection of reserpine induced a decrease of the release of DA and DOPAC without alteration of DA-specific activity when the specific activity of DOPAC was lowered. From these results it is concluded that the releasable compartment of the amine is located, in part, in vesicles different in nature from the vesicles containing the stored amine.(ABSTRACT TRUNCATED AT 250 WORDS)

In rats anaesthetized with urethane, push pull cannulae were stereotaxically introduced in the hippocampus (bilaterally) and Zn2+ assayed in the perfusate by atomic absorption spectrophotometry. When the cannula was located in the immediate vicinity of the mossy fiber zone, both spontaneous and K+ evoked release of Zn2+ were observed, this release was associated with a reduction in the histologically demonstrable Zn2+ as assessed by means of the Timm's stain. Neither spontaneous nor evoked release of Zn2+ was observed when the cannula was located in the medial part of CA1, the fimbria or the thalamus. These observations suggest that Zn2+ is released in the mossy fiber zone.

Dopamine can be released in the substantia nigra for the dendrites of nigrostriatal dopaminergic neurones, to be involved there in the self-regulation of the dopaminergic cells, to control the release of neurotransmitters from nigral afferent fibres and to influence the activity of nigral non-dopaminergic cells.