Eric A. Muth

Antidepressant biochemical profile of the novel bicyclic compound Wy-45,030, an ethyl cyclohexanol derivative

The novel bicyclic compound Wy-45,030 [1-2-(dimethylamino)-1-(4-methoxyphenyl)ethyl cyclohexanol, hydrochloride] exhibited a neurochemical profile predictive of antidepressant activity. Like the tricyclic antidepressants, it inhibited rat brain imipramine receptor binding and synaptosomal monoamine uptake (dopamine as well as norepinephrine and serotonin). It did not inhibit monoamine oxidase. Unlike the tricyclic antidepressants, it was not antimuscarinic in the guinea pig ileum, nor did it have any appreciable affinity for brain alpha-1 adrenergic or histamine-1 binding sites. Wy-45,030 was also without affinity for alpha-2 or beta adrenergic, benzodiazepine, serotonin-1, serotonin-2, dopamine-2, and opiate receptors. Such a profile is predictive of antidepressant activity devoid of the side-effects common to tricyclic therapy.

A mapping of the distribution of acetylcholine, choline acetyltransferase and acetylcholinesterase in discrete areas of rat brain

Acetylcholine (ACh) concentration, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in 60 discrete areas dissected from the rat forebrain. All 3 substances were detectable in every region examined. The range for ACh levels was approximately 9-fold, with highest levels in the striatal and mesolimbic areas. Wider ranges were found for ChAT and AChE. In addition to not having a uniform distribution ACh, ChAT and AChE did not always show proportional variations. ACh levels did not appear to relate to the activity of either enzyme in a simple manner. There was a better correlation (r = 0.902) between the activities of ChAT and AChE, with AChE activities always being higher. In some regions, AChE was disproportionately low or high relative to ChAT. In general, the biochemical results presented here are compatible with histochemical studies of AChE. Such measurements in small brain regions should prove valuable in future experiments designed to determine cholinergic function and localize cholinergic pathways.

Changes in central cholinergic neurons in the spontaneously hypertensive rat

The activity of choline acetyltransferase (ChAT) was measured in discrete areas of the brain in 4-, 8- and 12-week-old spontaneously hypertensive rats (SH rats) and age matched Wistar Kyoto (WKY rats) controls. The concentration of acetylcholine (ACh) was also measured in certain hindbrain nuclei of 12 week SH and WKY rats. An increase in the ChAT activity and ACh concentration in the locus coeruleus was detected in 12-week-old SH rats. Decreases in the ChAT activity were found in several hypothalamic nuclei of SH rats, specifically in the paraventricular nucleus of 4-week-old rats, in the dorsomedial nucleus at 8 and 12 weeks and in the posterior hypothalamic nucleus at 12 weeks. Changes in ChAT activity were also detected in 4- and 8-week-old SH rats in the anterior ventral thalamus and in the nucleus gigantocellularis. These results suggest that cholinergic nerve activity in certain rat brain areas, several of which play a role in cardiovascular control, is altered in spontaneously hypertensive rats.

DMI, Wy-45,030, Wy-45,881 and ciramadol inhibit locus coeruleus neuronal activity☆

Wy-45,030 and Wy-45,881 block the uptake of norepinephrine and serotonin in rat brain synaptosomal preparations and share several in vivo and in vitro effects with known tricyclic antidepressants. To further characterize their activity, these compounds were compared to desipramine and ciramadol in electrophysiological studies of their acute effects on noradrenergic neuronal activity. All four compounds inhibited locus coeruleus neuronal activity with a rank order of potency of desipramine greater than Wy-45,881 greater than Wy-45,030 greater than ciramadol. Administration of the alpha-adrenergic blocking drug, piperoxane, increased locus coeruleus firing rate after desipramine, Wy-45,030 and Wy-45-881. Pretreatment with naloxone prevented the reduction in locus coeruleus impulse flow observed after ciramadol administration but had no effect on the inhibition produced by Wy-45,030. Wy-45,030 and Wy-45,881, like classical antidepressants, appear to inhibit locus coeruleus neuronal firing by potentiating neuroinhibitory transmission of locus coeruleus neurons by blocking the uptake of norepinephrine into presynaptic terminals.

Stria medullaris: a possible pathway containing GABAergic afferents to the lateral habenula

The habenular complex (Hb) is an epithalamic structure located bilaterally along the dorsal edge of the third ventricle and consists of medial (MHb) and lateral (LHb) nuclei. The habenula has been associated with the limbic system 24 and functionally linked with a variety of roles related to this system4,5,z0, 30. The forebrain limbic structures are connected to the Hb through one of its major pathways, the stria medullaris (SM). Studies in several mammalian species have demonstrated that fibers from diverse sources pass via the SM and project to the Hb (for review see refs. 6, 17, 23). A paucity of knowledge exists concerning the identity of the neurotransmitter(s) which are released from these fibers. Further information along this line could improve our understanding of the role of the Hb in brain function. The use of histofluorescence, histochemical and sensitive microassay techniques have revealed in the Hb the presence of putative transmitter substances and their associated metabolic enzymes such as catecholamines11, 33, serotoninl, 3, choline acetyltransferase 27, acetylcholinesterase11, 33, glutamic acid decarboxylasO s,32 and substance pg. However, the amount of information on the compartmentalization of these substances in the Hb is relatively small, although the presence of cholinergic and substance P-containing cell bodies in the M H b was recently reported°,12,16, ~1. The present work will focus on the gamma-aminobutyric acid (GABA) system in the Hb, as manifested by the activity of its synthesizing enzyme glutamic acid decarboxylase (GAD), a marker of GABAergic neurons. In the habenular complex, G A D activity is primarily contained in the LHb is and to a large extent, probably, in GABAergic nerve terminals. The latter are characterized in other regions by a high GABA content 25 and G A D activity 7 as well as the capacity to accumulate exogenous GABA by a high affinity sodium and energydependent processes ~0. Afferent projections to the Hb through the SM have been reported to emanate from various regions such as the globus pallidus (GP) and ento-

Lack of biochemical evidence for a direct habenulo-raphe GABAergic pathway

Morphological observations have suggested that the habenular nuclei, particularly the lateral habenula (LHb), are a major source of afferents to the mesencephalic raphe region. These connections have been demonstrated earlier by a fiber degeneration technique iv, and more recently by autoradiography v and horseradish peroxidase tracing ~,ag,~° (see, however, ref. 16). In a recent electrophysiological study it was reported that habenular stimulation inhibited the firing of cells in the dorsal raphe (dr) nuclei, and that gamma-aminobutyric acid (GABA) might be the neurotransmitter contained within the habenulo-raphe projection el. The present work has been undertaken to biochemically test this suggestion. Glutamic acid decarboxylase (GAD), the GABA synthesizing enzyme, was used as a marker of GABAergic neurons. The activity of GAD in the dr was measured in rats with habenular lesions following different survival periods. Choline acetyltransferase (CHAT), a marker of cholinergic neurons, was measured in the interpeduncular nucleus (ip) of the same animals in order to verify the completeness of the lesion s,ae,14. The results of this study do not support the hypothesis that a direct GABAergic pathway exists between the LHb and the dr nuclei. Male Sprague-Dawley rats weighing about 300 g were obtained from ZivicMiller (Allison Park, Pa.). Bilateral radiofrequency lesions of the habenula were placed in animals anesthetized with Equi-Thesin (Jensen-Salsbery, Kansas City, Mo.). The lesions were made with stainless steel electrodes (0.5 mm in diameter) insulated with Epoxy resin except for 1.5 mm at the tip. The coordinates were according to Kanig and Klippel 1° and adjusted for body weight. The habenular lesion was extensive and included also the emerging part of the fasciculus retroflexus (FR) (Fig. 1) at 4.7

Biochemical and electrophysiological studies of the psychotropic compound, amperozide

Amperozide (AB Ferrosan, FG 5606), a new antiaggressive agent which exhibits a diverse preclinical profile of in vivo and in vitro activities, was examined to determine its acute effects on noradrenergic neurons of the locus coeruleus. The firing rates of all locus coeruleus neurons tested were increased by IV administration of amperozide. The amperozide-induced increase in locus coeruleus firing rate was similar in magnitude to that of an alpha-2 antagonist; however, amperozide was weaker than the alpha-2 antagonist yohimbine in reversing clonidine-induced inhibition of locus coeruleus neuronal activity and had weak affinity at the alpha-2 receptor (Ki = 3.5 microM). Biochemically, amperozide displayed the most significant in vitro affinity at serotonin-2 receptors (Ki = 26 nM) and had low affinities at all other receptors examined. These properties are discussed in the context of amperozides activation of the locus coeruleus as a part of its hypothetical mechanism of antiaggressive action.

Effects of ovarian hormones on levels of luteinizing hormone in plasma and on serotonin concentrations in discrete brain nuclei

Levels of serotonin were measureed in microdissected, individual brain nuclei in ovariectomized rats after treatment with ovarian hormones. Regions sampled included nuclei in the forebrain, rostral and medial hypothalamus, and midbrain tegmentum. Estradiol benzoate decreased levels of luteinizing hormone in plasma but did not affect serotonin levels in any region. Progesterone alone elevated serotonin content in the nucleus tractus diagonalis and ventral tegmental area. The combined estrogen plus progesterone regime produced a surge in plasma luteinizing hormone and also markedly elevated serotonin in the median eminence. These results may be of significance for ovarian hormonal regulation of gonadotropin secretion and reproductive behavior.

The effect of two anesthetic agents on norepinephrine and dopamine in discrete brain nuclei, fiber tracts, and terminal regions of the rat.

Catecholaminergic neurons have been implicated in the mechanism of general anesthesia, but previous attempts at measuring changes in adrenergic neuron function during anesthesia have been limited by techniques to whole brain. Microdissection techniques and sensitive radioisotopic-enzymatic assays were used to measure levels of catecholamines in 20 different nuclei, fiber tracts or nerve terminal regions in brains of rats anesthetized for 90-105 min with 1% halothane or 18% cyclopropane. These two anesthetics were chosen because of their diverse effects on the electroencephalogram and on the cardiovascular and respiratory systems. Of the areas examined, significant increases in norepinephrine content with both anesthetic agents were found only in the nucleus accumbens, locus coeruleus and central gray catecholamine areas. Only in the nucleus accumbens was the dopamine level increased by both anesthetics; cyclopropane, but not halothane anesthesia, also increased the dopamine content of the caudate nucleus, while halothane, but not cyclopropane anesthesia, significantly decreased the dopamine level of the ventral nucleus of the thalamus. Changes in levels of transmitters do not distinguish cause from effect of anesthesia, and further experiments are needed to delineate what role, if any, the specific areas play in muscle relaxation, analgesia, sleep or anesthesia. This study shows that a drug can affect one nucleus or region without significantly affecting other regions that contain the same transmitter substance, and that changes in transmitter levels can occur selectively in different regions of brain even if the nerve endings are derived from contiguous cell bodies.

Evidence that noradrenaline modulates the increase in striatal dopamine metabolism induced by muscarinic receptor stimulation

The effect of oxotremorine was studied on the concentration of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethylglycol (MHPG) in the corpus striatum of rats. At a dose of 1 mg/kg oxotremorine increased HVA levels by 68% and MHPG, by 51%. MHPG was also increased in the nucl. accumbens (58%) and neocortex (42%). Pretreatment with clonidine, 0.1 mg/kg abolished the increase in MHPG in the striatum and significantly inhibited the rise in HVA. 1-Propranolol 2.5 mg/kg, but not d-propranolol, had an effect similar to that of clonidine. A lower dose of oxotremorine (0.5 mg/kg) increased striatal HVA by 36% but did not alter MHPG levels. This increase in HVA was not reduced by 1-propranolol. Eight days after bilateral lesions of the locus coeruleus, there was a reduction in the basal concentrations of noradrenaline (41%) and MHPG (57%) in the striatum. The data suggest that at higher doses of oxotremorine (1 mg/kg), but not a lower dose (0.5 mg/kg), a noradrenergic pathway is stimulated to increase the rate of metabolism of noradrenaline in the striatum. Oxotremorine also appears to increase dopamine metabolism in the striatum by at least two separate mechanisms, one of which involves the mediation of noradrenaline.