Angelos E. Halaris

Effects of locus coeruleus lesions upon cerebral monoamine content, sleep-wakefulness states and the response to amphetamine in the cat

The purpose of the present study was to investigate the effects of complete lesions of the noradrenaline locus coeruleus neurons upon wakefulness and paradoxical sleep. Radiofrequency lesions of the nucleus were performed in 8 chronically implanted cats which were continuously recorded with an EEG for 5 days prior to and 21 days following the lesions, when they were sacrificed. In 3 of these animals amphetamine (2 mg/kg) was administered on one control day and on the 10th day post-lesion. Following sacrifice, monoamine content was assayed in discrete brain regions, and the lesion was examined in Nissl-stained sections of the pons. (1) The majority (x 69%) of the locus coeruleus was bilaterally destroyed by the lesions which only minimally exceeded the boundaries of the nucleus within the dorsolateral pontine tegmentum. Noradrenaline was depleted by a mean of 85% in the paleo- and neocortex and by a mean of 60% in the thalamus and midbrain. (2) EEG activation reappeared within 12-48 h following the lesion and represented a normal percentage of recording time on the 3rd and subsequent days post-lesion. The behavioral arousal and long-lasting EEG activation produced by amphetamine was qualitatively and quantitatively the same pre- and post-lesion. (3) Despite alteration of certain components, paradoxical sleep reappeared within 48 h and recovered to normal amounts by the second week post-lesion. Muscle atonia was permanently absent in 7 animals. Ponto-geniculo-occipital (PGO) spiking was acutely redistributed across all states and chronically reduced in frequency (by a mean of 50%) within paradoxical sleep. These results indicate that the noradrenaline locus coeruleus neurons are not necessary for the tonic maintenance of EEG activation that occurs in normal wakefulness and in amphetamine-produced arousal. Furthermore, these neurons are not necessary for the occurrence of paradoxical sleep, although they may be involved in modulation of PGO spiking.

Ascending projections of the locus coeruleus in the rat. I. axonal transport in central noradrenaline neurons

Axonal transport of protein and metabolites of L-[3H(G)]3, 4-dihydroxyphenylalanine ([3H]DOPA) was studied in the central noradrenaline neurons of the pontine nucleus locus coeruleus and was correlated with regional alterations of noradrenaline content following destruction of the nucleus. Unilateral lesions of the locus coeruleus produce a partial depletion of noradrenaline in the ipsilateral hypothalamus and telencephalon, indicating that these neurons project widely to the ipsilateral forebrain. Twenty-four to 48 h following local injections of 50 micronCi [3H]proline, locus coeruleus neurons take up labeled material and transport it, presumably as protein, to ipsilateral structures in the midbrain, diencephalon and telencephalon including the neocortex. Similarly 8 h after injection of 25 micronCi [3H]DOPA into the locus coeruleus, transport of material including catecholamines occurs to ipsilateral diencephalon and telencephalon. Axonal transport of proteins to telencephalic structures is greatly diminished by selective lesions of catecholamine terminals with 6-hydroxydopamine (6-OHDA) and following destruction of the medial forebrain bundle. These results provide further support for the view that noradrenaline neurons of the locus coeruleus nucleus project widely within the neuraxis to ipsilateral structures of the brain stem, diencephalon and telencephalon, including all cortical areas. In addition, evidence is presented for a contralateral projection with a similar distribution. The rate of axonal transport of labeled protein and metabolites of [3H]DOPA including [3H]catecholamines in central noradrenaline neurons is estimated to be 3-4 mm/h and is accordingly similar to that reported for noradrenaline neurons of the peripheral sympathetic nervous system.

Antidepressant drugs affect dopamine uptake

The catecholamine hypothesis of depression postulates a deficiency of norepinephrine at functional receptor sites. The evidential basis derives from studies on the turnover and uptake of norepinephrine and the blockade of reuptake by tricyclic antidepressants El 3]. Serotonin systems may also be involved in antidepressant drug action [4, 5]. Relatively little attention has been paid to the possible role of dopamine in the pharmacotherapy of depression. Although Horn et al. [6] included some tricyclic antidepressants in their study on regional catecholamine uptake by rat brain synaptosomes, it is not known whether inhibition of dopamine uptake is a common property of antidepressants currently in clinical use. Our laboratory has been investigating the mode of action of antidepressant compounds. We have reported time-dependent changes in serotonin metabolism after chlorimipramine: rat brain serotonin increased 30 min after an i.p. injection of the drug and this effect was interpreted as reflecting re-uptake blockade [7]. Since brain dopamine levels are also increased by chlorimipramine (unpublished observation), we investigated the possible effect of this and other compounds from the family of antidepressants on dopamine uptake. We report here that a number of widely used antidepressants inhibit dopamine uptake by rat brain nuclei-free homogenates. For comparative purposes, four stimulants were included in the study. Male Sprague Dawley rats (150-200g, Madison, Wis.) were killed by decapitation and their brains immediately removed. Brains were homogenized (12 strokes, I min) in 10 vol. ice-cold 0.32 M sucrose in Tri-R glass homogenizers with Teflon pestles (clearance 0.009 to 0-011 in.), and centrifuged at 1000g for 10 min in a Sorvall RC 2-B centrifuge. The supernatant nuclei-free suspension was separated from the pellet, gently stirred to make a uniform suspension and reserved for uptake and protein assays. We used the method of Snyder and Coyle I-8] with minor modifications. The incubation mixture was agitated at 37 ° in 4-0ml Kreb~Henselei t bicarbonate buffer (pH 7.4) in 15-ml Corex tubes. The drugs tested (all at 10 ~ M) were added to the incubation mixture prior to the addition of tritiated dopamine (sp. act. 9-8 Ci/m-mole, New England Nuclear Corp.); preincubation was omitted. At the end of the 8-min incubation period, the samples were placed in ice-water for 2 min. The tubes were centrifuged at 60409 for 20 min in a Sorvall RC 2-B centrifuge to form the tissue pellet. The incubation mixture was aspirated and the pellet rinsed once with 4-0 ml ice-cold saline. Solubilization of the pel-. lets was performed by incubation with 1 ml Soluene-350 (Packard) at 50 ° for 30 min. The solutions were transferred to glass counting vials, 10 ml toluene fluor was added, and radioactivity was measured in an Isocap/300 scintillation counter (Searle Radiographics, Inc.). The protein content of 0.2ml aliquots of the synaptosomal suspensions was determined by the method of Lowry et al. [9]. The aim in this pilot study was to detect whether inhibition of dopamine uptake is a common property of a number of antidepressants. Preliminary data expressing the variation of uptake of aH-dopamine with increasing substrate concentrations (0-01 to 1 pM) when graphed revealed two consecutive saturable curves. These data were resolved into two straight lines in Lineweaver Burk plots, suggesting the existence of two components responsible for the accumulation of 3H-dopamine into synaptosomal suspensions from whole brain. One component operated at low substrate concentrations with a Km at 1 x 10 v M and another component at high concentrations of 3H-dopamine with a K,, at 3.3 × 10 7 M (Fig. 1). Snyder and Coyle E8] have previously reported two affinity systems for dopamine uptake by homogenates from all brain regions except for the striatum which possesses only one uptake system. The value reported by these authors for high affinity uptake was 0-8 x 10 -v M; the corresponding value for low affinity uptake was 1.4 × 10 ~ M. The different value for low affinity uptake is probably due to the

Axonal transport in serotonin neurons of the midbrain raphe

The projections of serotonin-containing neurons of the midbrain raphe nuclei (nucleus raphe dorsalis, nucleus centralis superior) are studied by analysis of axonal transport of labeled amino acids. These results are correlated with regional alterations of serotonin content following midbrain raphe lesions which produce significant serotonin depletion in nearly all regions of the central nervous system. Twenty-four hours following injection of 100 muCi [3H]proline, raphe neurons have taken up labeled material and transported it, presumably as protein, to telencephalon, diencephalon, brain stem, the cerebellum and the spinal cord. This transport appears to take place predominantly in serotonin neurons. After injection of 100 muCi [3H]5-HTP into nucleus raphe dorsalis or nucleus centralis superior, the pattern of regional distribution of transported material is very similar to that obtained with tritiated proline. Selective lesions of serotonin terminals with 5.6-DHT result in greatly diminished axonal transport of proteins to all telencephalic, diencephalic and mesencephalic areas as well as to cerebellum, pons-medulla and spinal cord. Unilateral destruction of the medial forebrain bundle results in significant reduction in axonal transport of labeled material to ipsilateral telencehalon and thalamus. These results provide further support for the view that serotonin neurons of the midbrain raphe nuclei project widely throughout the neuraxis to telencephalon, diencephalon, brain stem, cerebellum and spinal cord.

Deficits in food and water intake after knife cuts that deplete striatal DA or hypothalamic NE in rats.

Knife cuts ventral or medial to the striatum were used to interrupt some of the principal connections of this structure. All of the cuts depleted striatal dopamine and produced aphagia and adipsia but there was no indication that the two classes of effects were always correlated. Cuts medial to the striatum produced the most severe DA depletions, persistent aphagia and adipsia, and the full complement of deficits in responding to glucoprivic and hydrational challenges that characterize rats that have recovered from lateral hypothalamic lesions. Cuts ventral to posterior portions of the striatum produced comparable periods of aphagia and adipsia (but few of the persisting impairments in responsiveness to regulatory challenges) even though their effect on striatal DA was relatively small (the average depletion was 51% compared to 89% for rats with cuts medial to the striatum). A second group of rats with cuts below more anterior aspects of the striatum sustained severe DA depletions (70%) but only very brief periods of aphagia and adipsia and only slight deficits in responding to osmotic challenges. The effects of the DA depleting cuts were compared with the behavioral consequences of coronal cuts in the midbrain tegmentum which selectively depleted hypothalamic norepinephrine. These cuts did not produce reliable effects on either food or water intake but abolished the normal feeding response to 2-deoxy-d-glucose without affecting the response to insulin. A correlational analysis of the biochemical and behavioral results of our cuts indicated a significant positive relationship between drinking in response to cellular thirst stimuli and hypothalamic NE as well as striatal DA. The postoperative body weights of our experimental animals were positively correlated with striatal dopamine and negatively related to hypothalamic norepinephrine.

A correlational analysis of the effects of surgical transections of three components of the MFB on ingestive behavior and hypothalamic, striatal, and telencephalic amine concentrations☆

A retractable wire knife was used to transect medial or lateral components of the MFB or its lateral projections to the striatum and amygdaloid complex. All cuts produced significant depletions of NE, DA, and 5-HT from telencephalon and striatum but little or no effect on hypothalamic NE or 5-HT. Two of our cuts resulted in aphagia and adipsia, the third in hyperphagia and obesity. A detailed correlational analysis of the magnitude and direction of the behavioral and biochemical consequences of our cuts indicated that the ingestive behavior of all of our experimental animals (including animals which had been aphagic and adipsic after surgery as well as animals which were hyperphagic and obese) was positively correlated with the concentration of DA in striatum and telencephalon and negatively correlated with telencephalic 5-HT. Less consistent evidence for facilitatory noradrenergic influences on food intake was also obtained. Our results suggest that the regulation of food intake may be the result of an interaction between telencephalic serotonergic mechanisms and dopaminergic pathways which exert opposite effects on ingestive behavior.

Effects of central norepinephrine depletion on the initiation and maintenance of maternal behavior in the rat.

The catecholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was used to test the hypothesis that increased transmission across selected noradrenergic synapses is involved in the initiation of maternal behavior. Specifically, 6-OHDA was infused intraventricularly either two days before parturition or four days after parturition. Control animals were infused with the vehicle alone. Among prepartum animals, NE depletion of more than 30% of control levels interfered with the initiation of maternal behavior. Among lactating animals, similar degrees of NE depletion had no significant effect on the maintenance of maternal behavior. Thus, NE appears to be involved in the initiation of maternal behavior, but not in the maintenance of the behavior once that behavior is established.

Determination of plasma 3-methoxy-4-hydroxyphenylglycol by pulsed electron capture gas chromatography

An improved method is described for determining picogram quantities of 3-methoxy-4-hydroxyphenylglycol (MHPG) in plasma of humans and of other species. The method makes use of gas-liquid chromatography and electron capture detection. Low level nonlinearity of detector response was corrected by operating the detector in the pulsed rather then the customary steady state mode. Detector overloading was prevented by heat coagulation of plasma proteins and subsequent ultrafiltration. Sensitivity was significantly enhanced by utilizing a derivatizing agent carrying a higher number of electrophores. Baseline conditions are described and control values for plasma MHPG of human volunteers, Rhesus monkeys and rats are presented.

Regulatory deficits after surgical transections of three components of the MFB: Correlation with regional amine depletions ☆

Parasagittal knife cuts along the lateral border of the diencephalon (PS), coronal cuts across the lateral (LMFB) or medial (MMFB) components of the medial forebrain bundle reproduce most of the persisting deficits in responding to glucoprivic and hydrational challenges that characterize rats with lateral hypothalamic lesions or intracranial injections of 6-hydroxydopamine (60HDA). Each of these cuts produced a differnet pattern of regulatory deficits, suggesting that individual components of the LH syndrome may be mediated by different neural substrates. This interpretation is supported by the results of our correlational analysis of the relationships between specific behavioral and biochemical effects of our cuts. For example, feeding responses to insulin were reliably correlated with striatal DA concentrations but feeding responses to 2-deoxy-d-glucose (2DG) were not. Water intake during periods of food deprivation was reliably correlated with striatal DA but water intake after an experimental osmotic challenge was not. Only one of the common persisting deficits (impaired feeding response to peripheral injections of insulin) was positively correlated with the duration of aphagia and adipsia.

Variations in the uptake of 3H-dopamine during the estrous cycle

Abstract The in vitro uptake of 3 H-Dopamine ( 3 H-DA) into nuclei-free homogenates from brain parts was studied during each phase of the rat estrous cycle. Across four time points (12:00, 2:00, 4:00 and 6:00 p.m.) each phase exhibits a unique pattern of uptake in the diencephalon. Differences in the level of uptake between phases are most marked at 2:00 p.m. with diestrus animals displaying the highest rate of uptake. Metestrus animals show the lowest rate of uptake (less than 50% of the diestrus rate), while estrus and proestrus show intermediate rates. At 12:00, 2:00 and 4:00 p.m. during proestrus, the average rate of uptake is significantly less than the average uptake rate of any other phase. At 6:00 p.m. proestrus, the rate of uptake is significantly elevated over the values found at the previous three time points. Kinetic analysis of the diencephalic uptake system reveals hourly changes in the kinetic properties of the uptake system (Km and Vmax) during all phases. In non-diencephalic regions (“brain minus diencephalon”), few variations in uptake are observed. The variations in the rate of uptake in the diencephalon are thought to reflect changes in the functional activity of the DA neurons. Thus, during proestrus the reduction of 3 H-DA uptake at 12:00, 2:00 and 4:00 p.m. suggests an increase in DA receptor activation.