J. Pecci Saavedra

Development of the laminated pattern of the chick tectum opticum

Several ontogenetic studies have been devoted to the structural organization of the developing tectum opticum. They disagree in many respects because they are based on histological preparations performed with differently oriented planes of section. According to our results the differences found in the literature mainly result from the fact that the developmental gradient axis undergoes remarkable positional changes with respect to both optic lobe and neural tube longitudinal anatomical axes during the early stages of development. The present work is a dynamic description of the tectum opticum lamination based on sections coinciding with the developmental gradient. Since this latter displays a curved disposition, several slightly modified planes of section had to be used to obtain a complete picture along the developmental gradient.

Immunofluorescence and glutaraldehyde fixation. A new procedure based on the Schiff-quenching method.

The immunofluorescence technique is one of the most useful methods for localizing antigens in several tissues, including the central nervous system. For immunohistochemical procedures, especially immunofluorescence methods, formaldehyde is commonly used as a fixative agent. But for some protocols, mainly in neurobiology, glutaraldehyde is necessary to recognize a number of small molecules (haptens) whose antisera have been raised using glutaraldehyde as the cross-linking agent. This is a severe limitation because glutaraldehyde gives rise to a strong autofluorescence on tissue that precludes the observation of specific immunofluorescence staining. In this paper we present a new method that allows the use of immunofluorescence techniques on glutaraldehyde-fixed tissues. The new method consists of a treatment of tissue sections with the Schiffs reagent (leucobasic fuchsin) followed by a reduction of the Schiff-dye with sodium borohydride. This reduced dye produces a quenching of glutaraldehyde-induced fluorescence on the tissue. The goal of the new method is to make possible the use of a great number of available glutaraldehyde-raised antisera for immunofluorescence techniques, a useful tool in both basic and clinical research.

Degeneration in the parvocellular portion of the lateral geniculate nucleus of the cebus monkey

SummaryThe synaptology of the Cebus lateral geniculate nucleus (LGN) was studied after varying (3–15 days) periods of survival following unilateral and bilateral eye enucleations. Part of the material was processed with the Glees and Nauta techniques for light microscopy while the rest was processed for electron microscope observation. The study revealed a variety of degenerated terminals in the parvocellular portion of the LGN and allowed the differentiation of the retinal from the extraretinal terminals. The most frequent synaptic type of retinal origin is a glomerular large central terminal (up to 20 μ long) which makes axodendritic and axoaxonic synaptic contacts with geniculate dendrites and peripheral small terminals. Simple axodendritic and axosomatic terminals of retinal and extraretinal origin were also found. The early changes affecting the geniculate neurons and astrocytes during the degenerative process are described.These results are discussed in relation to: 1) previous work on the LGN synaptology of cats and macaques; 2) the physiology of the LGN; 3) the phagocytic role of astrocytes; 4) the general problem of degeneration in the central nervous system. In addition, a correlation between the light and electron microscope observations is attempted.

2,4-dichlorophenoxyacetic acid through lactation induces astrogliosis in rat brain

Comparison of astroglial immunoreactivity in mesencephalon, cerebellum, and hippocampus of 25-d-old rat pups exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through the mothers milk was made using a quantitative immunohistochemical analysis. A glial reaction was detected at the level of serotonergic nuclei and extreme astrogliosis in the hippocampus and cerebellum. A quantitative analysis of reactive astrocytes was performed by using GFAP and S-100 protein as specific markers. The study showed a significant increase in their number, size, number of processes, and density of immunostaining in 2,4-D-exposed animals. Exposure to 2,4-dichlorophenoxyacetic acid on the first days of life modifies the astroglial cytoarchitecture in parallel to previously described neuronal changes.

Ultrastructure of cells and synapses in the parvocellular portion of the Cebus monkey lateral geniculate nucleus

SummaryThe ultrastructural study of the Cebus lateral geniculate nucleus shows the existence of a complex synaptic organization in which several synaptic types are recognized: a) axosomatic; b) simple axodendritic; c) glomeruli.Three main synaptic contacts were found in the glomeruli: 1. axodendritic 1, between central terminals and primary and secondary dendrites; 2. axodendritic 2, between peripheral terminals and dendrites and 3. axoaxonic, between central and peripheral terminals. At the axoaxonic junctions, the central are presynaptic to the peripheral terminals. Elliptical vesicles fill the peripheral terminals.These findings are discussed in relation with a) previous results of other authors in primates and cats; b) previous neurophysiological and pharmacological evidences for the existence of chemical synapses in the lateral geniculate nucleus; c) the possible functions of axoaxonic synapses.

In vivo and in vitro action of antisera against isolated nerve endings of brain cortex

Abstract Antisera against nerve endings of rabbit brain cortex (RANES) and cat brain cortex (CANES) were obtained by injection into rabbits of isolated nerve endings from the corresponding species. The titres for complement fixation test varied between 1 50 – 1 130 for RANES and 1 800 – 1 1500 for CANES and a thick precipitation band was obtained in the Outcherlony test. CANES applied to the visual cortex of the cat produced epileptiform spike-dome discharges recorded in the ECG. Both CANES and RANES in the presence of complement produced lysis and disintegration of isolated nerve endings incvitro and observed under the electron microscope. The validity of these findings, in spite of the apparent lack of specificity of the seralogical and inmuno-diffusion tests, is discussed.

Basis for the specificity of anti-5-HT-like antisera in immunocytochemistry applied to the central nervous system

SummaryRecently (Pecci Saavedra et al. 1982; Brusco et al. 1982, 1983) we have showed that the actual specificity of the rabbit anti-5-HT antibodies, is for the β-carboline derivatives of 5-HT as a result of cyclization of the lateral chain. We explained this as resulting from the use of formaldehyde which acted both as a fixative in the preparation of the tissues, and as the couplng agent in the preparation of the immunogen. Following this line we have fixed several brain stem specimens with 0.5% p-benzoquinone; 3% glutaraldehyde; 4% paraformaldehyde plus 0.25% glutaraldehyde and compare the results with tissues fixed in 4% paraformaldehyde. Glutaraldehyde and p-benzoquinone do not produce cyclization of 5-HT but immobilize monoamines in situ. As expected, the antibodies applied according to the PAP technique did not stain the neuronal bodies of the raphe system, known to contain 5-HT when 3–4% glutaraldehyde or 0.5% p-benzoquinone were used. Good staining was obtained with 4% paraformaldehyde alone or with 4% paraformaldehyde plus 0.25% glutaraldehyde.A quantitative assay of the spot test of Larsson (1981) was devised for measuring in vitro the inhibitory effects of 5-HT, of the 5-HT-BSA complex and of the cyclic derivative, 6-OH-1,2,3,4-tetrahydro-β-carboline. The results confirmed that the avidity of the antiserum is much greater for the cyclic derivatives contained in the 5-HT-BSA complex and for 6-OH-1,2,3,4-tetrahydro-β-carboline than for 5-HT. It is concluded that the formation of a new ring by the lateral chain of 5-HT is responsible of thein-vitro andin the tissue immunoreactivity of the anti-5-HT-antibodies.

Synaptic transmission in the degenerating lateral geniculate nucleus an ultrastructural and electrophysiological study

Abstract The functional and anatomical changes taking place at the main (optic) geniculate synapses after bilateral eye enucleation were studied using neurophysiological and ultrastructural techniques. Cessation of synaptic transmission in the lateral geniculate nucleus (LGN) took place between 48 and 72 hours after enucleation in two cats, and between 72 and 96 hours in five cats. During the period in which synaptic transmission was still preserved, the study of the discharges to orthodromic stimulation in the optic radiation and the analysis of the recovery cycle of the main geniculate neurons both indicated that a progressive deterioration of synaptic efficiency was taking place. The ultrastructural study of the main synapses of the LGN revealed, immediately after cessation of transmission, that the synaptic terminals were in the filamentous stage of degeneration. The gradual decrease in number of synaptic vesicles in the optic terminals of the LGN correlates well with the progressive deterioration of synaptic efficiency. It is suggested that these phenomena were due to a decrease in the amount of available transmitter for release at the arrival of each presynaptic spike. Possibly, the rate of transmitter synthesis was also altered.

CHANGES IN SEROTONIN-IMMUNOREACTIVITY IN THE DORSAL AND MEDIAN RAPHE NUCLEI OF RATS EXPOSED TO 2,4-DICHLOROPHENOXYACETIC ACID THROUGH LACTATION

Comparison of serotonin-immunoreactive (SER-IR) neurons in nucleus raphe dorsalis (NRD) and median raphe nucleus (MRN) of 25-d-old rat pups exposed to 70 mg/kg/d 2,4-dichloro-phenoxyacetic acid through mothers milk and control pups was made using an immunohistochemical analysis. Significant 2,4-D-treatment-related increase in size and density of SER-IR neuronal somata as well as in fiber length were observed. We postulate that exposure to 2,4-dichlorophenoxyacetic acid on the first day of life would modify the synthesis of 5-HT or the maturation of the brain serotonergic system.

Development of serotoninergic chick retinal neurons

Numerous neurotransmitters have been studied in detail in the developing retina. Almost all known neurotransmitters and neuromodulators were demonstrated in vertebrate retinas using formaldehyde‐induced fluorescence, uptake autoradiography or immunohistochemistry procedures. Serotoninergic (5HT) amacrine neurons were described in the inner nuclear layer (INL) of the retina with their dendrites spreading within the inner plexiform layer (IPL). The present work describes the morphological pattern of development of serotoninergic amacrine neurons with a stratified dendritic branching pattern in the chick retina from embryonic day 12 to postnatal day 7. Serotoninergic‐bipolar neurons are also described. 5HT‐amacrine neurons have round or pear‐shaped somata and primary dendritic trees oriented toward the IPL that runs through the INL, showing several varicosities. Secondary dendrites then go through the INL, without any collateral branch. At the outer and inner margin of the IPL the primary and secondary dendrites originate an outer and an inner serotoninergic network, respectively. When the primary dendritic tree reaches the IPL it deflects laterally in sublayer 1—the outer serotoninergic network. Tertiary branches then arise from the secondary dendrite and deflect in the innermost sublayer of the IPL— the inner serotoninergic network. The final pattern of branching of 5HT amacrine cells was present at embryonic day 14 and was completely developed at hatching. Serotoninergic (5HT) bipolar neurons were also present in the INL at hatching. They are weakly immunoreactive and are probably a subset of bipolar cells that accumulate serotonin from the intersynaptic cleft and are not “true” 5HT neurons.