Multiple N-methyltransferases for aromatic alkylamines in brain

This report describes our studies of the enzymatic N-methylation of tryptamine and β-phenylethylamine by fractions of homogenized rat brain with either 5-methyltetrahydrofolic acid (5-MTHF) or S-adenosyl-L-methionine (SAM) as the methyl donor. We found the pH optimum between 6.5 and 7.0 in reaction involving 5-MTHF and either substrate. (We confirmed reports that reactions involving SAM have a pH optimum of 7.9 with either substrate). Enzymatic activity in the presence of 5-MTHF was linear with time and protein concentration. Measures of enzymatic activity in the 100,000 x g supernate of whole rat brain homogenerate were enriched 15–25 fold after Sephadex G-100 fractionation of the 40–50% ammonium sulfate precipitate. Dialysis enhanced the enzyme activity in virtually all of the fractions. Blanks with tryptamine and boiled enzyme gave unexpectedly high counts (comparable to those without enzyme), suggesting possible non-enzymatic methylation in addition to enzymatic N-methylation. We controlled for this by using blanks of substrate plus boiled enzyme. Radioactive products were isographic with N-monomethyltryptamine and N,N-dimethyl tryptamine on thin layer chromatograms. In the presence of 5-MTHF there is low enzymatic affinity (Km = approximately 10^?3 M) for either substrate, suggesting that enzymatic N-methylation might occur only when amine concentrations are abnormally high. Assays in the presence of each donor in combination with each substrate suggested the possible existence of multiple N-methyltransferases, with one specific for tryptamine and the others non-specific for aromatic alkylamines.     

TRIPHOSPHOINOSITIDE PHOSPHODIESTERASE IN DEVELOPING BRAIN OF THE RAT AND IN SUBCELLULAR FRACTIONS OF BRAIN

Abstract— An assay system for the measurement of triphosphoinositide phosphodiesterase in homogenates of rat brain is described. With triphosphoinositide (TPI) as substrate, and in the presence of 0·1 m -KCI and saturating amounts of diethyl ether, the activity of phosphodiesterase in myelinated brain was 400–500 μmoles of TPI hydrolysed per g wet wt. per hr. One quarter of the adult level of the enzyme was present in rat brain one day after birth, with the remainder being added prior to and during the early stages of myelination. On subfractionation of brain homogenates, substantial activity of the enzyme was located in the soluble portion and in the paniculate fractions enriched in myelin and synaptosomes. The enzyme associated with the particulate fractions could not be detached from the membranes by any of several methods employed. There was a rough correlation between distribution of phosphodiesterase and that of 5'-nucleotidase, an enzyme associated with plasma membrane in a number of tissues. Some implications of the results are discussed.     

The [3H]Tryptamine Receptor in Human Brain: Kinetics, Distribution, and Pharmacologic Profile

Abstract: The kinetics and distribution of [³H]tryptamine binding sites in human brain were investigated. Specific [³H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus ≫ caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [³H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺) indicate stereospecific displacement as well as a rank-order potency profile that is different from that reported for the rat [³H]tryptamine binding site. This suggests the presence of distinct species-dependent [³H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine-mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [³H]-tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [³H]-tryptamine in human brain.     

EFFECTS OF LESIONS AND DRUGS ON BRAIN TRYPTAMINE

Abstract— The effects of various drugs and lesions on rat brain 5-hydroxytryptamine and tryptamine were determined. Monoamine oxidase inhibition caused a proportionately greater increase in tryptamine than in 5-hydroxytryptamine, reserpine depleted 5-hydroxytryptamine but had no effect on tryptamine while p -chlorophenylalanine lowered 5-hydroxytryptamine but increased tryptamine. α-Methyl- p -tyrosine reduced striatal dopamine with no effect on either 5-hydroxytryptamine or tryptamine. Increasing brain tryptophan by amphetamine administration. 24 h food deprivation or giving L-tryptophan did not increase brain tryptamine. However a high dose of ^L-tryptophan (100 or 200mg/kg) together with a monoamine oxidase inhibitor caused a proportionately much greater increase in tryptamine than in 5-hydroxytryptamine. Raphe lesions reduced 5-hydroxytryptamine by 64 per cent and tryptamine by only 29 per cent while intraventricular 6-hydroxydopamine lowered striatal dopamine (56 per cent), had no effect on 5-hydroxytryptamine but reduced tryptamine by 24 per cent, suggesting that tryptamine can be formed in both 5-HT and catecholaminergic neurones.
The results are discussed in relation to the formation, distribution, storage and possible transmitter function of tryptamine in rat brain.     

ENZYMATIC FORMATION OF TETRAHYDRO-β-CARBOLINE FROM TRYPTAMINE AND 5-METHYLTETRAHYDROFOLIC ACID IN RAT BRAIN FRACTIONS: REGIONAL AND SUBCELLULAR DISTRIBUTION

—In rat brain extract tryptamine is converted to 1,2,3,4-tetrahydro-β-carboline (THβJC) and N-methyltryptamine to 2-methyl-THβC in the presence of 5-methyltetrahydrofolic acid. We believe this occurs through enzymatic conversion of 5-methyltetrahydrofolic acid to formaldehyde and tetrahydrofolic acid, followed by spontaneous condensation of the radioactive formaldehyde with the substrate tryptamine (Donaldson & Keresztesy , 1961). The final products of the reactions have been identified by both thin layer chromatography and mass spectrophotometry. Subcellular fractionation shows more than 90 per cent of the formaldehyde-forming enzyme activity to be in the cytosol. Specific activities in fractions from 16 discrete regions of the brain and CNS range from 210·2 ± pmol of THβC/mg protein/h in corpus striatum to 62·9 ± 3·6 pmol of THβC/mg protein/h in corpus callosum.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Characterisation of a Myristoyl CoA:Glycylpeptide N-Myristoyl Transferase Activity in Rat Brain: Subcellular and Regional Distribution

Abstract: An enzyme activity in rat brain, capable of catalysing the transfer of myristic acid from myristoyl CoA to the amino terminus of synthetic peptides, has been characterised. The synthetic peptides used as substrates were one based on the N-terminal eight amino acids of cyclic AMP-dependent protein kinase and another hexadecapeptide based on the N-terminal sequence of p60^src. This N -myristoyl transferase (NMT) activity, which is both peptide dependent and heat labile, occurs in rat brain at levels at least three times those found in other rat tissues. In the presence of both ATP and CoA the enzyme catalysed the transfer of myristic acid, but not palmitic acid, specifically to the N-terminal glycine of the peptides. Both peptide substrates exhibited Mi-chaelis-Menten kinetics yielding K _m values of 100 μ M and 60 μ M , and V_max values of 5 and 14.8 pmol/min/mg for the cyclic AMP-dependent protein kinase peptide and sre-derived peptides, respectively. The majority of the NMT activity was present in the cytosol of the brain homogenates, and there was evidence of an NMT inhibitory activity in both the particulate fraction of brain homogenates and in brain cytosol. NMT activity could also be demonstrated in the 100,000 g supernatant of lysed synaptosomes, and the synaptosomal membranes also exhibited an inhibitory activity on the soluble enzyme. Different brain areas exhibited different levels of the N -myristoyl transferase activity and there was a fivefold difference in the activity found in the most active area, the hippocampus, compared to spinal cord.     

Studies of monoamine oxidases: properties of the enzyme in bovine and rabbit brain mitochondria

Brain mitochondria were prepared from rabbit and bovine cerebral cortex and the purity and intactness of the preparation assessed through the use of enzyme markers and electron microscopy. Enzymatic properties of monoamine oxidase were studied in the purified mitochondrial preparations which were essentially devoid of major contamination by other organelles, especially microsomes. Five substrates were used for characterization of the enzyme: dopamine, kynuramine, serotonin, tryptamine and tyramine. It was found that there was considerable substrate variation in the properties, but in general, the two species showed similar characteristics. The more pertinent findings were: (1) apparent K_m values ranged from 1.1 ± 10^?5m for tryptamine to 2.5 ± 10^?4m for dopamine; (2) substrate specificity from V_max values in decreasing order was tyramine > dopamine > kynuramine > serotonin > tryptamine for the bovine enzyme and tyramine > kynuramine > dopamine > serotonin > tryptamine for rabbit; (3) there appeared to be three distinct pH optima according to substrate: pH 7.5 for phenylethylamines, pH 8.2–8.5 for the indolylamines and pH 9.1 for kynuramine; and (4) the activity with tyramine was highly sensitive to increased oxygen tension while kynuramine showed no sensitivity. It is proposed that the properties of monoamine oxidase, a membrane-bound enzyme, might be influenced by the microenvironment and results are also discussed in terms of multiple forms or multiple activity sites on a single form.     

FACTORS INFLUENCING BRAIN AND TISSUE LEVELS OF TRYPTAMINE: SPECIES, DRUGS AND LESIONS

With a modification of the spectrophotofluorometric (SPF) method of HESS & UDENFRIEND (1959) (J. Pharmac. exp. Ther. 127 , 175-177), brain tryptamine levels in the rat (20.9 ng/g) and guinea-pig (20.7 ng/g) were found to be less than those in the dog (32.1 ng/g) and cat (52.2 ng/g). Regional distribution studies in the dog and cat showed that tryptamine was present in all major brain regions with highest concentrations in the spinal cord. Blood levels of tryptamine in the guinea-pig, dog and cat (6-7 ng/ml) were lower than brain levels. Pargyline significantly increased brain tryptamine in both the dog and cat; whereas, isocarboxazid (after 4 h) increased brain tryptamine levels in the dog but decreased brain levels in the cat. Reserpine (0.5-1.0 mg/kg per day for 1-4 days) did not significantly decrease brain, spinal cord or blood tryptamine levels in the dog. Spinal cord transection did not decrease tryptamine levels below the lesion in the chronic spinal dog.     

Distribution of 17β-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues

The interconversion of estrone (E₁) and 17β-estradiol (E₂), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3β,17β-diol is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD). The enzyme 17β-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17β-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17β-HSD mRNA in some human tissues. Enzymatic activity was measured using ¹⁴C-labeled E₁, E₂, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highestlevel of 17β-HSD activity, low but significant levels of E₂ as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E₂→E₁, T→4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17β-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.     

Structure-activity-relationships of certain hallucinogenic substances based on brain levels.

A structure-activity-relationship of a variety of behaviorally active and inactive compounds which are naturally occuring or closely related structurally to putative neurotransmitters has been assembled. For the first time comparisons of activity are based on actual brain levels instead of doses administered.A different and new SAR is obtained if minimal effective brain levels (MEBL), instead of administered doses, are used. Differences are due to the fate of the substance in the body and its availability to the CNS. Predictions of brain levels based on $\text{in}$$\text{vitro}$ data, blood levels and/or lipid solubility can be misleading.It is suggested that the “minimal effective brain level (MEBL)” or that concentration of a substance in the brain in moles/g at which time the first significant behavioral effect in a particular test situation can be detected, be used as the basis of comparison in SAR studies of behaviorally active substances. This is, of course, not the ultimate parameter which might be the number of molecules of a substance at a particular receptor, but it would eliminate a great number of “artifacts” which can be controlled with the present state of the art.The use of MEBLs will allow more valid correlations between behavioral activity and certain physico-chemical parameters of the compounds, and an attempt to use MEBLs was recently made by Houk and co-workers (53). Also, MEBLs in animals can serve as a better base for the interpretation of human studies and can make these behavioral studies in man more meaningful; for instance, the lack of behavioral activity in bufotenin in man observed recently (54) could perhaps be due to the fact that at the doses tested the compound did not reach the CNS in sufficient concentrations because of its extreme difficulty in crossing the BBB. Similarly, 3,4-dimethoxyphenylethylamine has been found to be inactive in man after oral administration (55); based on our studies (27) the compound would actually not be expected to be active by this route since it is too quickly metabolized and cannot reach the MEBL, thus, the behavioral activity of this compound in man remains unknown.Based on the criteria applied in this review, LSD and 5-methoxytryptamine are the most potent psychoactive substances followed by tryptamine (after MAO-inhibition) and pentamethoxyphenylethylamine. It is of interest to note that tryptamine and 5-methoxytryptamine are known to occur naturally in the mammalian brain. It remains to be determined what role, if any, these substances may play in the pathogenesis of abnormal behavior in man.     

MONOAMINE OXIDASE A AND B IN CULTURED CELLS

Abstract— Monoamine oxidase (MAO) activity against tryptamine was compared in a number of continuous rodent lines, including neuroblastoma, hepatoma, melanoma, nephroma, sarcoma and L cells. Activities against tryptamine varied over 300-fold in homogenates of different lines, being highest in hepatoma line MH₁C₁ and lowest in a neuroblastoma line lacking hypoxanthine phosphoribosyltransferase (HPRT) activity. The amount, but not the type, of MAO activity varied with the stage of growth in homogenates of neuroblastoma and hepatoma cells. Measurements of succinate-cytochrome c reductase (SCCR), another mitochondrial enzyme, also showed 20-fold variations between lines, being highest in neuroblastoma line N1E-115 and lowest in hepatoma line MH₁C₁; SCCR and MAO activities appeared to be regulated independently. The relative proportions of the A and B types of MAO activity were determined in homogenates and living cultures. Clorgyline inhibition of tryptamine deamination in homogenates indicated that in all lines except MH₁C₁, greater than 95% of the MAO activity was of the A type. In MH₁C₁ homogenates, using clorgyline or deprenyl, 40–70% of the activity appeared to be of the A type and 30-60% of the B type. In cultures of neuroblastoma N1E-115 cells, deamination of tryptamine and dopamine was sensitive to inhibition by low concentrations of clorgyline, indicating that the A type of activity is present intracellularly. as in homogenates. In MH₁C₁ hepatoma cultures, tryptamine deamination showed a biphasic sensitivity to clorgyline. We interpret this to mean that A and B types of MAO activity occur together in living hepatoma cells.     

SUBCELLULAR DISTRIBUTION AND SOME PROPERTIES OF ACETYL-COENZYME A HYDROLASE IN THE BRAIN

—The detailed subcellular distribution and some properties of acetyl-CoA hydrolase were studied in the rat brain. The brain homogenate (S₁) hydrolysed acetyl-CoA at a rate of approx 2·3 nmol/min/mg of protein at 37°C. The total activity of acetyl-CoA hydrolase was distributed in the following order: soluble > mitochondrial > microsomal, synaptosomal > myelin fraction. The order of the specific activity of the enzyme was: soluble, microsomal > mitochondrial > synaptosomal > myelin fraction. The synaptic vesicle fraction (D) had relatively high specific activity among the intraterminal particulate fractions, having two or three times higher specific activity than that of the synaptic cytoplasmic membrane fraction (F or G). Attempts to de-occlude acetyl-CoA hydrolase in the particulate fraction showed that only the enzyme activity in the myelin fraction was increased markedly by the treatment with ether or Triton X-100. Lineweaver-Burk plots gave straight lines for each subcellular fraction and apparent K_m values for acetyl-CoA were between 0·1 and 0·2 mM. Neither diisopropyl fluorophosphate nor physostigmine at the concentration of 0·1 mm inhibited the enzyme activity.     

The Presence of (+)-S-Adenosyl-l-Methionine in the Rat Brain and Its Lack of Effect on Phenylethanolamine N-Methyltransferase Activity

Abstract: (+)-S-Adenosyl- l -methionine [(+)-SAM] was isolated from rat brain and was quantified by HPLC followed by UV spectrophotometric measurements and by ¹H-NMR. Its estimated ratio in brain is 3% of total SAM. Because of its commercial unavailability, (+)-SAM was also prepared from chemically synthesized SAM by separation of the two diastereoisomers on a preparative reverse-phase Nucleosil C8 column. The (+) diastereoisomer thus obtained was then assayed in vitro both as an inhibitor and a substrate of phenylethanolamine N -methyltransferase. Enzymatic activity was measured by HPLC analysis. It was shown that (+)-SAM has no effect on phenylethanolamine N -methyltransferase activity; therefore, it is unlikely that (+)-SAM plays any possible role in regulation of adrenaline synthesis in the brain.     

THE DISTRIBUTION OF CALCIUM-DEPENDENT PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The properties of Ca2⁺-dependent phosphatidylinositol-phosphodiesterase in membrane fractions and supernatants prepared from rat brain have been examined with the aim of providing firm evidence for the existence of a membrane-bound activity distinct from the soluble enzyme found in the cytosol (EC 3.1.4.10). The soluble enzyme is either stimulated or inhibited at pH 7.0 by deoxycholate depending on the ratio of detergent to substrate. The effects of deoxycholate are pH dependent and result in a shift of the enzyme optimum to a higher pH if the enzyme is assayed in the presence of deoxycholate. The soluble enzyme cannot hydrolgse membrane-bound phosphatidylinositol (in ₃₂P-labelled rat liver microsomes) unless deoxycholate is present. The pH optimum is 6.7 for this detergent-dependent hydrolysis and this is probably dependent on the ionization of deoxycholic acid. The lactate dehydrogenase (EC 1.1.1.27) content of rat brain membrane fractions has been measured to estimate the contamination of these fractions by supernatant phosphatidylinositol-phosphodiesterase. No evidence has been found for phosphatidylinositol-phosphodiesterase activities that cannot be explained by such contamination. It is concluded that all the properties of calcium-dependent phospha-tidylinositol-phosphodicsterase in rat brain can be explained by the existence of only the solublc cyto-plasmic enzyme: no evidence confirming a distinct membrane-bound activity has been obtained.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 °C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected ³²P_i was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.     

[3H]Tryptamine Binding Sites Are Not Identical to Monoamine Oxidase in Rat Brain

Competition binding studies, subcellular distribution, and in vitro autoradiography were employed to compare the binding in rat brain of [3H]tryptamine with two radioligands for monoamine oxidase (MAO), [3H]pargyline, and [3H]1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine ([3H]MPTP). The MAO inhibitors pargyline, clorgyline, and deprenyl all yielded biphasic competition curves versus [3H]tryptamine. At low concentrations, these drugs stimulated binding by protecting the radioligand from MAO oxidation; at considerably higher concentrations, they inhibited binding by direct competition at the [3H]tryptamine binding site. In subcellular distribution studies, [3H]tryptamine was localized preferentially to the synaptosomal fraction, whereas [3H]pargyline showed greater binding to the mitochondrial fraction. Equilibrium binding studies revealed that the potencies of a series of seven compounds at inhibiting [3H]tryptamine binding were completely different from their potencies at inhibiting [3H]MPTP binding. Finally, the autoradiographic distribution of [3H]tryptamine binding in rat brain was different from that of [3H]MPTP and [3H]pargyline. We conclude that the [3H]tryptamine binding site in rat brain is not equivalent to MAO.     

Multiple substrate determination of monoamine oxidase distribution and iproniazid inhibition in rat brain

Abstract— —A variety of monoamine oxidase substrates (tyramine, dopamine, serotonin, tryptamine) have been used with and without Iproniazid inhibition to evaluate further the extent to which enzyme multiplicity may exist in various regions of rat brain. Levels of monoamine oxidase activity, as measured by ammonia production, were found to vary as a function of both brain area and kind of substrate used, in the absence as well as in the presence of Iproniazid, in vivo and in vitro. Similarity of substrate metabolizing patterns among the different brain areas, however, strongly suggests that only one kind of monoamine oxidase exists in rat brain.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: ¹⁴C-labeled tryptamine, dopamine and kynuramine. With dopamin as substrate, the enzyme activity showed decline during 24 and 48 h starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decreased after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

Regional distribution of EDRF/NO-synthesizing enzyme(s) in rat brain.

Stimulation of soluble guanylyl cyclase and increase in cyclic GMP in rat fetal lung fibroblasts (RFL-6 cells) was used as a bioassay to detect EDRF/NO formation. The cytosolic fraction of whole rat brain synthesized an EDRF/NO-like material in a process dependent on L-arginine and NADPH. The enzymatic activity was destroyed by boiling and inhibited by N omega-nitro-L-arginine. Hemoglobin and methylene blue blocked the effect of EDRF/NO. When different brain regions were analyzed in the presence of L-arginine and NADPH, the cytosolic fraction from cerebellum showed the highest EDRF/NO-forming activity (2-3 times higher than whole brain). Activity similar to whole brain was found in hypothalamus and midbrain. Enzymatic activities in striatum, hippocampus and cerebral cortex were about two thirds of whole brain. The lowest activity (less than half of whole brain) was found in the medulla oblongata.