A screening system of prodrugs selective for MAO-A or MAO-B

We synthesized several prodrugs of glycine and gamma-aminobutyric acid. In order to establish a screening system from the prodrugs of selective activity to MAO-A or MAO-B, we examined purification conditions such as solubilization with Triton X-100, precipitation with ammonium sulfate, gel filtration and anion exchange chromatography. MAO-B was purified from various tissues such as guinea pig brain, kidney and spleen. MAO-A from human placenta without MAO-B was unstable in above purifications and used as crude. At each purification step, we checked sensitivity of the enzyme to specific inhibitors by developing a convenient fluorescence assay, in which hydrogen peroxide produced by the enzyme was reacted with p-hydroxyphenylpropionic acid. A fluorescence microplate reader measured a fluorescence of the fluorescent product from p-hydroxyphenylpropionic acid with horseradish peroxidase. In comparison with milacemide, N,N-bis(carbamoylmethyl)-N-pentylamine was the best and exclusive substrate for MAO-B. 2-N-(phenylethylamino)-acetoamide was the good substrate for MAO-A and MAO-B same as milacemide. 4-N-(n-pentylamino)-butyric acid and 4-(N-phenylethylamino)-butyric acid were the moderate substrates for both enzymes, which should release gamma-aminobutyric acid. These drugs will be new leading compounds.     

Selective inhibition of MAO-A but not MAO-B activity increases rat pineal melatonin

Summary Clorgyline, a selective MAO-A inhibitor, increased (5 times) rat pineal melatonin and N-acetyl-serotonin (NAS) content, and decreased 5-HIAA level by 80%. Deprenyl, a selective MAO-B inhibitor, did not change melatonin or other pineal indoles content. The data obtained show that inhibition of MAO-A but not B enzyme is responsible for pineal melatonin increase caused by MAO inhibitors.It is suggested that the stimulation of melatonin synthesis caused by MAO inhibitors may contribute to their antidepressive effect.     

MAO-A and MAO-B localisation in human lung and spleen

Localisation of MAO-A and -B in human lung and spleen was studied by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3). Samples of lung and spleen were obtained from 6 routine autopsy cases. Both immunoreactivities showed a homogeneous distribution in lung, where all cell types had both MAO-A and -B staining. In spleen MAO-A and -B showed a very weak immunoreactivity, which was restricted to smooth muscle cells and reticular cells of the white pulp. These data represent the most comprehensive study of MAO-A and -B localisation in the two tissues.     

Effect of the selective MAO-A inhibitors brofaromine,clorgyline and moclobemide on human platelet MAO-B activity

Summary MAO-B activity was compared in healthy volunteers following oral treatment with clinically effective doses of the selective MAO-A inhibitors brofaromine (100 mg q.d. for 14 days), moclobemide (150 mg t.i.d. for 14 days) and clorgyline (5 mg t.i.d. for 10 days). Brofaromine and clorgyline did not alter platelet MAO activity. Following moclobemide treatment, MAO-B activity was reduced by 32% (p<0.05). It recovered during the 5 subsequent days after discontinuation of treatment. These results confirm earlier findings. The explanation for this finding may be that metabolities of moclobemide are active inhibitors of MAO-B.     

In vivo comparison of the effects of inhibition of MAO-A versus MAO-B on striatal L-DOPA and dopamine metabolism

Summary Utilizing the cerebral microdialysis technique, we have compared in vivo the effects of selective MAO-A, MAO-B, and nonselective MAO inhibitors on striatal extracellular levels of dopamine (DA) and DA metabolites (DOPAC and HVA). The measurements were made in rats both under basal conditions and following L-DOPA administration. Extracellular levels of dopamine were enhanced and DA metabolite levels strongly inhibited both under basal conditions and following L-DOPA administration by pretreatment with the nonselective MAO inhibitor pargyline and the MAO-A selective inhibitors clorgyline and Ro 41-1049. The MAO-B inhibitor deprenyl had no effect on basal DA, HVA, or DOPAC levels. Nervertheless, deprenyl significantly increased DA and decreased DOPAC levels following exogenous L-DOPA administration, a finding compatible with a significant glial metabolism of DA formed from exogenous L-DOPA. We conclude that DA metabolism underbasal conditions is primarily mediated by MAO-A. In contrast, both MAO-A and MAO-B mediate DA formation when L-DOPA is administered exogenously. The efficacy of newer, reversible agents which lack the cheese effect such as Ro 41-1049 are comparable to the irreversible MAO-A inhibitor clorgyline. The possible relevance of these findings for the treatment of Parkinson's disease is discussed.     

Histochemistry of MAO-A and MAO-B in the locus coeruleus of the Mongolian gerbil

Summary A coupled peroxidation technique for localization of monoamine oxidase (MAO-A and MAO-B), applied to post-mortem fixed tissue of the locus coeruleus of the Mongolian gerbil is demonstrated. Tyramine hydrochloride,-phenylethylamine and 5-hydroxytryptamine creatinine sulphate were used as substrates, l-deprenyl and clorgyline served as specific inhibitors. All three substrates stained the neurons of locus coeruleus in the absence of inhibitor. In the presence of 1-deprenyl, tyramine hydrochloride and 5-hydroxytryptamine creatinine sulphate were metabolized, whereas in the presence of clorgyline no reaction with either substrate could be observed. Immunocytochemical staining of tyrosine hydroxylase (TH) was employed as comparison.     

Behavioral and pituitary-adrenal response of adult squirrel monkeys to mother-infant separation

(1) The behavioral and pituitary-adrenal responsiveness of adult squirrel monkeys to brief mother-infant separation was assessed. (2) The plasma cortisol levels of males and non-maternal females in an adult group and of non-separated mothers (lactating females) in a mother-infant group were determined under undisturbed basal conditions as well as after 30 min exposure to three other conditions: infant removed from its mother and separated into a small cage either next to the social group cages (Adjacent Separation) or into a small cage and carried to a separate building (Total Separation); and presentation of a strange juvenile conspecific in a small cage next to the group cages (Stranger). (3) Males showed equivalent cortisol levels after all four conditions, while both types of females exhibited significant elevations over basal levels after both infant separation conditions but not after the Stranger condition. (4) Although lactating females showed cortisol values twice as great as those of non-lactating females, the patterns of elevations were identical for the two types of females: both separation conditions produced significant cortisol elevations. Therefore, females exhibit a marked sensitivity, independent of lactation, to social signals from the agitated mother as well as the crying infant. (5) The adult group was assessed for behavioral responses, and the only significant result was an avoidance by the males of the separated infant. (6) Relationships between social status and responsiveness to the test conditions were also examined in the adult group. The dominant animal of each sex usually showed the lowest cortisol value and greatest behavioral response, while the lowest ranking animal showed the highest cortisol value and least behavioral response.     

The physiological response to group formation in adult male squirrel monkeys

(1) Formation of bisexual groups during the nonbreeding season has been shown to induce breeding in primates. The hormonal response to group formation affects adult males differentially depending on the relative dominance status of the individual. (2) In this study, testosterone and cortisol levels in adult male squirrel monkeys were assessed in three conditions: prior to group formation, following formation of male groups and following introduction of females to the male groups. (3) Hormonal levels prior to group formation could not be used to predict subsequent dominance status, but following formation of male groups hormonal levels and dominance status were found to be correlated. (4) Formation of bisexual groups did induce breeding and further differentiated the males' hormonal state depending on the relative dominance status of each male. (5) The results indicate that the formation of social relationships influences each individual's physiological state and that existing relationships further determine the individual's hormonal response to the introduction of new group members.     

Gender differences in reactivity of adult squirrel monkeys to short-term environmental challenges

Evidence is presented to show that individual adult squirrel monkeys show gender-specific reactivity profiles to threatening stimuli under laboratory conditions, and that a putative anxiogenic drug, benactyzine hydrochloride, enhances the vocal response to threatening stimuli, but otherwise preserves the relative importance of the stimuli to both males and females. These data support the conclusion that screening of putative anxiolytic drugs in a primate model can be accomplished using efficient, ethologically based testing procedures in the laboratory.     

Modification of dopamine release by selective inhibitors of MAO-B

Chronic low dose deprenyl treatment in rats causes an increase in striatal extracellular dopamine level, without significant reduction in deaminated metabolite formation. This effect could be the result of increased endogenous levels of the MAO-B substrate beta-phenylethylamine, which is both a releaser of dopamine as well as an inhibitor of the neuronal membrane active dopamine uptake. In guinea pigs, however, striatal extracellular dopamine was not increased either by deprenyl or by clorgyline. Local infusion of the dopamine uptake inhibitor GBR-12909 caused a greater increase in striatal dopamine in microdialysate in rats than in guinea pigs. Intra-species differences in synaptic architecture or in density of dopamine transporter expression may account for these differences.     

Nerve injury-induced depletion of tachykinin immunoreactivity in the somatosensory cortex of adult squirrel monkeys

Cortical immunoreactivity for tachykinin neuromodulators was evaluated 5-21 days after median and ulnar nerve transections in adult squirrel monkeys. Contralateral to the deafferentations, immunoreactive cells were reduced by about 20-40% in layer IV of the hand representation compared to the face region in area 3b. Similar deafferentations have been shown to alter the pattern of neuronal activity in the somatosensory cortex. Unresponsive regions are produced that with time become reactivated by receptive fields served by intact nerves. The immunocytochemical results reported here suggest that the presence or pattern of somatosensory input regulates content of tachykinin neuropeptides in intrinsic cortical neurons.     

Neurophysiological correlates of hand preference in primary motor cortex of adult squirrel monkeys.

Variability in the functional topography of area 4 was examined in adult squirrel monkeys. Conventional intracortical microstimulation techniques were used to derive detailed maps (250 microns interpenetration distances) of distal forelimb movement representations in both hemispheres of six monkeys. Spatial features of these representational maps were then compared to the hand preferred by the individual animals during a motor task requiring skilled digit use. Beyond a few broad generalizations common to all area 4 motor maps, the local mosaic-like topography of individual distal forelimb representations was highly idiosyncratic. Using statistical procedures to determine the independent contributions of individual, side, and movement category to the total variation in motor maps, the results demonstrate statistically significant variation in representational topography among individuals as well as between hemispheres of the same individuals. In the dominant hemisphere (i.e., the hemisphere opposite the preferred hand), the distal forelimb representations generally were greater in number and larger in total area, and displayed a longer total boundary length and a greater index of spatial complexity. Because of the direct relationship between interhemispheric asymmetry and behavioral asymmetry, these studies suggest that a large source of variability found in the topography of motor maps in this and other studies derives from differences in the way particular movements and/or movement combinations are performed by individual animals.     

Latency variation of auditory N1m responses to vocal and nonvocal sounds

To investigate the temporal aspect of timbre processing, we recorded auditory-evoked neuromagnetic responses to periodic complex sounds, which were matched in all acoustic parameters except for two fundamental frequencies (F0s) and 12 spectral envelopes of vocal and nonvocal categories. Only for nonvocal sounds, a significant difference in N1m latency for F0 was detected in both hemispheres. A significant difference among stimuli was detected in both hemispheres for vocal and linear sounds, whereas only in the right hemisphere for instrumental sounds. Moreover, the results of paired comparison among F0s revealed that not only the vocal sounds but also some of the nonvocal sounds were F0-independent. This latency independence may be attributed to the relatively high power in the higher frequency spectrum.     

Effects of lesions of globus pallidus on species-typical display behavior of squirrel monkeys

In a study of brain mechanisms underlying species-typical communication, systematic testing has been conducted on the effects of cerebral lesions on the mirror display of squirrel monkeys. The mirror display is a highly predictable variation of a naturally occurring display used by male squirrel monkeys in a show of aggression, in courtship and in greeting. Of the 5 features of the display, vocalization, thigh-spreading and forward thrusts of the erect phallus are the major and most regularly occurring manifestations, constituting the so-called trump display. Testing has been performed on more than 100 animals with lesions in various structures of the brain. The present report describes the positive effects of electro-coagulation of certain parts of the globus pallidus. Lesions of the medial segment have resulted in an enduring elimination or fragmentation of the trump display. Recovery of the display may occur with lesions predominantly involving the external segment, while destruction of the caudalmost pallidum is without effect. A variety of evidence indicates that the behavioral changes are not due to a deterioration of health, motor disabilities, seasonal factors or motivation. When weighed against the negative or transitory effects of lesions of numerous other structures of the brain, the present findings support the hypothesis that the striatal complex plays a basic role in the organized expression of species-typical behavior.     

Variability in hand surface representations in areas 3b and 1 in adult owl and squirrel monkeys

Detailed microelectrode maps of the hand representation were derived in cortical areas 3b and 1 from a series of normal adult owl and squirrel monkeys. While overlap relationships were maintained, and all maps were internally topographic, many map features varied significantly when examined in detail. Variable features of the hand representations among different monkeys included a) the overall shapes and sizes of hand surface representations; b) the actual and proportional areas of representations of different skin surfaces and the cortical magnifications of representations of specific skin surfaces, which commonly varied severalfold in area 3b and manyfold in area 1; c) the topographic relationships among skin surface representations, with skin surfaces that were represented adjacently in some monkeys represented in locations many hundreds of microns apart in others; d) the internal orderliness of representations; e) the completeness of representations of the dorsal hand surfaces; and f) the skin surfaces represented along the borders of the hand representation. Owl monkey maps were, in general, internally more strictly topographic than squirrel monkey maps. In both species, area 3b was more strictly topographic and less variable than was area 1. The degree of individual variability revealed in these experiments is difficult to reconcile with the hypothesis that details of cortical maps are ontogenetically specified during a period in early life. Instead, we propose that differences in the details of cortical map structure are the consequence of individual differences in lifelong use of the hands. This conclusion is consistent with earlier studies of the consequences of peripheral nerve transection and digital amputation, which revealed that cortical maps are dynamically maintained and are alterable as a function of use or nerve injury in these monkeys (Merzenich et al., '83a,b, '84a; Merzenich, '86; Jenkins et al., '84; Jenkins and Merzenich, '87).     

Monoamine oxidase inhibition by the MAO-A inhibitors brofaromine and clorgyline in healthy volunteers

Summary The present study compared the extent and duration of MAO inhibition by the selective and reversible MAO-A inhibitor brofaromine with the selective and irreversible MAO-A inhibitor clorgyline using amine pressor tests and excretion of urinary amine metabolites (MHPG, tryptamine). The pharmacological characterization of clorgyline as an irreversible and brofaromine as a reversible MAO-A inhibitor in clinically effective doses was confirmed in humans.     

Modulation of adult hippocampal neurogenesis by thyroid hormones: implications in depressive-like behavior

Hormonal imbalances are involved in many of the age-related pathologies, as neurodegenerative and psychiatric diseases. Specifically, thyroid state alterations in the adult are related to psychological changes and mood disorders as depression. The dentate gyrus of the hippocampal formation undergoes neurogenesis in adult mammals including humans. Recent evidence suggests that depressive disorders and their treatment are tightly related to the number of newly born neurons in the dentate gyrus. We have studied the effect of thyroid hormones (TH) on hippocampal neurogenesis in adult rats in vivo. A short period of adult-onset hypothyroidism impaired normal neurogenesis in the subgranular zone of the dentate gyrus with a 30% reduction in the number of proliferating cells. Hypothyroidism also reduced the number of newborn neuroblasts and immature neurons (doublecortin (DCX) immunopositive cells) which had a severely hypoplastic dendritic arborization. To correlate these changes with hippocampal function, we subjected the rats to the forced swimming and novel object recognition tests. Hypothyroid rats showed normal memory in object recognition, but displayed abnormal behavior in the forced swimming test, indicating a depressive-like disorder. Chronic treatment of hypothyroid rats with TH not only normalized the abnormal behavior but also restored the number of proliferative and DCX-positive cells, and induced growth of their dendritic trees. Therefore, hypothyroidism induced a reversible depressive-like disorder, which correlated to changes in neurogenesis. Our results indicate that TH are essential for adult hippocampal neurogenesis and suggest that mood disorders related to adult-onset hypothyroidism in humans could be due, in part, to impaired neurogenesis.     

Telemetrically recorded neuronal activity in the inferior colliculus and bordering tegmentum during vocal communication in squirrel monkeys (Saimiri sciureus)

In order to find out whether the inferior colliculus, in addition to its auditory decoding function, also has an auditory gating function in the sense that it treats self-produced sounds differently from external ones, we have explored the inferior colliculus and bordering tegmentum for neurones reacting differently to self-produced vocalizations and vocalizations produced by conspecifics. The experiments were made in the squirrel monkey (Saimiri sciureus), using a telemetric extracellular recording technique which allowed to register neuronal activity in freely moving animals during natural vocal communication. The results show that the neurones of the central nucleus of the inferior colliculus do not react differently to self-produced and group mate vocalizations of the same type. In the external nucleus of the inferior colliculus, in addition to classical auditory neurones, neurones are found which react to the vocalizations of group mates, but not to self-produced vocalizations. In the paralemniscal area just below the inferior colliculus, there are neurones which are active during self-produced vocalization, but not during vocalization produced by other animals. The results suggest that the external nucleus of the inferior colliculus and bordering tegmentum are involved in vocalization-dependent auditory gating processes.