Immunohistochemical estimation of brain choline acetyltransferase and somatostatin related to the impairment of avoidance learning induced by thiamine deficiency.

We have found that thiamine-deficient (TD) rats show significant impairment of avoidance learning on the 25th day after the start of TD diet, as measured by passive-avoidance task. Administration of physostigmine (0.1 mg/kg, i.p.) from the 14th day after the start of TD diet improved the impairment of avoidance learning to the pair-fed (PF) control level by the 25th day. However, the recovery effect of physostigmine did not occur on the 25th day when the treatment was begun on the 21st day. To ascertain the correlation between the cholinergic neuronal function in rat brain and the avoidance learning impairment induced by TD, the immunohistochemical distribution of brain choline acetyltransferase (ChAT) was determined by fluorescence intensity using two-dimensional microphotometry. The intensity of the ChAT fluorescence started to decrease in the cortex and hippocampus on the 14th day and showed a marked decrease in the cortex, hippocampus and thalamus on the 25th day of TD feeding in comparison with PF controls. The intensity of the somatostatin (SST) fluorescence was unchanged on the 14th day of TD feeding, but on the 25th day, SST was significantly decreased in comparison with PF controls. Furthermore, physostigmine treatment from 14th day after the start of TD diet reversed SST fluorescence intensity to the control level by the 25th day. These results suggest that the impairment of avoidance learning induced by TD may involve not only cholinergic but also somatostatinergic systems.

Immunocytochemical characterization of quisqualic acid- and N-methyl-D-aspartate-induced excitotoxicity in the retina of chicks.

A single, large dose of N-methyl-D-aspartate (NMDA) or quisqualic acid (QA) injected into the chick eye has been shown previously to destroy many retinal amacrine cells and to induce excessive ocular growth accompanied by myopia. The purpose of this study was to identify distinct populations of retinal cells, particularly those believed to be involved in regulating ocular growth, that are sensitive to NMDA or QA. Two pmol of NMDA or 0.2 micromol of QA were injected unilaterally into eyes of 7-day-old chicks, and retinas were prepared for observation 1, 3, or 7 days later. Retinal neurons were identified by using immunocytochemistry, and cells containing fragmented DNA were identified by 3'-nick-end labelling in frozen sections. NMDA and QA destroyed many amacrine cells, including those immunoreactive for vasoactive intestinal polypeptide, Met-enkephalin, and choline acetyltransferase, but they had little effect upon tyrosine hydroxylase-immunoreactive cells. Other cells affected by both QA and NMDA included those immunoreactive for glutamic acid decarboxylase, gamma-aminobutyric acid, parvalbumin, serotonin, and aminohydroxy methylisoxazole propionic acid (AMPA) receptor subunits GluR1 and GluR2/3. Cells largely unaffected by QA or NMDA included bipolar cells immunoreactive for protein kinase C (alpha and beta isoforms) and amacrine cells immunoreactive for glucagon. DNA fragmentation was detected maximally in many amacrine cells and in some bipolar cells 1 day after exposure to QA or NMDA. We propose that excitotoxicity caused by QA and NMDA induces apoptosis in specific populations of amacrine cells and that these actions are responsible for the ocular growth-specific effects of QA and NMDA reported elsewhere.

Localization of choline acetyltransferase-expressing neurons in the larval visual system of Drosophila melanogaster.

Choline acetyltransferase (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4 kb of 5' flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig's organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig's nerve and a neuron close to the insertion site of the optic stalk. This neuron's axon ran in parallel with Bolwig's nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig's organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.

Influence of tongue myoblasts on rat dissociated hypoglossal motoneurons in culture.

Hypoglossal motoneurons of 1-2 day-old newborn rats were retrogradely labelled following an injection of fluorescent latex microspheres and carbocyanines. Motoneurons were identified among the cell population of the hypoglossal nuclei during the dissociation and culture procedures. DiI labelled motoneurons could be maintained in culture on poly-L-lysine coating and Dulbecco Minimum Essential Medium with Ham F12 complement, supplemented with additives and 3% fetal calf serum. Neuronal survival as well as extension of neurites, identified by their content in DiI or by the presence of choline acetyltransferase immunoreactivity, was increased in the presence of myoblastic satellite cells originating from tongue muscular explants. Co-cultures of dissociated hypoglossal cells with tongue myoblasts revealed the presence, after 10-15 days in culture, of structures morphologically similar to neuromuscular junctions. Such re-innervated muscular fibres exhibited muscular contractions which were blocked by curare and augmented by glutamate applications, demonstrating the functionality of the observed re-innervations.

Differential activation and survival of basal forebrain neurons following infusions of excitatory amino acids: studies with the immediate early gene c-fos.

These experiments investigated, by studying patterns of c-fos expression, the distribution of neurons activated or destroyed by the infusion into the basal forebrain of various excitatory amino acids at toxic and subtoxic doses. The results of experiment 1 showed that N-methyl-D-aspartic acid (NMDA), quisqualic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) differentially increased the expression of c-fos in magnocellular cholinergic nucleus basalis, dorsal and ventral pallidal neurons. AMPA was the most, and NMDA the least, effective in inducing FOS in nucleus basalis magnocellularis (nbM) neurons, with quisqualic acid having an intermediate effect, whereas the reverse was true in terms of the induction of FOS in pallidal neurons. In experiment 2, it was demonstrated that, in animals with ibotenic acid-induced lesions of the basal forebrain that were targetted on the nbM, virtually no pallidal neurons could be visualized that expressed FOS following AMPA-induced excitation of the dorsal and ventral striatum. By contrast, in animals with AMPA-induced lesions of the nbM, excitation of the striatum was followed by the expression of FOS in many dorsal and ventral pallidal neurons. Thus, infusions of AMPA into the basal forebrain appears preferentially to activate or destroy, depending on the concentration infused, cholinergic nbM neurons, whereas ibotenic acid or NMDA preferentially destroys or activates neurons of the dorsal and ventral pallidum. These results provide novel and complementary information regarding the organization of the basal forebrain and allow a clearer understanding of the different behavioural consequences of NMDA agonist-induced and non-NMDA agonist-induced excito-toxic lesions of this area.

Cholinergic neurons in the pedunculopontine tegmental nucleus are involved in the mediation of prepulse inhibition of the acoustic startle response in the rat.

The amplitude of the acoustic startle response (ASR) is markedly reduced when the startle eliciting pulse is preceded by a weak, non-startling stimulus at an appropriate lead time, usually about 100 ms. This phenomenon is termed prepulse inhibition (PPI) and has received considerable attention in recent years as a model of sensorimotor gating. We report here on experiments which were undertaken in order to investigate some of the neural mechanisms of PPI. We focused on the characterization of the cholinergic innervation of the pontine reticular nucleus, caudal part (PnC), an obligatory relay station in the primary startle pathway. The combination of retrograde tracing with choline acetyltransferase-immunocytochemistry revealed a cholinergic projection from the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) to the PnC. Extracellular recording from single PnC units, combined with microiontophoretic application of the acetylcholine (ACh) agonists acetyl-beta-methylcholine (AMCH) and carbachol revealed that ACh inhibits the majority of acoustically responsive PnC neurons. Neurotoxic lesions of the cholinergic neurons of the PPTg significantly reduced PPI without affecting the ASR amplitude in the absence of prepulses. No effect on long-term habituation of the ASR was observed. The present data indicate that the pathway mediating PPI impinges upon the primary acoustic startle circuit through an inhibitory cholinergic projection from the PPTg to the PnC.

Accumulation of circulating endogenous and exogenous immunoglobulins by hypothalamic magnocellular neurons.

Rat monoclonal antibodies, used in immunocytochemistry of normal rat brain, result in a granular reaction product within neurons innervating areas lacking a blood-brain barrier. Immunocytochemical characterization shows that the staining is independent of the primary antibody and exclusively dependent on the presence of anti-rat immunoglobulin. This granular staining could be selectively eliminated by pre-adsorption of the anti-rat immunoglobulin with purified rat immunoglobulin or disruption of microtubule retrograde transport systems by intraventricular injection of colchicine. A dependence on retrograde transport and complete independence from local synthesis was further substantiated by the rapid uptake and accumulation of intravenously administered rabbit or rat [125I]immunoglobulins by the supraoptic-neurohypophysial system. Immunoelectron microscopy was used to identify the endogenous rat immunoglobulin within lysosome-like organelles in the cytoplasm of magnocellular neuroendocrine cells. The uptake and incorporation of plasma macromolecules into the lysosomal system of magnocellular and other neurons projecting to regions with a weak blood-brain barrier may represent a novel mode of blood-central nervous system interactions.