Suzanne Knapp

Histologic and enzymatic studies of the mesolimbic and mesostriatal serotonergic pathways

Selective lesions of the dorsal (B7), median (B8), or lateral (B9) raphe nuclei were made stereotaxically in male rats 4 weeks before sacrifice. The extent of damage to each raphe nucleus was quantified histologically by means of a simplified formaldehyde histochemical method for visualization of serotonin in cryostat sections. A detailed mapping of the distribution of the yellow-fluorescent raphe perikarya provided the basis for quantification. Tryptophan hydroxylase activity was measured in 6 forebrain regions from each animal, and the results were correlated with the per cent damage to each raphe nucleus. Tyrosine hydroxylase was also assayed in 5 of these regions; it was not significantly affected by any of the raphe lesions. Dorsal raphe lesions reduced tryptophan hydroxylase activity in the striatum, thalamus, cortex, and hypothalamus, but not in the septal nuclei or hippocampus. Damage to B8 resulted in decrements in this serotonergic enzyme in the septal nuclei, hippocampus, cortex, and hypothalamus, but not in the striatum or thalamus. Lesions of the scattered B9 cells had no significant effect on enzyme activity in any region examined. These data suggest that the dorsal and median raphe nuclei provide two distinct though perhaps overlapping serotonergic systems innervating different parts of the forebrain: a mesostriatal pathway originating in B7 and a mesolimbic system derived from B8. Behavioral studies on the animals, which are presented in a companion paper, indicated that damage to the median nucleus is responsible for many of the behavioral effects previously reported after combined lesions of both major raphe nuclei.

Short- and Long-Term Lithium Administration: Effects on the Brain's Serotonergic Biosynthetic Systems

Short-term treatment with lithium chloride stimulates the uptake of tryptophan and its conversion to serotonin by striate synaptosomes. Preincubation of striate synaptosomes with L-tryptophan and in vivo administration of L-tryptophan appear to act in a similar manner. Midbrain tryptophan hydroxylase activity is reduced in temporal continuity with the lithium-induced activation of tryptophan uptake and conversion. By 10 days, conversion of tryptophan to serotonin in nerve endings becomes a joint function of the maintained increased uptake of tryptophan and a decreased level of tryptophan hydroxylase activity in nerve endings. The occurrence of this delayed alteration corresponds in time to the previously described axoplasmic flow rate for tryptophan hydroxylase.

Narcotic Drugs: Effects on the Serotonin Biosynthetic Systems of the Brain

The effects of short- and long-term administration of morphine on the activity of two measurable forms of rat brain tryptophan hydroxylase were studied. Morphine administration produced an immediate decrease and a longterm increase in the nerve ending (particulate) enzyme activity but did not change the cell body (soluble) enzyme activity. Cocaine administration demnonstrated a short-term decrcease in measurable nerve eniding enzyme activity that was due to the inhibition of the high affinity uptake (the Michaelis constant, Km is 10-5 molar) of trytophan, the serotonin precursor. Cocaine did not aflect the low affinity uptake Km = 10-5 molar) of tryptophan. Both the uptake of the precursor and the enizymiie activity appeared to be drug-sensitive regullatory processes in the biosynthlesis of serotonin.

Parachlorophenylalanine - its three phase sequence of interactions with the two forms of brain tryptophan hydroxylase

Abstract Tryptophan hydroxylase was studied in two discrete regions of rat brain: midbrain and septal areas. The selection of these regions was based upon the histochemical evidence (1) demonstrating a high concentration of serotonin cell bodies in the midbrain and serotonin nerve endings in the septal area, as well as our choice of these two regions as representative of the two forms of the enzyme (Fig. 1). This enzyme, rate-limiting in the biosynthesis of serotonin, appeared to be influence by PCPA in a linked series of temporarily related events following its acute administration: (1) Competition with substrate for entry into the nerve ending; (2)Reversible competitive inhibition of the enzyme for substrate; (3) Irreversible inhibition by incorporation into the enzyme during new protein synthesis in the cell body. The defective enzyme formed in the latter event was later seen in the nerve endings of the septal area due to a slow axonal transport of 1–2 mm/day. Since all three processes may decrease brain serotonin, it becomes important to establish the manner of action by which PCPA is operating at the time of studies of neurochemical mechanisms. Studies of this enzyme with drugs (2) or with steroid (3) which have been so characterized by conflictual reports (4), have not taken this sequence of mechanisms into consideration. It is possible that specific brain regions, subcellular fractions and/or time after drug manipulation become critical determinance of effects on the serotonin biosynthetic system in the brain.

Calcium activation of brain tryptophan hydroxylase

Abstract Using both radioisotopic and fluorometric techniques to measure the activity of midbrain soluble enzyme, we have demonstrated that calcium activates tryptophan hydroxylase. The observed activation apparently results from an increased affinity of the enzyme for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH 4 ). The calcium activation of tryptophan hydroxylase appears to be specific for both enzyme and effector: other brain neurotransmitter biosynthetic enzymes, such as aromatic amino acid decarboxylase(s) and tyrosine hydroxylase, are not affected by calcium (at concentrations ranging from 0.01 mM to 2.0 mM); other divalent cations, such as Ba ++ , Mg ++ , and Mn ++ , have no activating effect on tryptophan hydroxylase. This work suggests that increases in brain serotonin biosynthesis induced by neural activation may be due to influx of Ca ++ associated with membrane depolarization and resulting activation of nerve ending tryptophan hydroxylase.

Some factors in the regulation of central serotonergic synapses

Abstract For a number of years release, reuptake, vesicular storage, and degradation via monoamine oxidase were the exclusive foci in chemical studies of the regulation of central serotonergic synapses. This brief review describes some other factors, including the brain levels of tryptophan and uptake processes in the nerve ending relevant to the substrate, the activity and amount of tryptophan hydroxylase in cell bodies and nerve endings, and a potential regulatable parasynaptic inactivation process, N-methylation. In chronic and acute drug studies some of these factors appear to function to compensate for acute perturbations in central serotonergic synaptic processes.

The effects of environmental isolation on behavior and regional rat brain tyrosine hydroxylase and tryptophan hydroxylase activities.

Regional brain levels of tyrosine hydroxylase and tryptophan hydroxylase were determined in rats isolated for intervals of up to 16 days. The activity of midbrain and striatal tyrosine hydroxylase was found to be elevated in the isolated rats when compared to grouped controls. In contrast, septal tryptophan hydroxylase activity was significantly reduced and midbrain tryptophan hydroxylase remained unchanged. Animals isolated for as little as 5 days were found to exhibit an increased level of spontaneous motor activity. In addition, amphetamine-induced behavioral excitation appeared to be additive with that produced by isolation.

Lithium and chlorimipramine differentially alter bilateral asymmetry in mesostriatal serotonin metabolites and kinetic conformations of midbrain tryptophan hydroxylase with respect to tetrahydrobiopterin cofactor.

Abstract Lithium decreased and the tricyclic drug chlorimipramine (CMI) differentially altered hemispheric asymmetries in striatal and hippocampal concentrations of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) in the rat. The kinetic functions of tryptophan hydroxylase from the two halves of the midbrain, demonstrated by pteridine cofactor (BH 4 ) kinetic functions, also changed differentially in response to the two drugs. Bilateral asymmetries in 5-HIAA and kinetic functions with regard to BH 4 after CMI administration suggest that this drug may alter the neurophysiological activity (5-HT cell discharge rate) of the 5-HT system in one hemisphere relative to the other. In contrast, lithium evoked similar tryptophan hydroxylase kinetic functions with regard to BH 4 from both hemispheres and reduced asymmetries in metabolites, which is more suggestive of a bilaterally simultaneous biochemical “field” mechanism.

Methamphetamine-induced alteration in the physical state of rat caudate tyrosine hydroxylase.

Abstract Methamphetamine was found to induce a shift of a portion of rat striate (and not midbrain) tyrosine hydroxylase activity from the 11,000 g supernatant to the “synaptosomal” and “mitochondrial” particulate fractions with no change in total measurable enzyme activity, although the particulate enzyme demonstrated a greater affinity for the synthetic cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin (DMPH4). The magnitude of this shift appeared dose-related up to 5 mg/kg. It demonstrated a shortest latency of 10 min and a longest duration of 8 hr. It could not be produced in vitro by homogenizing the striate area in varying concentrations of methamphetamine or catecholamines. The effect could not be abolished by pretreatment with cycloheximide or intraventricular colchicine. It could not be induced by the administration of imipramine, footshock, electroconvulsive shock, or behaviorally activating intraventricularly-infused norepinephrine (NE). However, this change could be produced by the administration of α-methyltyrosine and reserpine. The ressrpine-induced change differed in that in addition to the shift of activity from the soluble to the particulate fraction, there was an increase in total measurable enzyme activity as well. It was suggested that a drug-induced reduction in intraneuronal catecholamines may be the common stimulus for the observed relative increase in rat caudate particulate tyrosine hydroxylase. The particulate state of caudate tyrosine hydroxylase generally resisted narrow clearance homogenization, hypotonic shock and sonication but varied in a complex way with varying ion concentrations (especially Ca2 and Mg2) in vitro. Varying membrane binding of soluble tyrosine hydroxylase via the manipulation in vitro of divalent cations led to apparent decreases in the specific activity of tyrosine hydroxylase. However, other recent studies in our laboratory have demonstrated that membrane binding of tyrosine soluble hydroxylase activates the enzyme allosterically for DMPH4 binding as well as for the competitive inhibition of the affinity of this cofactor site by dopamine (DA) and norepinephrine (NE). It is within this context that the amphetamine-induced alteration in physical state of tyrosine hydroxylase assumes potential regulatory significance.

Conformational influences on brain tryptophan hydroxylase by submicromolar calcium: Opposite effects of equimolar lithium

Tryptophan hydroxylase from rat midbrain, EGTA-pretreated and dialyzed, manifested allosteric properties with respect to its substrate tryptophan, cofactor tetrahydrobiopterin, and the calcium ion. Kinetic studies suggest two preferred enzyme conformations in the presence of low concentrations of the cosubstrates: a higher affinity form manifesting hyperbolic substrate kinetics, induced by submicromolar (0.4–0.8μM) calciumin vitro and cocainein vivo, and a lower affinity form exaggerating cooperativity with respect to substrate, induced by submicromolar (0.4 to 0.8μM) lithiumin vitro and lithiumin vivo. Lithiums effect on serotonin biosynthesis may be due to its antagonism of the positive effector influence of calcium on tryptophan hydroxylase, either as a negative effector or by blocking the calcium site.