DA Terminals Research Articles

Effects of Acute and Chronic Morphine on DOPAC and Glutamate at Subcortical DA Terminals in Awake Rats

Effects of morphine and naloxone on the levels of 3,4-dihydroxy-phenylacetic acid (DOPAC) and glutamate in the striatum and nucleus accumbens of awake rats were studied with in vivo microdialysis. Acute morphine (50 mg/kg, IP) treatment increased the levels of DOPAC and glutamate in the striatum and nucleus accumbens, but both decreased from the elevated levels when naloxone (10 mg/kg, IP) was given 2 h later. Chronic morphine treatment, twice daily for 5 days in incremental doses (5, 10, 20, 40 and 50 mg/kg, IP), increased the level of DOPAC but decreased that of glutamate in the striatum and nucleus accumbens. When naloxone was given 2 h later, the reverse of the above phenomena are found. After given repeated morphine treatment and experiencing naloxone-precipitated withdrawal, the rats with an intact cortex and the rats with ibotenic acid (5 μg/0.5 μl/2.5 min) lesions on the medial prefrontal cortex and sulcal cortex have similar alternations in the levels of DOPAC and glutamate in the striatum. However, in the nucleus accumbens, the level of DOPAC dropped more and the level of glutamate increased more in the intact rats than the lesioned rats during the withdrawal stage. These data suggested that the intact cortex ordinarily exerted an inhibitory role to influence the level of DOPAC in the striatum and nucleus accumbens during chronic morphine treatment. In conclusion, morphine seems to activate different pathways in dependent and non-dependent rats.

Independent mediation of unconditioned motor behavior by striatal D1 and D2 receptors in rats depleted of dopamine as neonates.

The effects of systemic administration of DA receptor antagonists suggest that unconditioned motor behavior in rats depleted of DA as neonates continues to be dependent upon dopaminergic transmission, yet the specific contribution of D1 and D2 receptors to these behaviors has been altered. The purpose of the present study was to determine whether these depletion-induced receptor changes are occurring at the level of striatal DA terminals and their targets. The ability of bilateral intrastriatal injections (0.5 microliter) of DA receptor antagonists to induce motoric deficits was determined in adult rats treated with vehicle or 6-OHDA (100 micrograms, intraventricular) on postnatal day 3. Administration of the D1-like antagonist SCH 23390 (0.5-2.0 micrograms) or the D2-like antagonist clebopride (1.0-4.0 micrograms) induced dose-dependent akinesia, catalepsy, and somatosensory neglect in vehicle-treated controls. In contrast, neither antagonist produced deficits in rats depleted of forebrain DA as neonates. However, combined administration of SCH 23390 + clebopride induced similar akinesia, catalepsy, and somatosensory neglect in both controls and DA depleted animals. Animals depleted of DA were more sensitive than controls to the low doses of this combined D1 + D2 antagonism. These results demonstrate that activation of striatal DA receptors remains necessary for unconditioned motor behavior in rats depleted of DA as neonates. However, the specific contributions of D1- and D2-like receptors to these behaviors differ between intact animals and those depleted of DA as neonates. The ability of endogenous DA acting at either D1 or D2 receptors to support spontaneous motor behavior in rats depleted of DA as neonates may contribute to their relative sparing from parkinsonian deficits.

Long-term effects of amygdaloid kindling on striatal dopaminergic terminals

The present study assessed the effects of kindling on striatal DA terminals. Kindled and control rats were tested for DA transporter density using [ 3H]GBR-12935 binding to striatal membranes and for amphetamine and KCI-induced [ 3H]DA release from striatal slices. Kindling decreased the maximal number of [ 3H]GBR-12935 binding sites in the dorsal striatum of rats sacrificed either 2 h or 4 weeks after the last seizure but had no effect on stimulated fractional [ 3H]DA release. These findings suggest a minor damage to DA terminals in the dorsal striatum. At the same postseizure time points, kindling augmented the hyperlocomotion associated with novel environment. Explanation of this effect requires in vivo measures of striatal DA functioning.

Catecholamine-CRF synaptic interaction in a septal bed nucleus: Afferents of neurons in the bed nucleus of the stria terminalis

Projections of catecholamine neurons to the bed nucleus of the stria terminalis (BST), especially its corticotropin releasing factor (CRF)-producing neurons, are implicated as being major contributors to the neurochemically mediated central regulation of the stress response. The purpose of the present study was to examine in the BST of the rat brain the morphological characteristics of interactions between two neuron populations of the brain, catecholaminergic and CRF neurons. A double-label immunocytochemical, light and electron microscopic technique allowed the demonstration of the synaptic interaction between dopamine (DA i.e, tyrosine hydroxylase-containing) and norepinephrine (NE i.e., dopamine-beta-hydroxylase-containing) axons and CRF neurons in the BST. DA terminals formed synapses with dendrites and soma of CRF neurons in the dorsolateral BST. NE terminals formed synapses with dendrites of CRF neurons in the ventrolateral BST. In conclusion, catecholamine afferents can directly affect the contribution of CRF neurons of the BST to an animals response to stress.

Assessment of the role of TRH in the release of [ 3H]-dopamine from rat nucleus accumbens-lateral septum slices

We have studied [ 3H]-dopamine ([ 3H]-DA) release from rat nucleus accumbens lateral septum slices in response to various paradigms aimed at increasing endogenous or exogenous thyrotropin releasing hormone (TRH) concentrations in the extracellular space. High KCl concentrations significantly enhanced [ 3H]-DA release by fourfold. TRH (10 −4 or 5 × 10 −4 M) did not affect [ 3H]-DA release. The release of [ 3H]-DA was not stimulated by TRH either in the presence of N-l-carboxy-2-phenylethyl (N imbenzyl)-histidylβnaphthylamide, a specific pyroglutamyl peptidase II inhibitor, or that of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I. None of the peptidase inhibitors modified the [ 3H]-DA release by themselves. These results suggest that the TRH stimulation of [ 3H]-DA release in vitro observed in previous studies is not due to peptide inactivation but may be due to a nonspecific effect. TRH enhancement of DA release in nucleus accumbens in vivo may not be the result of a direct effect of TRH on DA terminals.

Autoradiographic localization of dopamine D 1 and D 2 receptors in rat cerebral cortex following unilateral neurotoxic lesions

Relative to dopaminergic innervation of cortex, dopamine D 1 and D 2 receptors may be located on presynaptic terminals and/or postsynaptically on cortical neurons. To assess the relative distribution of these sites, quantitative in vitro receptor autoradiography was performed following injection of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle (MFB; which lesions presynaptic DA terminals) and ibotenic acid into the prefrontal and anterior cingulate cortices (which lesions neurons whose cell bodies are intrinsic to cortex). Receptor autoradiography was performed ten days after injection of neurotoxins with [ 3H]SCH 23390 (a D 1 probe) and [ 125I]epidepride (a D 2 probe). Both DA receptor subtypes were found in all layers of anterior cingulate and prefrontal cortices but were concentrated in deeper layers V and VI. Ibotenic acid lesion of cortex reduced D 1 and D 2 receptors by 55–80%, although the concentrations of DA and its major metabolite dihydroxyphenylacetic acid (DOPAC) were unchanged. Lesion of MFB produced no significant change in D 1 and D 2 receptors, but was associated with a 49–52% decrease in DA and DOPAC levels relative to the contralateral side. These results suggest that the majority of D 1 and D 2 receptors in prefrontal and anterior cingulate cortices are located postsynaptically on neurons intrinsic to the cortex.

Is the Release of Dopamine from Medial Prefrontal Cortex Modulated by Presynaptic Receptors? Comparison with Nigrostriatal and Mesolimbic Terminals

Results obtained from our in vitro studies employing superfused slices obtained from three functionally different brain regions rich in DA axon terminals were discussed. Striking qualitative and quantitative similarities were found for the modulation of DA release from the nucleus caudate and the OT of the rabbit. However, the PFC DA terminals showed important differences from the nigrostriatal and mesolimbic DA terminals. Although release modulatory D2 DA autoreceptors could also be demonstrated in superfused slices of the PFC, our results suggest that the cortical nerve terminals may have a lower number of functional autoreceptors or a reduced efficiency of coupling between receptors and inhibition of release. Either possibility could explain (a) the poor inhibitory efficacy of the agonists, (b) the small facilitatory effect of the antagonists, (c) the disproportionate increase in transmitter overflow produced by neuronal uptake inhibitors, and (d) the lack of synergism between uptake inhibitors and DA antagonists. When the efficacy of the autoreceptor mechanisms was evaluated at stimulation frequencies comparable to the in vivo firing rates reported for each of the three neuronal groups, it was found that DA release from the striatum and the OT was tightly modulated by presynaptic D2 DA receptors; whereas release from PFC was not. We propose that the autoreceptor-mediated control of DA release from PFC may not function in vivo, even though modulation of release by presynaptic D2 DA receptors from PFC terminals could be demonstrated under specific experimental conditions in vitro. However, it is envisaged that if in vivo firing rate of the PFC DA neurons is reduced, the inhibitory actions of DA agonists on DA release may be regained. From these and other studies it is apparent that drug effects on autoreceptors are highly dependent on the rate and duration of stimulation applied to a specific neuronal group. We propose that the basal status of activity of a specific neuronal target could determine the type and magnitude of the effect produced by a therapeutic agent acting at release modulatory receptors. The neuronal activity (firing rate and pattern) may be affected by physiological status, disease, and by current or previous drug treatments. The mechanisms by which PFC DA terminals release a larger proportion of their storage pool compared to other mesotelencephalic DA terminals is unknown and may represent a compensatory mechanism to the continuous rapid firing rates at which these neurons are exposed in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

The functional output of the mesolimbic dopamine system.

In summary, the nucleus accumbens, located at the interface of the limbic projections from the amygdala, hippocampus, and cingulate cortex, and receiving extrapyramidal fibers from midbrain DA-containing nuclei, is well situated to form neural circuitry that mediates the behaviorally activating properties of several stimulants. Efferent GABAergic fibers projecting from the nucleus accumbens to the ventral pallidum translate integrated limbic and extrapyramidal information to lower motor circuitry; some of this information appears to be carried by ventral pallidal efferent fibers projecting to the dorsomedial nucleus of the thalamus. It seems very possible that activation of this circuitry by positive reinforcing environmental stimuli, through the release of endogenous DA or opiate compounds, might contribute to motivated behavior. Indeed, environmentally generated locomotor activity can be blocked by disruption of this circuitry following destruction of N. Acc. DA terminals. It is also tempting to speculate that pathological changes in activity within this system might disrupt normal reinforcement contingencies, and contribute to the affective components of both psychiatric and neurologic disease states.

Amphetamine, but not reserpine, protects mice against dopaminergic neurotoxicity of MPTP

In mice, long-term reductions in striatal DA produced by the neurotoxin MPTP were completely prevented by its combined administration with amphetamine. Depletion of DA from DA terminals by cotreatment with reserpine did not suppress but rather enhanced MPTP-induced DA decrements in striatum. Amphetamine protection against DA neurotoxicity of MPTP in mice is probably not due to increases in DA release and may be related to inhibition of MPTP transport into nigrostriatal terminals via the DA reuptake system.

Neurochemical and Histochemical Characterization of Neurotoxic Effects of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine on Brain Catecholamine Neurones in the Mouse

Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused a rapid and long-lasting reduction of both 3,4-dihydroxyphenylalanine (dopamine, DA) and noradrenaline (NA) in mouse brain, as observed histo- and neurochemically. The depleting effects were more pronounced after repeated MPTP administration and the most marked reductions were observed after 2 X 50 mg MPTP/kg s.c., when DA in striatum and NA in frontal cortex were reduced by greater than 90% 1 week after MPTP. Mice with such catecholamine depletions were markedly sedated and almost completely immobilized. The behavioural syndrome after MPTP resembled that seen after reserpine, a monoamine-depleting drug. MPTP also caused a long-lasting reduction of catecholamine uptake in striatal DA and cortical NA nerve terminals and reduced tyrosine hydroxylase activity in these regions. There was no evidence that MPTP caused any marked DA and NA cell body death. MPTP given acutely transiently elevated serotonin levels. The results are compatible with a neurotoxic action of MPTP on both DA and NA nerve terminals. The nigro-striatal DA and the locus coeruleus NA neurone systems appeared to be most susceptible. Synthesis and utilization of residual striatal DA and cortical NA were increased, as often observed in partially denervated monoamine-innervated brain regions. Both DA and NA showed a gradual recovery, which took months to become complete and may have been related to a regrowth of catecholamine nerve terminals.

A role for striatal beta-adrenergic receptors in the regulation of dopamine release

The effect of (−) isoproterenol on the spontaneous release of [ 3H]dopamine ([ 3H]DA) continuously formed from [ 3H]tyrosine has been studied both in vitro using rat striatal slices and in vivo in halothane-anaesthetized cats implanted with push-pull cannulae in each caudate nucleus and each substantia nigra. In vitro, (−) isoproterenol (10 −6M) stimulated [ 3H]DA release. This effect was stereospecific since (+) isoproterenol (10 −6M) was without effect. (±) Propranolol (10 −6M), which did not block the acetylcholine (10 −5M)-induced release of [ 3H]DA, completely prevented the stimulatory effect of (−) isoproterenol on [ 3H]DA release. (±) Practolol (10 −5M) significantly reduced the (−) isoproterenol-induced release of [ 3H]DA whereas phentolamine (10 −5M) was uneffective. These results suggest that (−) isoproterenol acts on [ 3H]DA release through β 1-receptors. Since the adrenergic agonist still stimulated [ 3H]DA release in the presence of tetrodotoxin, the β-receptors involved may be located on DA terminals. Confirming in vitro results, (−) isoproterenol (10 −6M) enhanced the local release of [ 3H]DA when applied into one caudate nucleus. This effect was biphasic and prevented by (±) propranolol (10 −6M). The β-antagonist by itself slightly reduced [ 3H]DA spontaneous release suggesting that noradrenergic neurons which may innervate the caudate nucleus could exert a tonic facilitatory influence on DA release. No change in [ 3H]DA release occured in other structures studied during the application of (−) isoproterenol into one caudate nucleus. These various results indicate that striatal β-adrenergic receptors play a role in the regulation of DA release from DA nerve terminals and suggest an interaction between noradrenergic and dopaminergic neurones in the caudate nucleus.

The effect of drugs altering striatal dopamine levels on apomorphine induced stereotypy.

Drugs that increase or decrease striatal dopamine levels appear to affect apomorphine induced stereotypy. This finding was unexpected, as it has previously been maintained that drugs which exert any action on striatal DA terminals exclusively would affect only indirect dopaminergic agonists, as opposed to apomorphine which induces stereotypy by acting directly on postsynaptic dopamine receptors. Specifically, inhibiting intrastriatal dopamine levels inhibits this behavior. This effect is explained in terms of apomorphine having a greater intrinsic activity and agonist affinity for striatal dopamine receptors than dopamine itself. Thus, dopamine and drugs which promote its release, may diminish the central behavioral effects induced by apomorphine relative to drugs which inhibit dopamine release centrally.

Possible DA agonist properties of naloxone.

Naloxone is widely if not universally considered to be a pharmacologically 'pure' opiate (mu) receptor antagonist virtually devoid of agonist action when administered in moderate dosages. However, naloxone (NX) appears to possess a striking number of DA agonist properties. Thus, some investigators have found NX capable of inducing stereotyped rearing and locomotor activity in habituated rats (a controversial finding), and decrease serum prolactin levels, improve Parkinsonism, enhance copulatory performance in sexually sluggish animals, and increase striatal HVA levels, in mimicry of centrally acting DA agonists. NX can also significantly potentiate the central effects induced by DA agonists including DA agonist (d-amphetamine) induced 3H-dopamine release, and antagonize a number of the central effects elicited by DA release inhibiting agents. Finally, virtually all of the central effects of morphine reversible by NX have also been found to be antagonized by a variety of dopamine agonists; while DA release inhibiting agents can abolish the ability of NX to antagonize morphine induced effects. Thus, NX may be exerting its central effects through a dopaminergic mechanism. Since NX does not bind DA receptors, it is quite likely that NX may ultimately antagonize the central effects of morphine by enhancing DA release from DA terminals upon which opiate receptors are localized. The same opiate receptors, shown to be localized on DA nerve terminals, have already been implicated in opiate mediated modulation of DA release.

Genetic variations in midbrain dopamine cell number: Parallel with differences in responses to dopaminergic agonists and in naturalistic behaviors mediated by central dopaminergic systems

Mice of the inbred strain BALB/cJ have more midbrain dopaminergic cell bodies and greater activity of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the nigrostriatal and mesolimbic dopaminergic systems than mice of the CBA/J strain. This difference in cell number and TH activity in the midbrain dopaminergic systems are paralleled by differences in drug responses and behaviors which are dependent on the release of dopamine in midbrain dopaminergic systems. BALB/cJ mice showed greater locomotion and stereotypy than CBA/J mice after d-amphetamine (2–20 mg/kg, i.p.). There was no difference in the amount of amphetamine accumulated in brain at the peak of drug response or in the duration of drug effect, suggesting that the differences in behavioral effect were not due to strain differences in pharmacokinetic distribution of the drug. In contrast to the greater stereotypy to d-amphetamine, BALB/cJ mice showed less stereotypy after apomorphine (2–10 mg/kg, i.p.) than CBA/J mice. BALB/cJ mice also showed more exploration than CBA/J mice, measured as locomotion and rearing in a novel open field and investigation of a novel object. Genetically determined differences in the number of midbrain dopaminergic cell bodies and in the relative density of innervation of DA terminals in target fields and in TH activity in the nigrostriatal and mesolimbic dopaminergic systems are paralleled by differences in behavioral responses mediated by release of dopamine. The number of cells of a particular neurochemical class may dictate the magnitude of behaviors, drug-induced or spontaneous, mediated by those neurons.

Efferents and afferents of the ventral tegmental-A10 region studied after local injection of [ 3H]leucine and horseradish peroxidase

The efferents and the afferents of the VMT-A10 region were studied by using anterograde ([ 3H]leucine) and retrograde (HRP) tracing techniques. In order to produce very small injections in various parts of the VMT-A10 region, a slow diffusion technique for [ 3H]leucine labelling and a microiontophoretic injection for horseradish peroxidase labelling were developed. According to the histochemical and biochemical data, the [ 3H]leucine anterograde results were separated into three main types of projections. (1) Projections to regions rich in DA terminals. These projections certainly correspond to the efferents of the dopaminergic A10 neurones. According to various injection sites, we have been able to identify mesolimbic projections originating from the VMT-A10, pars medialis and mesostriatal-mesolimbic projections originating from the VMT-A10, pars lateralis. The mesolimbic projections include the prefrontal cortex, the medial part of the lateral septum, the interstitial nucleus of the stria terminalis, the accumbens nucleus and the olfactory tubercle. The mesostriatal-mesolimbic projections include the anteromedial part of the caudate nucleus, the cingular cortex, the entorhinal cortex, the amygdaloid complex, the accumbens nucleus, the olfactory tubercle and the piriform cortex to a lesser extent. (2) Projections to regions suspected of containing DA terminals. These ascending and descending projections which could represent the dopaminergic efferents of the VMT-A10 neurones have been demonstrated. Ascending projections originating either from the VMT-A10 pars medialis or pars lateralis region were found in the claustrum, the nucleus of the tractus diagonalis, the olfactory nuclei, the lateral habenula, the medial hypothalamus and the median eminence. The projections observed in the medial hypothalamus included the periventricular region, the arcuate nucleus, the ventral part of the ventromedial nucleus and the dorsomedial nucleus. The labelling of the anteromedial part of the dorsal hippocampus appeared to originate from the VMT-A10, pars posterior. The projections to the medial hypothalamus, median eminence and hippocampus may have a great functional significance, but further proof of their dopaminergic nature is needed. Descending projections were found ipsilateally to the dorsal raphe and to the cerebellum, and bilaterally to the locus coeruleus. The projections to the cerebellum are distributed to the nuclei interpositus and dentatus and to the Purkinje cell layer and granular layer of the cortex. These results raise the problem of descending dopaminergic projections from the A10 neurones. (3) Projections to regions not known to contain DA terminals. Anterior projections were found ipsilaterally to the supraoptic nucleus and bilaterally to the anterodorsal thalamic nucleus. Posterior projections were traced ipsilaterally to the limbic midbrain area, including the median raphe, the ventral and dorsal tegmental nucleus and the central gray. The horseradish peroxidase experiment supplied some clues as to the posterior afferents of the VMT-A10 region. Some labelled cells were found ipsilaterally in the substantia nigra, the medain raphe and the ventral tegmental nucleus. Numerous cells were labelled ipsilaterally in the dorsal raphe nucleus, and nuclei interpositus and dentatus of the cerebellum, and contralaterally in the locus coeruleus. These structures are likely to play an important role in the modulation of the activity of VMT-A10 neurones. The results of [ 3H]leucine and HRP experiments permitted us to demonstrate reciprocal connections between VMT-A10 region and anterior raphe nuclei, locus coeruleus and cerebellum.

STIMULATORY EFFECT OF l‐GLUTAMATE AND RELATED AMINO ACIDS ON [3H]DOPAMINE RELEASE FROM RAT STRIATUM: AN IN VITRO MODEL FOR GLUTAMATE ACTIONS

Abstract— The effects of l‐glutamate and a number of structural analogues on the spontaneous release of [3H]dopamine from slices of rat striatum were examined. Glutamate, and other excitatory amino acids produced a marked stimulation of [3H]DA release which was Ca2+‐dependent and unaffected by either procaine or tetrodotoxin. The glutamate‐stimulated release was abolished in kainate‐lesioned striatum. The action of glutamate was effectively antagonised by glutamamate diethylester and 2‐amino‐4‐phosphonobutyric acid, but only weakly by l‐methionine‐dl‐sulfoximine. Other proposed amino acid antagonists were inactive. The likely site of the releasing action of l‐glutamate on presynaptic sites on nigro‐striatal DA terminals is discussed.

Isolated catecholaminergic projections from substantia nigra and locus coeruleus to caudate, hippocampus and cerebral cortex formed by intraocular sequential double brain grafts

Sequential intraocular grafting of defined areas from fetal rat brain to adult host rats was used to explore the possibility that such double grafts would become interconnected. Norepinephrine- containing neurons of the locus coeruleus were grafted together with either parietal cerebral cortex, hippocampus, or the caudate nucleus. Dopamine-containing neurons of the substantia nigra were transplanted together with either parietal cerebral cortex or the caudate nucleus. The brainstem grafts showed good survival and development in oculo, using both histochemical and electrophysiological criteria. Locus coeruleus neurons were found to innervate cerebral cortex, hippocampus, and the caudate nucleus. Substantia nigra neurons invaded cerebral cortex abundantly, with a terminal distribution typical of cortical DA terminals in situ. The innervation of the caudate nucleus from substantia nigra transplants was variable, but areas of dense confluent terminals were observed. We conclude that sequential brain grafting in oculo permits generation of isolated yet defined catecholaminergic projections, which are suitable for electrophysiological, pharmacological, and histochemical studies.

The role of presynaptic receptors in the release and synthesis of 3H-dopamine by slices of rat striatum

Striatal slices were continuously superfused with L-3,5-(3)H-tyrosine (50 muCi/ml) and 4H-H2O [index of 3H-dopamine (3H-DA) synthesis] and 3H-DA estimated in 0.5 ml (2.5 min) superfusate fractions. Depolarization with 50 mM K+ for 7.5 min induced a marked increase in 3H-DA release and a biphasic effect on synthesis (slight increase in the first fraction followed by a significant decrease in the third and fourth fractions). The decrease in the rate of 4H-H2O formation induced by K+ was not related to modifications of the specific activity of tyrosine in tissues. The possibility that the inhibition of synthesis was due to alterations in DA concentration in the synaptic cleft was examined. Benztropine in a concentration which produced inhibition of DA uptake (10(-6) M) increased the K+ induced overflow of 3H-DA but failed to alter the inhibition of synthesis. On the other hand, when the powerful neuroleptic fluphenazine was added to the superfusion medium in a concentration which only weakly blocked 3H-DA uptake (10(-6) M) it potentiated 3H-DA release and prevented the inhibition of synthesis both in the absence or presence of benztropine. A similar effect was seen following the in vivo treatment of rats with fluphenazine (2 mg/kg; 1 1/2 h before sacrifice). The addition of exogenous DA (0.6 X 10(-6) M) or NA (10(-6) M) to the superfusion medium increased 3H-DA outflow and reduced DA synthesis while isoproterenol (10(-6) M) was without effect. The DA inhibitory effect on synthesis was still observed in the presence of benztropine (10(-6) M) while the NA effect was prevented. This concentration of benztropine blocked both DA and NA uptake. The administration of fluphenazine (10(-6) M) significantly prevented the decrease in 3H-DA synthesis induced by exogenous DA and partially prevented the effect of NA. In addition, the effect of exogenous DA on the inhibition of synthesis was still seen in the presence of 2-amino-4-hydroxy-6,7-dimethyl-5,6,-7,8-tetrahydropteridine hydrochloride (DMPH4) (to protect against end-product inhibition). The present results provide direct support for the concept that activation of presynaptic DA receptors located on DA terminals in the striatum of the rat results in an inhibition of synthesis and release of the transmitter.

Ultrastructural identification of catecholamine neurones in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects

Summary The basal hypothalamus of the rat, with special reference to the external and internal layer of the median eminence, the arcuate nucleus and the periventricular area, was studied with the formaldehyde fluorescence technique of Falck and Hillarp and with electron microscopy. The presence in boutons of so-called small granular vesicles,i.e., sinaptic vesicles with a diameter of about 50 nm containing an electron-dense core, was used as a criterion for identification of monoamine boutons at the ultrastructural level. After a routine glutaraldehyde-OsO4 fixation procedure no such boutons could be identified. However, after KMnO4 fixation small granular vesicles were found in boutons in the internal layer of the median eminence, the arcuate nucleus and in the periventricular region,i.e., in regions assumed to contain mainly NA axon terminals. However, after administration of exogenous amines such as 5-hydroxydopamine (5-OH-DA) or 6-hydroxydopamine (6-OH-DA), eitherin vivo by intraventricular injections or to brain slicesin vitro, it was also possible to identify boutons with small granular vesicles in the external layer of the median eminence, a region containing predominantly DA terminals. When exogenous amines were used such boutons were also demonstrated in all regions studied after glutaraldehyde-OsO4 fixation although in our hands this procedure gave less distinct dense cores as compared with KMnO4 fixation. The quantitative study revealed that the density of monoamine boutons is highest in the superficial, lateral parts of the median eminence, where about 33% of all boutons, corresponding to a density of about 120 such boutons/500 sq.μm area, belong to monoamine neurones. In the arcuate nucleus, on the other hand, the corresponding values were 2.6% and 2/500 sq.μm. These results demonstrate that in the external layer,i.e., the neurohaemal contact zone, about two-thirds of all boutons belong to non-monoamine neurones, which tentatively may be supposed to store and release the releasing and/or inhibitory factors controlling the hormone secretion from the anterior pituitary. Several studies have indicated an important functional role of DA in the external layer of the median eminence, mainly in the control of gonadotropin secretion. However, the present results do not give a definite answer as to the site of action of the amine. With the techniques used, including ethanolic phosphotungstic acid impregnation, no typical ‘synapses’ between axon profiles could be identified. Such synapses would strongly have supported the view of an axo-axonic influence of DA on the axon terminals containing the releasing and/or inhibitory factors.

Barbiturates and meprobamate: Decreases in catecholamine turnover of central dopamine and noradrenaline neuronal systems and the influence of immobilization stress

Summary The effects of barbiturates and meprobamate on the central DA and NA neurones of non-stressed and stressed rats were studied using histochemical and biochemical analysis of DA and NA. Changes induced by the different drug and/or stress treatments in the rate of depletion of the CA stores 4 h after tyrosine hydroxylase inhibition were regarded as changes in turnover. Barbiturates, but not meprobamate, clearly decreased the turnover of NA in the cortical NA nerve terminals. No clear effects were found in the hypothalamus with the two drugs. Stress increased NA turnover in all parts of the brain studied, and these increases could be counteracted by both barbiturates and meprobamate. The net effect on DA turnover in the nerve terminals of the neostriatum and the median eminence was a reduction after 4 h of immobilization stress. The stress-induced decrease in turnover of the DA terminals in the neostriatum was further decreased by pretreatment with barbiturates or meprobamate. In the median eminence, on the other hand, these drugs accelerated DA turnover in the external layer as demonstrated histochemically. The present results indicate that the effects observed on CA turnover are related to the sedative and anti-stress actions of barbiturates and meprobamate. It is unlikely that the specific differences in turnover changes observed between different areas of the brain are the result of a general depressant action on the central nervous system. The effects of barbiturates and meprobamate on CA turnover found in the present study are essentially similar to the effects of the minor tranquillizers of the benzodiazepine group as found in a previous study.