Recombinant inbreeding in mice reveals thresholds in embryonic corpus callosum development.

The inbred strains BALB/cWah1 and 129P1/ReJ both show incomplete penetrance for absent corpus callosum (CC); about 14% of adult mice have no CC at all. Their F(1) hybrid offspring are normal, which proves that the strains differ at two or more loci pertinent to absent CC. Twenty-three recombinant inbred lines were bred from the F(2) cross of BALB/c and 129, and several of these expressed a novel and severe phenotype after only three or four generations of inbreeding - total absence of the CC and severe reduction of the hippocampal commissure (HC) in every adult animal. As inbreeding progressed, intermediate sizes of the CC and the HC remained quite rare. This striking phenotypic distribution in adults arose from developmental thresholds in the embryo. CC axons normally cross to the opposite hemisphere via a tissue bridge in the septal region at midline, where the HC forms before CC axons arrive. The primary defect in callosal agenesis in the BALB/c and 129 strains is severe retardation of fusion of the hemispheres in the septal region, and failure to form a CC is secondary to this defect. The putative CC axons arrive at midline at the correct time and place in all groups, but in certain genotypes, the bridge is not yet present. The relative timing of axon growth and delay of the septal bridge create a narrow critical period for forming a normal brain.

An anterograde and retrograde tract-tracing study on the projections from the thalamic gustatory area in the rat: distribution of neurons projecting to the insular cortex and amygdaloid complex.

Projections from the thalamic gustatory nucleus, i.e. the parvicellular part of the posteromedial ventral thalamic nucleus (VPMpc) to the forebrain regions were studied in the rat by the tract-tracing methods with anterograde tracer (biotinylated dextran amine, BDA) and anterograde/retrograde tracer (wheat-germ agglutinin-horseradish peroxidase, WGA-HRP). After BDA injection into the VPMpc, terminal labeling was observed in the insular cortex, amygdaloid complex, and fundus striati. The terminal labeling in the amygdaloid complex was distributed in dorsolateral area of the rostral part of the lateral amygdaloid nucleus and the rostral part of the lateral subdivision of the central amygdaloid nucleus. The terminal labeling in the central amygdaloid nucleus extended to the fundus striati. The retrograde tracing study with WGA-HRP revealed that the projection fibers from the VPMpc to the amygdaloid complex originated from the medial part of the VPMpc and also from the thalamic area medial to the VPMpc. In the rats injected with Fluoro-Gold and WGA-HRP, respectively into the insular cortex and amygdaloid complex, no double-labeled neuronal cell bodies were found in the VPMpc, although neurons labeled singly with Fluoro-Gold were intermingled with those singly labeled with WGA-HRP in the medial part of the VPMpc. The results indicated that VPMpc neurons projecting to the amygdaloid complex constituted a population different from VPMpc neurons projecting to the insular cortex.

Timing and origin of the first cortical axons to project through the corpus callosum and the subsequent emergence of callosal projection cells in mouse.

A precise knowledge of the timing and origin of the first cortical axons to project through the corpus callosum (CC) and of the subsequent emergence of callosal projection cells is essential for understanding the early ontogeny of this commissure. By using a series of mouse embryos and fetuses of the hybrid cross B6D2F2/J weighing from 0.36 g to 1.0 g (embryonic day E15.75-E17.25), we examined the spatial and temporal distribution of callosal projection cells by inserting crystals of the lipophilic dye (DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) into the contralateral white matter just lateral to the midsagittal plane. Around 0.4 g or E15.8, retrogradely labeled cells were found restricted to a discrete cluster continuously distributed from the most ventral part of presumptive cingulate cortex to the hippocampus. During subsequent development, however, the tangential distribution of these labeled cells in ventromedial cortex did not extend further dorsally, and in fetuses where the CC became distinct from the hippocampal commissure (HC), labeled axons of cells in the ventral cingulate cortex were observed to intersect the callosal pathway and merge with labeled axons of the HC derived from cells in the hippocampus. The first cortical axons through the CC crossed the midline at about 0.64 g or E16.4, and these axons originated from a scattered neuronal population in the dorsal to lateral part of the presumptive frontal cortex. The earliest callosal cells were consistently located in the cortical plate and showed an immature bipolar appearance, displaying an ovoid- or pearl-shaped perikaryon with an apical dendrite coursing in a zig-zagging manner toward the pial surface and a slender axon directed toward the underlying white matter. Callosal projection cells spread progressively with development across the tangential extent of the cerebral cortex in both lateral-to-medial and rostral-to-caudal directions. In any cortical region, the first labeled cells appeared in the cortical plate and their number in the subplate was insignificant compared to that in the cortical plate. Thus, these results clarify that the CC is pioneered by frontal cortical plate cells, and the subsequent ontogeny of callosal projection cells proceeds according to the gradient of cortical maturation.

Innervation of blood vessels in the rat incisor pulp: a scanning electron microscopic and immunoelectron microscopic study.

Although two types of nerve endings have been proposed to innervate blood vessels in the dental pulp, the precise innervation pattern is not well understood. This is mainly due to the lack of information regarding the positional relationships of nerve fibers with blood vessels at the electron microscopic level. The rat incisor pulp was investigated by scanning electron microscopy (SEM) after connective tissue digestion and by transmission electron microscopy after immunohistochemical localization with polyclonal anti-PGP 9.5 antibody. SEM specimens revealed that unmyelinated nerve fibers passed through the tunica adventitia of the blood vessel in the center of the pulp and that these fibers then entered the tunica media of the smaller arterioles. The nerve fibers divided into many collaterals, and these terminated on the surface of smooth muscle cells and endothelial cells. The fibers extended toward smaller vessels. The terminal fibers then reached the subodontoblastic capillary plexus and terminated on pericytes, while the odontoblastic capillary plexus had no direct innervation. In the pulpal venules, nerve fibers were located adjacent to the pericytes and endothelial cells, and they extended toward postcapillary venules. The same results were confirmed by immunoelectron microscopy. The present study demonstrated that in the rat incisor pulp the microvasculature as well as larger vessels were directly innervated by free nerve endings, suggesting that the local regulation of blood flow could take place not only at larger vessels but also at the level of the microvasculature in this tissue.

Blood vessels and nerve fibers in rat incisor pulp. Immunoelectron microscopic observation with anti-substance P antibody.

The distribution of nerve fibers with special reference to their positional relationship with blood vessels in the pulp of the rat mandibular incisor was investigated by light and electron microscopic immunohistochemistry using polyclonal anti-substance P antibody. In the center of the pulp, numerous immunoreactive nerve fibers with varicosities were found along larger blood vessels. In the periphery of the pulp, there were two layers of capillary plexus; an odontoblastic and a subodontoblastic. Many nerve fibers, showing varicosities, were located in the subodontoblastic region, and some revealed a close association with pericytes of capillaries of the subodontoblastic plexus. A few nerve fibers entered the odontoblast layer, but no nerve fibers were seen to be associated with the odontoblastic capillary plexus. The results suggest that the local regulation of blood flow takes place not only in larger blood vessels but also in the capillaries of the rat incisor pulp.

Experimental odontogenic cysts induced by in vitro 4-nitroquinoline 1-oxide (4NQO) treatment of F344 rat incisor tooth germs.

This study was designed to establish an experimental animal model for elucidating the early stages of odontogenic cysts and tumors. It involves the in vitro treatment of tooth germs with 4-nitroquinoline 1-oxide (4NQO) at the early bell stage and their subsequent transplantation into the kidney subcapsular space. While all tooth germ transplants of the control group not exposed to the carcinogen showed continued tooth development with no pathological lesions, 21 of 23 4NQO-treated tooth germs developed into similar appearing keratinized cysts with or without associated tooth structures. The remaining two transplants failed to develop cysts and formed only a tooth. The present experimental procedure was effective in inducing keratinized cystic lesions that exhibit some similarities to human odontogenic keratocysts or primordial cysts.

Genetic controls of susceptibility and resistance to 4-nitroquinoline 1-oxide-induced tongue carcinomas in rats.

We analyzed the incidence of infiltrative mass-type tongue carcinomas (IMTC) induced in 550 rats by continuous oral administration of 0.001% 4-nitroquinoline 1-oxide solution for 180 days. The study included various crosses of susceptible Dark-Agouti rats (DA) and resistant Wistar/Furth rats (WF). DA showed a 93.6% incidence of IMTC measuring more than 5 mm in their largest diameter, while WF showed only a 4% incidence. Reciprocal F1 and F2 hybrids mated by DA and WF showed 47.5% and 45.8% incidences, respectively. Meanwhile, reciprocal backcrossed hybrids to DA and WF showed 73.7%, and 24.6% incidences, respectively. Segregation of the incidences suggests that there are two autosomal dominant genes, one linked to the susceptibility of DA and the other to the resistance of WF.

Cortical axon trajectories and growth cone morphologies in fetuses of acallosal mouse strains.

Hereditary absence of the corpus callosum (CC) provides an ideal experiment of nature for exploring mechanisms of axon guidance. In this study the prenatal development of CC axons in the acallosal mouse strains BALB/cWah1 and 129/ReJ or J was compared with normal hybrid mice by using the lipophilic dyes DiI and DiA. A few I/LnJ mice were also examined. The time of emergence and growth rate of CC axons from four cortical regions (frontal, parietal, temporal, occipital) were normal in acallosal strains. Their CC axons arrived at midplane on schedule but then often looped back to form the longitudinal Probst bundle. The frequency of formation of the Probst bundle was highest for axons from frontal cortex, which arrived at midplane first, and lowest for occipital axons, which arrived last. Once a few CC axons found a path to the other side via the hippocampal commissure, those that arrived later then crossed relatively normally. Some axons from the Probst bundle also managed to traverse midline in this manner. When no CC axons crossed, almost all of them entered the Probst bundle and eventually left it within a few hours to proceed in the ipsilateral white matter, never turning back toward midplane. Growth cones approaching midplane ipsilaterally and those that had crossed midline and entered contralateral white matter, as well as CC axons in the Probst bundle, expressed a normal range of size and complexity. These results demonstrate that the problem with callosal agenesis resides not in the cells of origin or the axons or growth cones themselves but in the substrates of axon guidance at the midsagittal plane.

Prenatal formation of the normal mouse corpus callosum: a quantitative study with carbocyanine dyes.

Judgment of abnormalities in fetal cortical axon development is more sensitive when a good standard of normal ontogeny is established. The recent availability of postmortem tract tracing methods has greatly improved the observation of axon extension and growth cone morphology in mouse fetuses, which allows much stronger statements about the timing of crucial steps in the formation of the corpus callosum in particular. The first outgrowth and crossing of midplane by axons of the corpus callosum (CC) were examined in 153 normal mouse embryos and fetuses of the hybrid cross B6D2F2/J with carbocyanine dyes applied to brains fixed by perfusion. In most brains a crystal of DiI was inserted into either frontal, parietal, temporal, or occipital cortex in one hemisphere, and a crystal of DiA was placed into a different site in the opposite hemisphere. Although dye diffusion obscured the emergence of axons, linear regression analysis revealed that the first callosal axons emerged from their cortical cells of origin at about 0.4 g body weight or 15.5 days after conception for all four sites. Subsequent axon growth rate was substantially faster for those from frontal cortex (3.2 mm/day) than occipital cortex (1.8 mm/day). Axons from frontal cortex crossed the cerebral midplane first (0.69 g, E16.3), followed by those from parietal (0.74 g), temporal (0.77 g) and occipital cortex (0.92 g, E16.9). Prior to crossing midplane, the pioneering CC axons were usually 200 microns or less in advance of the main bundle, but when they crossed midplane and encountered CC axons growing from homotopic sites in the opposite hemisphere, the pioneering axons were often 0.5 to 2.5 mm ahead of the main bundle. Growth cones were usually large and complex until they had crossed midplane and were thereafter smaller with simple and flat morphologies. The topography of axons in the CC at midplane was organized according to cortical region of origin from the very beginning, when the CC was only a small cap over the hippocampal commissure and dorsal septum. The quantitative results provide a convenient standard for normal callosal development in mice and should facilitate comparative studies.

Deficient corpus callosum in hybrids between ddN and three other abnormal mouse strains.

The mouse strain ddN from Japan was crossed with three other inbred strains prone to absence of the corpus callosum (BALB/cWah1, I/LnJ and 129/ReJ), and at least one brain with abnormally small corpus callosum was observed in offspring from each F1 hybrid cross. Data for several polymorphic protein markers revealed that the four strains are not closely related genetically. Nevertheless, they share common genetic causes of an absent corpus callosum, which helps to understand why anatomical studies of ddN and BALB/c have yielded similar results. The hippocampal commissure is abnormally small in I/LnJ mice and the anterior commissure is often malformed in BALB/c mice, but both commissures in hybrids were normal, which suggests a different genetic basis for these defects and the absent corpus callosum.

Cytochrome P-45011 beta in rat brain.

The presence of cytochrome P-45011 beta in rat brain was studied by immunohistochemistry using polyclonal rabbit antibodies raised against purified bovine adrenocortical P-45011 beta, which is involved in the steroid 11 beta-hydroxylation and glucocorticoid formation. The results showed that cytochrome P-45011 beta immunoreactivity is selectively localized to the tracts of myelinated fibers throughout the brain. The specificity of immunohistochemical stainings with P-45011 beta antibodies was established by control tests including nonimmune rabbit immunoglobulin Gs and P-45011 beta antibodies absorbed with purified antigen. Western immunoblots of homogenates from different brain areas with P-45011 beta antibodies, together with biochemical enzymatic assays for cytochrome P-45011 beta monooxygenase activity in these homogenates, confirmed the selective localization of this enzyme observed with immunohistochemistry. Cytochrome P-45011 beta and 11 beta-hydroxylase activity were detected in a homogenate from the cortical white matter (brain area rich in myelinated fibers) as in that from the rat adrenal, but were not detectable in a homogenate from the cerebral cortex (brain area poor in myelinated fibers). Furthermore, quantitation of the P-45011 beta bands on the immunoblots by the areal density revealed that the cortical white matter contains approximately 1.4 pmol of cytochrome P-45011 beta/mg of tissue protein, the value of which was about one sixth of the corresponding value estimated in the rat adrenal. This relatively high content of cytochrome P-45011 beta was also reflected in a relatively high level of 11 beta-hydroxylase activity measured in a homogenate of this brain area by biochemical enzymatic assays using [4-14C]-11-deoxycorticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative study of the development of the optic nerve in rats reared in the dark during early postnatal life.

Male rats were placed in complete darkness from birth until 30 days of age, followed in some cases by a 35 days period of rehabilitation in control lighting conditions. Groups of control and experimental animals were killed at 30 and 65 days of age by perfusion with buffered 2.5% glutaraldehyde. The right optic nerve was dissected out from each animal and processed for embedding in Epon. Quantitative stereological procedures were used to estimate the total number of both myelinated and non-myelinated optic nerve fibres and their mean minimum diameters. There were no significant differences in the total number of optic nerve fibres between dark- and light-reared rats. However dark-reared rats had myelinated and non-myelinated fibres with significantly larger fibre diameters than those in age-matched light-reared rats. The proportion of optic nerve fibres which were myelinated increased with age in both groups of animals. However by 65 days of age the degree of myelination was slightly but significantly greater in the previously dark-reared rats than in the light-reared controls. These results indicate that rats reared in complete darkness for the first 30 days of postnatal life show morphological changes in the optic nerves. The possible significance of these changes is discussed.

Studies of the immunohistochemical and biochemical localization of the cytochrome P-450scc-linked monooxygenase system in the adult rat brain.

Immunohistochemical and biochemical studies were performed on the brains of adult female and male rats using a specific antibody against bovine adrenocortical cytochrome P-450scc. The results showed that in both male and female rats, the myelinated regions of the white matter are selectively immunostained throughout the brain and that even in rats pretreated with colchicine, there is never positive staining of neuronal cell bodies and their dendrites in any brain region. Western immunoblotting with the P-450scc antibody and enzymatic assays revealed that P-450scc and cholesterol side-chain cleavage activity were present in a homogenate derived from the cortical white matter, but not detectable in that from the cerebral cortex. Furthermore, quantitation of the P-450scc protein in the immunoblots indicated that the concentration of P-450scc in the cortical white matter of both female and male rat brains is approx. 3-4 pmol per mg tissue protein. Thus it could be concluded that in the adult rat brain, P-450scc and cholesterol side-chain cleavage activity are selectively localized only in the myelinated region of the white matter.

Afferent fibers in the hypoglossal nerve: a horseradish peroxidase study in the cat.

The existence of afferent fibers in the cat hypoglossal nerve was studied by transganglionic transport of horseradish peroxidase (HRP). Injections of wheat germ agglutinin-conjugated HRP (WGA-HRP) into the hypoglossal nerve resulted in some retrograde labeling of cell bodies within the superior ganglia of the ipsilateral glossopharyngeal and vagal nerves. A few labeled cell bodies were also present ipsilaterally within the inferior ganglion of the vagal nerve and the spinal ganglion of the C1 segment. Some of the labeled glossopharyngeal and vagal fibers reached the nucleus of the solitary tract by crossing the dorsal portion of the spinal trigeminal tract. Others distributed to the spinal trigeminal nucleus pars interpolaris and to the ventrolateral part of the medial cuneate nucleus by descending through the dorsal portion of the spinal trigeminal tract. In the spinal cord these descending fibers, intermingling with labeled dorsal root fibers, distributed to laminae I, IV-V and VII-VIII of the C1 and C2 segments. Additional HRP experiments revealed that the fibers in laminae VII-VIII originate mainly from dorsal root of the C1 segment.

Ipsilateral corticocortical projections of fibers which course within Probst's longitudinal bundle seen in the brains of mice with congenital absence of the corpus callosum: a study with the horseradish peroxidase technique.

In the brains of mice with congenital absence of the corpus callosum, Probst's longitudinal bundle is always present in both cerebral hemispheres. In this research, the ipsilateral corticocortical projections by the fibers in this bundle were studied by iontophoretical injections of horseradish peroxidase into different loci in the neocortex of acallosal mouse brains. Our results show that the 'intrinsic' association fibers seen in normal mice are preserved unchanged in acallosal mice and that in these mice, a considerable portion of the fibers arising from a particular cortical region and coursing in Probst's bundle terminate ipsilaterally in the same area as do the 'intrinsic' association fibers from that region. Furthermore, careful comparison of the labeling patterns in acallosal and normal mice revealed that ipsilaterally labeled cells in acallosal mice are distributed as in similarly injected normal mice. This fact strongly suggests that in spite of the presence of ipsilateral corticocortical projections by the fibers in Probst's bundle, the overall pattern of intrahemispheric connections in the neocortex of acallosal mice is not different from that of normal mice. Thus, from the present findings it can be concluded that in congenitally acallosal mouse brains, the ipsilateral corticocortical projections of fibers which arise from a given cortical region and course within Probst's longitudinal bundle are formed only in areas that also have the 'intrinsic' association projections from that region.

Freeze-mount microautoradiographic study in the mouse hippocampus after intravenous injection of tritiated 2-deoxyglucose and glucose.

Differences in the uptake of tracers from radioactive 2-deoxyglucose ([1,2-3H] and [2,6-3H]), and glucose ([1-3H], [3-3H]) into hippocampal regions were investigated by freeze-mount microautoradiography after 45 min for 2-deoxyglucose, and after 15 and 45 min for glucose. Silver grains were assessed quantitatively by an image analyser. (1) The radioactivity (silver grains/mm2) in the stratum lacunosum-moleculare of Ammon's horn from 2-deoxyglucose autoradiograms was significantly higher than that in other hippocampal regions (P less than 0.01), while lowest in the hilus fascia dentata (P less than 0.01). (2) Autoradiograms of [1-3H]glucose and 15 min of [3-3H]glucose showed the radioactivity in the dentate molecular layer to be significantly higher than that in other regions, excepting the stratum lacunosummoleculare (P less than 0.05). (3) The 2-deoxyglucose and 45-min glucose autoradiograms showed intensely labeled perikarya of pyramidal cells in the CA3a sector. (4) Radioactivity in the dentate granular layer from the 45-min autoradiogram of [3-3H]glucose was significantly higher than that in the molecular layer (P less than 0.05). The results imply that the metabolic fate of glucose, i.e. whether it is mainly used for energy production or amino acid synthesis, depends on each structure of the hippocampus.

The fibers which course within the Probst's longitudinal bundle seen in the brain of a congenitally acallosal mouse: a study with the horseradish peroxidase technique.

The congenital absence of the corpus callosum, a brain anomaly frequently noted in humans, has been recently found to occur in some mice of the ddN strain in our laboratory. In the brains of these mice, the Probst's longitudinal bundle is always present on both cerebral hemispheres. In this research, the neuroanatomical features of the constituent fibers of this bundle were studied by iontophoretical injections of horseradish peroxidase into different loci in the neocortex of acallosal mouse brains. The results revealed that (1) certain cortical fibers of the Probst's bundle terminate in the ipsilateral neocortex; (2) some commissural fibers in the longitudinal bundle originate from the cells in the wide neocortical regions, and project to the opposite hemisphere in homotopic as well as heterotopic regions over the ventral hippocampal commissure; (3) the fibers from different cortical regions are arranged in a topographic manner within this bundle. The present data clearly demonstrate that a good portion of fibers in the Probst's longitudinal bundle seen in the congenitally acallosal mouse brain are corticocortical in nature, which indicates that this bundle has an ipsilateral neocortical association function.

The fibers which leave the Probst's longitudinal bundle seen in the brain of an acallosal mouse: a study with the horseradish peroxidase technique.

The congenital absence of the corpus callosum, a brain anomaly frequently noted in humans, has been recently found to occur in some mice of the ddN strain in our laboratory. In the brains of these mice, the Probst's longitudinal bundle is always present on both cerebral hemispheres and gives rise to some aberrant fibers toward the midline. In this research, the neuroanatomical features of these fibers were studied by iontophoretical injections of horseradish peroxidase (HRP) into the neocortex of acallosal mouse brains. The results revealed that the fibers which leave the Probst's longitudinal bundle are, at least, of 3 kinds: namely, the fibers that run out from the anterior portion of the bundle and take a U-turn ipsilaterally without crossing the midline through the septal tissue to go back again into the longitudinal bundle at the level where they have left it; the commissural fibers that leave the bundle from its middle portion and cross through a tiny bridge of tissue associated with the ventral hippocampal commissure to the opposite hemisphere; and the fibers that arise from the posterior portion of the bundle and accumulate as an anomalous fascicle below the cingulum. The observation that no labeled fibers were seen within the anterior commissure in the present HRP materials suggests that the axons from neocortex which are prevented from crossing the midline in mice with congenital absence of the corpus callosum cannot find an alternative pathway via the anterior commissure.

Differential effects of kainic-acid-induced hippocampal lesions on the acquisition of the jump-box avoidance task in mice.

This research examined the effects of kainic-acid (KA)-induced hippocampal lesions on the acquisition of a jump-box avoidance task in mice. When the hippocampal lesion was unilateral, mice with lesions in the lateral portion of the CA1 region showed deficits in acquisition while those with lesions in the CA3 region and/or the medial portion of the CA1 region did not. When the hippocampal lesion was bilateral, on the other hand, mice with lesions in the CA3 region and/or the lateral portion of the CA1 region were deficient in acquisition while those with lesions in the medial portion of the CA1 region were not. Among the mice with various KA-induced hippocampal lesions avoidance responses were not correlated with the level of open field activity, indicating that acquisition differences observed are not dependent upon, nor secondary to, levels of spontaneous activity. These results strongly suggest that there exist regional differences in the involvement of hippocampal pyramidal cell layers in acquisition of the jump-box avoidance task: both the CA3 region and the lateral portion of the CA1 region are involved in acquisition, while the medial portion of the CA1 region is not.

[Effects of 4-(o-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane) on the synthesis of acetylcholine in anemic hypoxia].

Pretreatment of mice with 4-(o-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane) protected against effects of anemic hypoxia. Befemelane delayed the loss of the righting reflex (from 17.8 +/- 1.3 to 21.9 +/- 1.2 min, p less than 0.05) and death (from 19.6 +/- 1.3 to 23.3 +/- 1.1, p less than 0.05) in mice with anemic hypoxia (induced with NaNO2). Pretreatment with bifemelane ameliorated the reduction in the synthesis of acetylcholine from labeled precursors in anemic hypoxia. Namely, it reduced the inhibition of acetylcholine synthesis from labeled choline (from 3.8 +/- 0.5 to 9.4 +/- 1.2 pmole/mg protein at 30 mg/kg, p less than 0.01), but not significant at 15 mg/kg. However it (15 mg/kg) caused a significant increase in the incorporation of [U-14C] glucose into acetylcholine compared to the value for hypoxic animals (from 5 +/- 0.5 to 9 +/- 1 dpm/mg protein, p less than 0.001). Under normal conditions, concentrations of acetylcholine and glucose in the brain were significantly increased by the 30 mg/kg of bifemelane, while the synthesis of acetylcholine from choline was significantly decreased. This reduction of synthesis might be caused by the increased acetylcholine concentrations in the brain. Fifteen mg/kg of bifemelane significantly increased the concentrations of glucose, 14C-acid soluble fraction and the synthesis of acetylcholine from [U-14C] glucose. In the in vitro experiments, cholinesterase activity was significantly inhibited by the bifemelane (1.47 microM). However, its inhibitory effects were about 1/9000 of physostigmine sulfate, which might be too weak to increase the acetylcholine concentration in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)