We have made an evaluation of mutation detection techniques for their abilities to detect mosaic mutations. In this study, Sanger sequencing, single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HD), protein truncation test (PTT), and denaturating high-performance liquid chromatography (DHPLC) were compared with parallel sequencing. In total DNA samples from nine patients were included in this study. Mosaic mutations were artificially constructed from seven of these samples, which were from heterozygote mutation carriers with the mutant allele present at 50%. The mutations analyzed were as follows: c.646C>T, c.2626C>T, c.2828C>A, c.1817_1818insA, c.2788dupA, c.416_419delAAGA, and c.607delC in the APC gene. The lowest degree of mutant alleles detected with SSCP/HD and DHPLC varied between 5% and 25%, and between 15% and 50% for Sanger sequencing. Three of the mutations were analyzed with PTT with considerable variations in detection levels (from 10 to 100%). Using parallel sequencing a detection frequency down to 1% was reached, but to achieve this high sensitivity sufficient coverage was required. Two patients with natural mosaic mutations were also included in this study. These two mutations had previously been identified with Sanger sequencing (NF2 c.1026_1027delGA) and SSCP/HD (APC c.2700_2701delTC). In conclusion, all the evaluated methods are applicable for mosaic mutation screening even though combinations of the conventional methods should be used to reach an adequate sensitivity. Sanger sequencing alone is not sensitive enough to detect low mosaic levels. Parallel sequencing seems to be the ultimate choice but the possibilities to use this technique is today limited by its complexity, economics, and availability of instruments.

Previous experiments in the rat have demonstrated that field CA1 and the subiculum project to the prefrontal cortex and that this direct unilateral pathway is excitatory. In the present study, anatomical and electrophysiological approaches were used to determine the transmitter mediating the excitatory responses in prefrontal cortex neurons to low-frequency stimulation of the hippocampus. The method of selective retrograde d-[3H]aspartate labelling was used to identify putative glutamatergic and/or aspartatergic hippocampal afferent fibres to the prefrontal cortex. Unilateral microinjection of d-[3H]aspartate into the prelimbic area of the prefrontal cortex resulted in the retrograde labelling of a fraction of hippocampal neurons. Some labelled cell bodies were distributed in field CA1 and the subiculum but larger numbers of neurons were detected in the ventral and intermediary subiculum. In a second series of experiments, the excitatory transmission from the hippocampus to the prefrontal cortex was pharmacologically analysed to provide further evidence for the involvement of glutamate and/or aspartate in the pathway. All prefrontal cortex neurons responding to the stimulation of the hippocampus were activated by selective agonists of the glutamate receptor subtypes alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-d-aspartate (NMDA), and these effects were selectively antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (APV) respectively. Most of the excitatory responses of prefrontal cortex neurons to single and paired-pulse stimulation of the hippocampus were antagonized by CNQX. APV only affected the excitatory response in a few cells. These results suggest that the hippocampal input to the prefrontal cortex utilizes glutamate and/or aspartate as a transmitter. Even though prefrontal cortex neurons responding to the stimulation of the hippocampus appear to have both AMPA and NMDA receptors, low-frequency stimulation of the hippocampo-prefrontal cortex pathway activates cortical neurons mostly through AMPA receptors.

Cerebellar climbing fibres react by collateral sprouting after subtotal lesions of the inferior olive, and the newly formed branches are able to reinnervate neighbouring denervated Purkinje cells. In the present paper, we used the Phaseolus vulgaris leucoagglutinin (PHA-L) tracing technique to label the climbing fibres and study their plasticity in detail at the light microscopical level. The specific objectives were to study the time course and morphological aspects of their sprouting, to estimate their extent of growth, and to compare the newly formed terminal plexuses with normal climbing fibres. Intraperitoneal injection of 3-acetylpyridine induced degeneration of the majority of the olivary neurones, which terminate as climbing fibres in the cerebellar cortex. Regularly, small numbers of neurones survived in the inferior olive. In the cerebellar cortex scattered surviving climbing fibres were found, which were devoid of any sign of injury. Already 3 days after the lesion, surviving climbing fibres had emitted collateral branches, which elongated for some distance through the molecular layer and ended with a number of varicosities and very fine branchlets. By 7 days, it was possible to recognize new developing arbours which grew in the molecular layer with the same orientation as normal climbing fibres. At longer survival times, extensive terminal arbours had developed and double labelling experiments confirmed that they terminated around the proximal dendrites of Purkinje cells. The newly formed terminal plexuses resembled, in all essential aspects, normal climbing fibres. In addition, from 1 month onward, it was evident that every surviving climbing fibre was able to form several new terminal plexuses reinnervating a number of neighbouring Purkinje cells. The result of this process was the formation of large clusters of newly formed plexuses around the parental arborization. Quantitative estimates indicated that the domain of innervation of single surviving climbing fibres could be increased by more than six times. It is concluded that climbing fibres surviving a subtotal olivary lesion are capable of extensive sprouting, axonal growth, and formation of new terminal plexuses, which resemble normal climbing fibres. Previous electrophysiological evidence indicates that this reinnervation is functional. The high specificity with which sprouting olivary axons reinnervate the proximal Purkinje cell dendrites suggests the existence of precise interactions between the growing fibres and their target. This example of "homotypic" collateral sprouting and reinnervation may thus provide a useful model for the study of nerve-target interactions.

The distribution of monoaminergic cell bodies in the brainstem of the cat has been examined with Falck-Hillarp fluorescence histochemical technique. Quantitative determinations indicate that the cat brainstem contains about 60,300 indolaminergic (IA) cells. The majority of these (about 46,700, or 77.5%) are located within raphe nuclei. The largest number is contained within nucleus raphe dorsalis (RD), accounting for around 24,300 IA cells, while raphe pallidus (RP) holds about 8,000 raphe centralis superior (RCS) 7,400, raphe magnus (RM) 2,400, raphe obscurus (RO) 2,300, linearis intermedius (LI) 2,100, and the raphe pontis (RPo) only some 280 IA cells. The IA cells represent, however, only part of the neuronal population of raphe nuclei, which, in addition, hold varying numbers of other medium-sized and small-sized neurons. Thus, quantification in Nissl-stained material indicate that the IA cells make up about 70% of the medium-sized cells in RD, 50% in RP, 35% in RCS and RO, 25% in LI, 15% in RM, and only 10% in RPo. The substantial numbers of small-sized perikarya observed in all raphe nuclei may represent interneurons. Significant numbers of IA cells were consistently located outside the raphe nuclei at all brainstem levels. In all, these amounted to approximately 13,600, or 22.5% of the total number of IA cells. Thus, IA cells occurred in the myelinated bundles, and sometimes in reticular formation, bordering the raphe nuclei; in the ventral brainstem forming a lateral extension from the ventral raphe (RP, RM, RPo, RCS, and LI) to the position of the rubrospinal bundle; in the periventricular gray and subjacent tegmentum of dorsal pons and caudal mesencephalon; in the locus coeruleus (LC) complex; around the motor trigeminal nucleus; caudal to the red nucleus; and in the interpeduncular and interfascicular nuclei. The wide distribution of IA cells leads to a considerable mixing with catecholaminergic (CA) cell groups. Our observations on CA cell distribution are essentially in accordance with previous reports. Quantifications indicate that the LC complex contains about 9,150 CA cells, unilaterally. A previously unnoticed group of scattered CA cells was found in relation to the vestibular nuclei and extending dorsally toward the deep cerebellar nuclei.

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The olivocerebellar climbing fiber system was investigated in the rat with anterograde Phaseolus vulgaris leucoagglutinin (PHA-L) tracing. The specific objective of the study was to find morphological evidence of climbing fiber collaterals innervating the cerebellar nuclei. Small iontophoretic injections of PHA-L were placed in different parts of the inferior olivary complex, and labelled olivocerebellar fibers could be traced to their termination as climbing fibers in sagittal zones of the contralateral cerebellar cortex. Reaching the cerebellum via the restiform body, the labelled olivocerebellar axons entered the deep cerebellar white matter anterior to the cerebellar nuclei. Most of these thicker, nonterminal axons continued dorsally around the nuclei, but some ran through them. Bundles of fibers could be followed into the folial white matter toward their cortical zones of termination. Depending on which part of the olivary complex that was injected with PHA-L, labelled axons were seen to converge on different regions of the cerebellar nuclei, where dense plexuses of thin varicose terminal fibers appeared. Quantitative estimates of the innervation ranged from 1.7 to 4.3 million boutons per mm3 in the fastigial (FN), interposed, and main parts of the lateral cerebellar (LCN) nuclei, whereas the parvicellular portion of LCN demonstrated 15-20 million varicosities per mm3. Frequently, thicker olivocerebellar axons, which seemed directed toward the cerebellar cortex, were seen to send a fine collateral branch toward these areas of nuclear innervation. As controls, PHA-L was injected into the degenerated olivary complex of 3-acetylpyridine-treated rats. Neither cortical climbing fiber terminals nor nerve terminal plexuses in the nuclei appeared in these experiments. In cases with injection sites extending into the reticular formation, substantial mossy fiber labelling was present bilaterally in the cortex, but the cerebellar nuclei were devoid of labelled innervation or demonstrated only a few larger diameter fibers. The projection of the inferior olivary complex to the cerebellar nuclei was strictly topographically organized and agreed in principle with the organization described in the cat by Groenewegen et al. ('79). The caudal medial accessory olive (MAO) projected to FN, the rostral MAO to the posterior interposed nucleus (NIP), the rostral part of the dorsal accessory olive (DAO) to the anterior interposed nucleus (NIA), and the principal olive (PO) to LCN.(ABSTRACT TRUNCATED AT 400 WORDS)

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