Flavio Keller

A membrane glycoprotein associated with the limbic system mediates the formation of the septo- hippocampal pathway in vitro

The ability of a neuronal surface glycoprotein to mediate the formation of neuronal connections was tested in an explant culture system. A monoclonal antibody against the limbic system-associated membrane protein (LAMP) was used in co-cultures containing cholinergic neurons of the septum and their hippocampal target neurons. Antibody treatment had no effect on general axon outgrowth, but significantly diminished the ability of septal cholinergic axons to invade and collateralize in the hippocampus. The results suggest that factors regulating general axon outgrowth may be distinct from those regulating the patterns of outgrowth that define the formation of neural circuits.

Development of cholinergic projections in organotypic cultures of rat septum, hippocampus and cerebellum

ChAT and AChE activity in the hippocampus originate primarily in axons from cholinergic neurons located in the medial septum. The development of cholinergic projections in organotypic explant cultures of rat septum, hippocampus, cerebellum and habenula was studied using AChE histochemistry and biochemical ChAT and AChE determinations. Hippocampal and cerebellar explants cultured without a septum contain negligible amounts of ChAT after 6 days of culture. When the hippocampus was cultured for several days in the presence of a septal explant, a massive increase in ChAT was observed in the hippocampal explant. When co-cultures were stained for AChE, AChE-positive projections were seen to grow out from the septum to invade the hippocampal explant. To a certain extent this ingrowth of septal cholinergic fibers into the hippocampus is target-specific, since cerebellar explants cultured with septum showed neither an ingrowth of AChE-containing septal fibers, nor an increase in ChAT activity. Also, habenular AChE-positive fibers fail to grow into a co-cultivated hippocampal explant. Further, in septal explants co-cultivated with hippocampal explants an increase in ChAT activity was seen as compared to septal explants cultivated alone. The possible factors responsible for the observed specificity and the increase in ChAT activity under co-culture conditions are discussed.

Selective kainic acid lesions in cultured explants of rat hippocampus

SummaryThe influence of the excitotoxin kainic acid (KA) on cultivated explants of rat hippocampus was investigated. Addition of 3 μM KA to the culture medium over 24–48 h induced a destruction of the pyramidal cells in the CA3 region, whereas the CA1 pyramidal cells and the granule cells were left undamaged. Higher concentrations (10–100 μM) of KA destroyed also the latter cell groups. The selectivity of the KA lesion at 3 μM was further indicated by the fact that the acetylcholinesterase-positive neurons in the hippocampus were not destroyed through KA administration and that the stereoisomer dihydrokainic acid was ineffective in inducing lesions. Application of tetrodotoxin did not protect the CA3 pyramidal cells from KA lesion, whereas γ-glutamylaminomethylsulphonic acid (GAMS) only offered a very small, statistically not significant, protection. Baclofen protected the cultures slightly from KA lesions but not when added together with GAMS. Possible mechanisms responsible for the KA lesions in these cultures are discussed.

Inhibition of PC12 Cell Attachment and Neurite Outgrowth by Detergent Solubilized CNS Myelin Proteins

Adhesion and neurite outgrowth of PC12 cells, as well as the spreading of 3T3 fibroblasts, were inhibited in a dose dependent manner by detergent solubilized mouse central nervous system myelin proteins as a tissue culture substrate. These inhibitory effects could be neutralized by the monoclonal antibody IN‐1 directed against the neurite growth inhibiting proteins NI‐35 and NI‐250. Separation of the detergent soluble proteins of bovine spinal cord by an anion exchange column showed that the peaks of inhibitory activity for the two cell lines overlapped, such that the PC12 cells were inhibited by a larger number of fractions comprising those inhibitory for 3T3 cells. Neurite outgrowth of PC12 cells was not influenced by the myelin associated glycoprotein, MAG.

Choline acetyltransferase in organotypic cultures of rat septum and hippocampus

Choline acetyltransferase (CAT) activity in the hippocampus originates almost exclusively in axons from neurons located in the medial septum. In the rat, the development of CAT in the hippocampus takes place during the first 3 weeks after birth. The development of CAT was studied in organotypic cultures of fetal rat septum and early postnatal rat hippocampus. In some septal explants, enzyme activity increased up to 10-fold during the first 3-4 weeks in vitro. Acetylcholinesterase (AChE) histochemistry showed the presence of AChE-positive cells and fibers in many explants. Thus it appears that septal cholinergic neurons develop CAT and AChE activity even without making contact with their target cells. However, the development of CAT was accelerated by the presence of hippocampal tissue. No CAT activity was found in the hippocampal cultures, confirming that there are few, if any, intrinsic cholinergic cell bodies in the hippocampus.

Strain-specific development of the mossy fiber system in organotypic cultures of the mouse hippocampus

The postnatal development of the hippocampus of the inbred mice strains BALB/c, C57BL/6, and DBA/2 was studied in organotypic explant cultures using the roller-tube technique. In vivo, mice exhibit strain-specific mossy fiber distribution patterns. As a main result we found, that after cultivation of 3-4 weeks, similar strain-specific patterns became apparent in vitro, as visualized by a modified Timm staining. From this experiment we can conclude that a postnatal extrinsic influence cannot be the cause of the strain-specific hippocampal features.

Organotypic culture of central histamine neurons

Organotypic cultures of histaminergic tuberomammillary (TM) neurons were grown using explants obtained from newborn rats. The cultures were examined after immunohistochemical localization of the histamine synthetic enzyme, L-histidine decarboxylase (HDC). The morphological properties of the somata, dendrites and axons of HDC-immunoreactive TM neurons in organotypic culture were virtually indistinguishable from those seen in situ. Extensive plexuses of HDC-immunopositive axons, including growth cones, were seen within the hypothalamus, the plasma surrounding the explant and co-cultured hippocampus. Organotypic cultures of TM histamine neurons, and co-cultures with their targets, provide a useful model system for studying several aspects of central histaminergic neurobiology.

Choline and acetylcholine metabolism in slice cultures of the newborn rat septum.

The incorporation of [3H]choline into acetylcholine and other choline-containing compounds was investigated in slice cultures of the septal area of newborn rats. At choline concentrations in the range of the high affinity transport mechanism (0.1-1 microM) most of the labeled choline was incorporated into phosphorylcholine, followed by lipids, acetylcholine and the free choline pool. Hemicholinium-3 (1-10 microM) lead to a marked decrease of acetylcholine synthesis, whereas choline accumulation or phosphorylcholine synthesis were not decreased. Both basal and K+-induced release of acetylcholine were Ca2+ dependent. The efflux of choline was not stimulated by high K+. When choline was absent from the incubation medium, the slices were able to liberate significant amounts of the [3H]choline previously incorporated into phospholipids, and were also able to synthesize some acetylcholine. In choline-free medium, acetylcholine synthesis was greatly enhanced by depolarization. During the period in culture, there was a decrease of the incorporation rate of [3H]choline into phosphorylcholine and an increase of the incorporation rate into acetylcholine. The tissue structure was well preserved after several weeks in culture. After staining for acetylcholinesterase, the cholinergic neurons in the cultures showed a similar morphology to that seen in situ. The main conclusions of the present study are: cholinergic neurons in slice cultures develop and behave in a manner which is very similar to their in situ counterparts; the main divergence from previous studies of choline metabolism in tissue culture is the substantial incorporation rate of choline into acetylcholine at choline concentrations in the range of the high affinity uptake mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification in macroscopic autoradiography with carbon-14—An evaluation of the method

Macroscopic autoradiograms can generally be quantified by exposing previously calibrated standard sources together with the labeled sections and measuring the silver grain density by photometric methods. Variation in section thickness is a real problem with isotopes like 14C, with beta-particles of higher energy than those of 3H. Differences in self-absorption between tissues may be neglected at low section thickness. A simple fitting procedure for non-linear relationship between radiation dose and optical density is described. The combined effect of the examined errors for the practical evaluation of autoradiograms is discussed.

Slice cultures confirm the presence of cholinergic neurons in the rat habenula

The opinion that the medial habenular nuclei contain cholinergic perikarya has recently been questioned, mainly on the basis of the difficulty to detect choline acetyltransferase (ChAT) immunoreactivity in cell bodies of these nuclei. We decided therefore to determine ChAT activity in long-term cultures of the embryonal rat habenula. In these cultures, all extrinsic fiber systems are expected to degenerate a few days after explanation. ChAT activity increased markedly during the first 3 weeks. Control cultures of adjacent thalamic tissue, which is devoid of intrinsic cholinergic neurons, displayed a 150-fold lower ChAT activity. Immunocytochemical staining with a monoclonal antibody revealed the presence of tightly packed, ChAT-containing cell bodies in the habenular slices. These two findings, together with the observation that habenular cultures show an extensive outgrowth of acetylcholinesterase-containing fibers, lead us to the conclusion that at least some cholinergic perikarya must be present in the habenular nuclei.