Aphagia and adipsia after preferential destruction of nerve cell bodies in hypothalamus.

Microinjections of the excitatory neurotoxin kainic acid into the lateral hypothalamus of rats produced a period aphagia and adipsia. Kainate-treated rats displayed transient motor effects during the first hours after the injection but did not show the persisting sensory-motor and arousal disturbances typically observed in animals with electrolytic lesions in this part of the hypothalamus. Histological examination revealed a significant reduction in the number of nerve cell bodies in the lateral hypothalamus. Silver-stained material indicated no evidence of damage to fiber systems passing through the affected region. Assays of dopamine in hypothalamus, striatum, and telencephalon did not indicate significant differences between experimental and control animals. These results are in agreement with recent reports of the anatomical and biochemical effects of intracerebral kainic acid injections and suggest that the observed effect on feeding behavior is related to the destruction of neurons in the lateral hypothalamus.

Nerve terminal effects of indoleamine psychotomimetics on 5-hydroxytryptamine.

The mode of action of indoleamine psychotomimetics has been closely linked to 5-HT. Early work showed increases in rat brain levels of 5-HT which were later localized to the nerve-ending fraction. With improved methodology, the 5-HT increment was further detected in the synaptic vesicle fraction. These effects were obtained with several indoleamine hallucinogens but not with mescaline. LSD has been most thoroughly studied and has served as the prototypical compound in ascertaining the mode of action of these drugs. Pretreatment with reserpine abolished the 5-HT effects of LSD in the vesicular fraction. However, a new compartment, termed "juxtavesicular," displayed 5-HT increases following reserpine and LSD. A soluble binding site for 5-HT within the synaptoplasm has been postulated in confirmation of independent results by other groups of investigators. The origin of the 5-HT increment appears to be associated with newly synthesized amine. This was deduced from experiments involving various 5-HT synthesis blockers. To ascertain whether inhibition of raphé neuronal firing is responsible for the accumulation of 5-HT at the nerve terminal, two sets of experiments were performed. Destruction of the raphé cell bodies by radiofrequency lesions failed to abolish the LSD-induced 5-HT increase early after the lesion. Destruction of cortical 5-HT neurons with the neurotoxin 5,7-dihydroxytryptamine completely abolished the 5-HT effect of LSD. It was concluded that an intact nerve terminal is necessary for the expression of the LSD-mediated increases in 5-HT. A LSD "autoreceptor" is postulated, possibly identical to a 5-HT presynaptic receptor inhibiting the release of 5-HT.

Apparent phase advance in diurnal MHPG rhythm in depression.

Several investigators have proposed that diurnal rhythms, particularly that of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), show abnormalities in affective disorders. The present study compared diurnal MHPG rhythms in the plasma of 18 male depressed patients and 12 healthy male volunteers. A diurnal rhythm of MHPG closely fit to a cosine model was observed in volunteers and, to a lesser extent, in depressed patients. Patients, especially those with endogenous depression (N = 11), demonstrated an earlier acrophase (mean +/- SD = 12.53 +/- 3.38 hours), and treatment with desipramine was associated with a significant (3-hour) phase delay. This study confirms and extends previous reports of apparent phase advances in circadian noradrenergic rhythms in depressed patients.

Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat.

The organization of the brainstem serotonin neuron projection to the hippocampal formation was analyzed in the rat. This projection arises in the raphe nuclei of the midbrain. Following destruction of the midbrain raphe nuclei, chiefly nucleus centralis superior, there is a 72% decrease in hippocampal serotonin content. Injection of tritiated amino acid into the midbrain raphe nuclei results in transport of tritiated protein to the hippocampal formation and this transport is blocked in animals pretreated by intraventricular administration of 5,6-dihydroxytryptamine (5,6-DHT). Autoradiographic analysis indicates that the transport reaches the hippocampal formation primarily via two major pathways, the cingulum and the fornix. Cingulum fibers terminate predominantly in the dorsal hippocampus whereas the fornix distributes throughout the entire hippocampal formation. Some fibers reach the ventral hippocampus from the entorhinal area. Within the hippocampus there is dense labeling in a restricted lamina of the CA1 stratum lacunosum-moleculare with moderate labeling in stratum radiatum. Stratum oriens is sparsely labeled in CA1 and moderately so in CA2 and CA3. Stratum radiatum and stratum lacunosum-moleculare are moderately densely labeled in CA2 and Ca3. The area dentata is sparsely to moderately labeled in the molecular layer and heavily labeled in a thin lamina of the hilar zone immediately beneath the granule cell layer. The remaining hilar zone is moderately labeled. All of the discrete labeling of the hippocampus and area dentata described above is absent in animals pretreated with 5,6-DHT. These observations indicate that serotonin neurons of the midbrain raphe provide a highly organized innervation of the hippocampal formation in the rat.

Serotonin neurons of the midbrain raphe: ascending projections.

The ascending projections of serotonin neurons of the midbrain raphe were analyzed in the rat using the autoradiographic tracing method. Axons of raphe serotonin neurons ascend in the ventral tegmental area and enter the medial forebrain bundle. A number of fibers leave the major group to ascend along the fasciculus retroflexus. Some fibers enter the habenula but the majority turn rostrally in the internal medullary lamina of the thalamus to innervate dorsal thalamus. Two additional large projections leave the medial forebrain bundle in the hypothalamus; the ansa peduncularis-ventral amygdaloid bundle system turns laterally through the internal capsule into the striatal complex, amygdala and the external capsule to reach lateral and posterior cortex, and another system of fibers turns medially to innervate medial hypothalamus and median eminence and form a contrelateral projection via the supraoptic commissures. Rostrally the major group in the medial forebrain bundle divides into several components: fibers entering the stria medullaris to terminate in thalamus; fibers entering the stria terminalis to terminate in the amygdala; fibers traversing the fornix to the hippocampus; fibers running through septum to enter the cingulum and terminate in dorsal and medial cortex and in hippocampus; fibers entering the external capsule to innervate rostral and lateral cortex; and fibers continuing forward in the medial olfactory stria to terminate in the anterior olfactory nucleus and olfactory bulb.

CSF and endocrine studies of premenstrual syndrome.

Eight women with prospectively documented premenstrual syndrome (PMS) underwent multiple samplings for estradiol, progesterone, prolactin, cortisol, and plasma 3-methoxy-4-hydroxyphenylglycol (MHPG) during an asymptomatic midcycle (late follicular) and a symptomatic premenstrual (late luteal) phase of the menstrual cycle. Cerebrospinal fluid (CSF) was collected for analysis of MHPG, norepinephrine (NE), 5-hydroxyindoleacetic acid (5-HIAA), dihydroxyphenylacetic acid (DOPAC), gamma-aminobutyric acid (GABA), homovanillic acid (HVA), tyrosine, tryptophan, beta-endorphin, prostaglandins, adrenocorticotropic hormone (ACTH), and arginine vasopressin (AVP). In subsequent months, a dexamethasone suppression test (DST) and a thyrotropin-releasing hormone (TRH) stimulation test were performed during midcycle and premenstrual phases. Significant results included increased CSF concentrations of MHPG in the premenstrual, as compared with the midcycle, phase of the cycle, and increased plasma cortisol concentrations during the midcycle phase. The DST showed a 62% overall rate of nonsuppression, irrespective of menstrual cycle phase. Though there were no abnormalities of thyrotropin-stimulating hormone (TSH) after TRH stimulation, the mean delta maximum prolactin values after TRH stimulation were higher than reported normal values both at midcycle and premenstrually. These pilot data suggest hormonal axes that might be worthy of further systematic investigation in future studies of PMS.

The raphe neuronal system and serotonergic effects of LSD.

Earlier work from this laboratory had shown that LSD caused significant increases in rat brain serotonin (5-HT). The increase was later localized to a subsynaptosomal fraction consisting largely of synaptic vesicles. However, the source of the increase and the mechanism by which LSD caused the enhanced 5-HT binding or retention had not been elucidated. The present study was undertaken to evaluate the serotonergic effects of LSD following destruction of raphe nuclei with radiofrequency lesions. When LSD was given to animals with large midbrain raphe lesions, it caused significant increases in forebrain of cortical 5-HT up to 48 hr, or 7 days post-lesion, respectively. It was concluded that an intact cell body is not necessary for the expression of the LSD-mediated increases in 5-HT occurring in the nerve-ending. The possible mechanisms by which LSD could act directly at the nerve-ending are discussed.

Evaluation of the safety and efficacy of bupropion in depression.

A placebo-controlled double-blind study was conducted to test the antidepressant effects of bupropion at dosage levels of 300 or 450 mg/day. Subjects were 30 hospitalized primary major depressives who were treated for 4 weeks. Physical and behavioral measures were obtained at baseline and at the end of each experimental week. The combined results of the two bupropion groups were significantly better than placebo. Preliminary results showed a significant antidepressant effect of the 300 mg/day dose, but not the 450 mg/day dose, compared to placebo. Anxiety symptoms were also somewhat reduced by the 300 mg/day dose. The results are compared with those of a previous study, which utilized higher dosages.

Bupropion in depression: a tri-center placebo-controlled study.

In a double-blind, placebo-controlled, variable-dose study of 59 hospitalized nonpsychotic depressed patients, bupropion was significantly (p less than .05 to less than .001) more effective than placebo on measures of depression, anxiety, and global improvement. Statistically significant drug-placebo differences appeared as early as day 5 of treatment and increased on subsequent assessments. In an evaluation of Baseline X Treatment interactions, bupropion was particularly more effective than placebo in those patients with more severe depression. Placebo and bupropion groups had similar frequencies and severity of side effects. Laboratory data showed minimal differences between the two treatments. The most common adverse experience was mild dry mouth (20% of patients). Compared to placebo, bupropion was found to be effective in the treatment of depression and to have a favorable safety profile.

Effects of neuropeptide Y on alpha 1-and beta-adrenoceptor-stimulated second messenger systems in rat frontal cortex.

Neuropeptide Y (NPY) (1 microM) significantly reduced the basal cAMP concentration in slices of rat frontal cortex. However, NPY (10(-9)-10(-6)M) did not alter the isoproterenol-stimulated (10(-9)-10(-5) M) accumulation of cAMP in the frontal cortical slices, showing that Y2 NPY receptors do not modulate the beta-adrenoceptor-stimulated adenylase cyclase activity. NPY (10(-8)-2.5 x 10(-5) M) was also demonstrated to stimulate inositol phosphate accumulation in rat frontal cortex slices in a dose-dependent manner. However, NPY (1 microM) did not potentiate the ability of phenylephrine (5 X 10(-8)-10(-4) M), an alpha 1-adrenoceptor agonist, to stimulate inositol phosphate hydrolysis. The combined effects of phenylephrine and NPY (1 microM) on inositol phosphate hydrolysis were additive, suggesting that the alpha 1-adrenoceptor and NPY Y1 receptor sites are located on different postsynaptic sites in rat frontal cortex. This study demonstrates the existence of both Y2 and Y1 NPY receptors in the rat frontal cortex based on second messenger systems, but there does not appear to be an interaction of NPY with either alpha 1- or beta-adrenoceptors.

Interactions between neuropeptide Y and alpha 2-adrenoceptors in selective rat brain regions.

Neuropeptide Y significantly reduced the potassium-stimulated release of [3H]norepinephrine [( 3H]NE) from slices of rat hippocampus, hypothalamus and frontal cortex but not from slices of parieto-occipital cortex. The NPY-induced inhibition of [3H]NE release from frontal cortical slices was concentration dependent, reaching statistical significance at 10 nM. The alpha 2-adrenoceptor partial agonist, clonidine, also reduced the potassium-stimulated release of [3H]NE. The combination of NPY and clonidine in hippocampal slices produced a greater reduction of stimulated [3H]NE release than either of the two compounds alone, suggesting a potentiation of their activity, whereas in frontal cortical slices, the effect was additive. When NPY and clonidine were added to frontal cortical slices, they independently produced a significant concentration-dependent reduction in forskolin-stimulated cAMP accumulation. However, NPY and clonidine combined did not produce a further reduction in forskolin-induced cAMP accumulation than either compound when used alone. These results suggest that the ability of NPY to potentiate alpha 2-adrenoceptor-induced inhibition of [3H]NE release in discrete brain regions does not depend on the reductions in cAMP.

Platelet I1-imidazoline binding sites are decreased by two dissimilar antidepressant agents in depressed patients.

Previous studies have indicated that there may be a dysregulation of alpha 2-adrenoceptors and imidazoline receptors in depression. This study compares the effects of chronic antidepressant treatment with a serotonin reuptake inhibitor (fluoxetine) versus a noradrenaline reuptake inhibitor (desipramine) on the binding parameters of the platelet imidazoline binding site (subtype I1) and of the platelet alpha 2-adrenoceptor in depressed patients. After 6 weeks of treatment with either antidepressant, platelet I1 binding sites became normalized (i.e. downregulated). A negative correlation was obtained between plasma epinephrine concentrations and platelet alpha 2-adrenoceptor Bmax values within the samples, but no correlation was obtained between any plasma catecholamine and a platelet I1 binding parameter. An additional finding was the increased affinity of alpha 2-adrenoceptors for p125I-clonidine in untreated depressed patients compared to healthy subjects. Because of the density of platelet I1 binding sites was downregulated by both of the antidepressants, we postulate that a decrease in platelet I1 binding site density may be related to an improved state from depression that these antidepressants produce.

Chronic desipramine treatment reduces regional neuropeptide Y binding to Y2-type receptors in rat brain.

Chronic treatment of rats with desipramine and imipramine (5 mg/kg/twice daily/i.p.) for 14 days caused a significant reduction in the binding of [3H]propionyl NPY to membranes prepared from frontal cortex, nucleus accumbens, hypothalamus and hippocampus. There was no change in binding of [3H]propionyl NPY in the parieto-occipital cortex, striatum or amygdala. Scatchard analysis of binding data from frontal cortical and hippocampal membranes showed that [3H]propionyl NPY bound to a single site with a Kd of approximately 0.3 nM. The loss of [3H]propionyl NPY binding in hippocampal and frontal cortical membranes revealed that chronic tricyclic antidepressant treatment produced a reduction in the number of binding sites with no change in the affinity for the ligand. Chronic desipramine treatment did not alter the ability of NPY (0.01-25 microM) to stimulate inositol phosphate accumulation in rat frontal cortical slices as compared to saline-treated animals. The lack of change of NPY-induced inositol phosphate accumulation following chronic desipramine treatment showed that there was no change to Y1 NPY-type receptors which are linked to the hydrolysis of inositol phospholipids. However, the ability of NPY (0.05-0.5 microM) to inhibit forskolin (1 microM) stimulated adenylate cyclase via Y2 NPY-type receptors in rat frontal cortical slices was significantly reduced following chronic desipramine treatment. This finding suggests that the reduction of [3H]proprionyl NPY binding in selective brain regions may be the result of an antidepressant-induced loss of Y2-type NPY receptors which are negatively linked to adenylate cyclase.

Compartmentation of catecholamines in rat brain: effects of agonists and antagonists.

The subsynaptosomal distributions of dopamine (DA) in striatum and of norepinephrine (NE) in hypothalamus and cerebral cortex were examined. Isolated nerve-endings from each region were osmotically disrupted and subfractionated into a soluble cytoplasmic fraction (end supernatant, Se) and a synaptic vesicle fraction (P2V). DA and NE were measured in the crude homogenate and in subcellular fractions by a radioenzymatic assay. Levels of NE and DA were 3--5 times higher in the nerve-ending cytoplasm than in the synaptic vesicles, suggesting that catecholamines within the nerve-endings are predominantly in soluble form. Amphetamine increased DA levels in the tissue homogenate and in the nerve-ending cytoplasm but not in synaptic vesicles. Pargyline and gamma-butyrolactone (GBL) increased DA levels in all fractions with the greatest increase occurring in the cytoplasmic fraction. Both 6-hydroxydopamine (6-OHDA) and alpha-methyltyrosine (AMT) caused uniform DA decreases in all fractions. Hypothalamic levels of NE in the two nerve-ending compartments were also reduced to a similar extent after AMT. Reserpine produced uniform depletions of striatal DA in both nerve-ending fractions while the rate of DA repletion was more rapid in the vesicular compartment. Levels of hypothalamic NE were also uniformly depleted by reserpine at the times examined. The cytoplasmic storage compartment is discussed in terms of a possible anatomical correlate such as the smooth endoplasmic reticulum.

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.

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.

Loss of body weight as a predictor of reserpine-induced amine depletion.

Loss of body weight was proved to be a convenient and reliable way to predict the degree of reserpine-induced amine depletion after i.p. injections of reserpine (2.5, 5 and 10 mg/kg) and it thus helps reduce variability. Two populations of animals were sharply distinguished lying on either side of the 5% cutoff point within the first 24 hr. One group (approximately 20% of all animals) lost less than 5% of their body weight, showed absence of physiological and behavioural effects, a moderate 5-HT depletion and a moderate 5-HIAA increase. These animals were called 'partially reserpinized'. Another group always lost 5% or more of body weight, exhibited the reserpine syndrome as well as a larger extent of 5-HT depletion and 5-HIAA increase. This group comprised 'reserpinized' rats. There were significant correlations between the percent weight loss and the degree of 5-HT depletion and 5-HIAA increase in the group of 'partially reserpinized' animals. The two groups differed also in the time course of amine changes. Dose had no effect in the differentiation of the two groups but did affect the extent of amine depletion. When reserpine was given i.v. or s.c., all animals were 'reserpinized'. The phenomenon of partial reserpinization is discussed.

Indoleamine compartmentation in human blood.

Improved methods are described for the fluorimetric determination of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and tryptophan (TRP) in plasma, platelet and RBC blood compartments. The methods for 5-HT and 5-HIAA utilize ion exchange chromatography and allow simultaneous determinations to be made on the same blood sample. The tryptophan assay is a modification of an existing extraction method. All of the 5-HT in whole blood was found to be associated with the platelet fraction (122.6 ng/ml), whereas all 5-HIAA was distributed between the plasma (78.3 ng/ml) and RBC (124.0 ng/ml) compartments. Tryptophan was found in all three compartments although the majority of this amino acid was in the plasma (4.3 microgram/ml) and RBC (1.7 microgram/ml) fractions.

Determination of plasma 3-methoxy-4-hydroxyphenyl-glycol 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.