Jorge Pecci Saavedra

Progesterone neuroprotection in the Wobbler mouse, a genetic model of spinal cord motor neuron disease

Motor neuron degeneration characterizes the spinal cord of patients with amyotrophic lateral sclerosis and the Wobbler mouse mutant. Considering that progesterone (PROG) provides neuroprotection in experimental ischemia and injury, its potential role in neurodegeneration was studied in the murine model. Two-month-old symptomatic Wobbler mice were left untreated or received sc a 20-mg PROG implant for 15 days. Both light and electron microscopy of Wobbler mice spinal cord showed severely affected motor neurons with profuse cytoplasmic vacuolation of the endoplasmic reticulum and/or Golgi apparatus and ruptured mitochondria with damaged cristae, a profile indicative of a type II cytoplasmic form of cell death. In contrast to untreated mice, neuropathology was less severe in Wobbler mice receiving PROG; including a reduction of vacuolation and of the number of vacuolated cells and better conservation of the mitochondrial ultrastructure. In biochemical studies, we determined the mRNA for the alpha3 subunit of Na,K-ATPase, a neuronal enzyme controlling ion fluxes, neurotransmission, membrane potential, and nutrient uptake. In untreated Wobbler mice, mRNA levels in motor neurons were reduced by half compared to controls, whereas PROG treatment of Wobbler mice restored the expression of alpha3 subunit Na,K-ATPase mRNA. Therefore, PROG was able to rescue motor neurons from degeneration, based on recovery of histopathological abnormalities and of mRNA levels of the sodium pump. However, because the gene mutation in Wobbler mice is still unknown, further studies are needed to unveil the action of PROG and the mechanism of neuronal death in this genetic model of neurodegeneration.

Neuroglial interactions in a model of para-chlorophenylalanine-induced serotonin depletion

Serotonin (5HT) is involved in the development and plasticity of the CNS through the release of S-100beta, a glial trophic factor which stabilizes synapses and neuronal cytoskeleton and promotes neuronal development. S-100beta is released from glial cells after activation of glial 5HT(1A) receptors. We present in this paper the effects upon neurons and glia of a 5HT depletion induced by 14 days of treatment with para-chlorophenylalanine (PCPA) in adult rats. S-100beta, 5HT, 5HT-transporter (5HT-T) and neurofilaments (Nf-200 and Nf-68) expressions were studied by immunohistochemistry and image analysis in striatum, hippocampus, parietal and frontal cortex. Immediately after ending PCPA treatment we found increased intracellular S-100beta immunoreactivity in glial cells, reduced 5HT immunolabelling, reduced density of 5HT-T, Nf-200 and Nf-68 fibers and morphological alterations in neuronal cytoskeleton. One week after PCPA treatment S-100beta immunoreactivity decreased towards control levels, 5HT was normalized in dorsal raphe nucleus, but not in innervation areas; 5HT-T, Nf-200 and Nf-68 fiber densities increased but some neuronal cytoskeletal alterations were still present in striatum. Two weeks after PCPA treatment S-100beta had returned to control levels in most studied regions; 5HT immunoreactivity was normalized, meanwhile 5HT-T, Nf-200 and Nf-68 fiber densities increased reaching values over the control level. We propose that S-100beta could be accumulated in glial cells during the 5HT depletion period, to be released once 5HT levels have recovered. Neuronal cytoskeletal alterations and reduced fiber density may be the expression of decreased extracellular availability of S-100beta. Conversely, increased 5HT-T, Nf-200 and Nf-68 expressions, once S-100beta is normalized, may be the biological response to the growth factor release.

Ultrastructural changes in nitric oxide synthase immunoreactivity in the brain of rats subjected to perinatal asphyxia: neuroprotective effects of cold treatment

Striatal and cortical neurons containing nitric oxide synthase (NOS) were studied in adult rats subjected to different periods of perinatal asphyxia (PA) using immunohistochemistry at both light microscopy (LM) and electron microscopy (EM). Another group was subjected to PA + hypothermia to study its neuroprotective effect. Quantitative image analysis was performed on the striatum and neocortex in order to count the number of immunoreactive neurons and to compare the pattern of staining between the different groups. Six-month-old rats that suffered subsevere and severe PA demonstrated, at LM, cytomegaly of the striatal and neocortical neurons containing NOS. Control and hypothermic neurons were more weakly immunostained than PA neurons. Subsevere and severe asphyctic rats showed an important neuronal loss that was reduced by hypothermic treatment. The PA group disclosed, at EM, dense electronic bodies distributed in terminals surrounding synaptic vesicles and in dendrites. Non-NOS-containing neurons showed signs of degeneration, such as dark cytoplasm and shrunken nuclei. Surrounding the blood vessels, we observed a clear edema. The immunolabeling in hypothermic rats resembled that observed in controls. These data suggest that subsevere and severe PA induces chronic changes in the neuronal content of NOS in the striatum and neocortex. Degeneration observed in neurons surrounding cytomegalic NOS-containing cells may be due to the excess of NO in their environment. Moreover, the chronic alterations produced by PA seem to be prevented by hypothermia.

Increased nitric oxide synthase activity in a model of serotonin depletion

Serotonin (5HT) containing cell bodies are localized in mesencephalic and rhombencephalic raphe nuclei. It has been proposed that 5HT could be involved in neuronal development and plasticity. In the central nervous system, nitric oxide (NO) has been postulated as a neurotransmitter and neuromodulator, and has been implicated in neurotoxicity as well as in neuroprotection. Using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) technique, NO synthesizing neurons were described in raphe nuclei. By immunohistochemistry, nitric oxide synthase (NOS) was found colocalized with 5HT in some dorsal raphe nucleus (DRN) neurons. In a model of inhibition of 5HT synthesis produced by daily administration of parachlorophenilalanine during 14 days, we have studied the relationship between 5HT and NO systems after 5HT depletion by histochemical and immunocytochemical methods. After the treatment, we observed an important reduction of 5HT immunostaining in the DRN and enhanced NOS activity demonstrated by NADPH-d technique, especially in the dorsomedial and ventromedial subgroups. In spite of the increased NOS activity, we could not observe significant changes in the NOS-immunoreactivity in the DRN after 5HT depletion. These results could indicate that 5HT depletion is concomitant with changes in NOS activity without affecting NOS expression in the DRN.

Neural and astroglial effects of a chronic parachlorophenylalanine-induced serotonin synthesis inhibition

Serotonin (5HT) is one of the classical neurotransmitters expressed earlier in the embryonic rat brain, and it was proposed as a developmental signal in the central nervous system. In the adult brain, 5HT seems to be involved in neuronal plasticity. It was postulated that S-100 protein, a glial neurotrophic factor, could be modulated by 5HT probably through the glial 5HT1A receptors. In a model of chronic inhibition of endogenous 5HT synthesis produced by the daily administration of parachlorophenylalanine (PCPA) for 2 wk, we have studied by immunohistochemical methods and digital morphometric analysis the expression of two proteins present in rat brain astrocytes: glial fibrillary acidic protein (GFAP) and S-100 protein. The effectiveness of the PCPA treatment was tested by the use of specific anti-5HT antibodies that showed absence of 5HT fibers in 5HT innervation areas like frontal cortex and hippocampus. Different effects of PCPA treatment on serotoninergic raphe nuclei were observed: dorsal raphe nucleus (DRN) seemed to be more sensitive to the PCPAs action than ventral raphe nucleus (VRN). In DRN and in the two 5HT innervation areas studied, glial cells responded to the 5HT depletion induced by PCPA showing astrocytes with large and tortuous processes. Astrocytes from 5HT-depleted regions showed higher immunostaining for S-100 protein than controls. There was not any modification in optical density of S-100 protein immunostaining in VRN, the area less sensitive to PCPA treatment. These observations indicated that astrocytes are sensitive to the 5HT level, and in presence of low 5HT concentration in the intercellular space, astrocytes could react by synthesizing glial proteins like GFAP and S-100 protein.

Neuronal and inducible nitric oxide synthase immunoreactivity following serotonin depletion.

Serotonin (5HT) modulates the development and plasticity of its innervation areas in the central nervous system (CNS). Astrocytic 5HT(1A) receptors are involved in the plastic phenomena by releasing the astroglial-derived neurotrophic factor S-100beta. Several facts have demonstrated that nitric oxide (NO) and the nitric oxide synthase enzyme (NOS) may also be involved in this neuroglial interaction: (i) NO, S-100beta and 5HT are involved in CNS plasticity; (ii) micromolar S-100beta concentration stimulates inducible-NOS (iNOS) expression; (iii) neuronal NOS (nNOS) immunoreactive neurons are functionally and morphologically related to the serotoninergic neurons; (iv) monoamines level, including 5HT, can be modulated by NO release. We have already shown that 5HT depletion increases astroglial S-100beta immunoreactivity, induces neuronal cytoskeletal alterations and produces an astroglial reaction, while once 5HT level is recovered, a sprouting phenomenon occurs [Brain Res. 883 (2000) 1-14]. To further characterize the relationship among nNOS, iNOS and 5HT we have analyzed nNOS and iNOS expression in the CNS after 5HT depletion induced by parachlorophenylalanine (PCPA) treatment. Studies were performed immediately after ending the PCPA treatment and during a recovery period of 35 days. Areas densely innervated by 5HT fibers were studied by means of nNOS and iNOS immunoreactivity as well as NADPH diaphorase (NADPHd) staining. All parameters were quantified by computer-assisted image analysis. Increased nNOS immunoreactivity in striatum and hippocampus as well as increased NADPHd reactivity in the striatum, hippocampus and parietal cortex were found after PCPA treatment. The iNOS immunoreactivity in the corpus callosum increased 14 and 35 days after the end of PCPA treatment. These findings showed that nNOS immunoreactivity and NADPHd activity increased immediately after 5HT depletion evidencing a close functional interaction between nitrergic and serotoninergic systems. However, iNOS immunoreactivity increased when 5HT levels were normalized, which could indicate one of the biological responses to S-100beta release.

Serotoninergic Innervation of the Monkey Basal Ganglia: An Immunocytochemical, Light and Electron Microscopy Study

Normal functioning of the basal ganglia system depends upon the proper balance among several substances controlling information transfer within the constituent neuronal circuits. Most of the components of this system are considerably richer than other regions of the central nervous system in these putative neurotransmitters and/or neuromodulators. This quality makes the basal ganglia an optimal group of structures in which to study the position of the neuropil elements with respect to each other, and the alterations induced by surgical, toxicological or pharmacological manipulations. The advent of immunocytochemical techniques which allow observations at the ultrastructural level (Moriarty and Halmi, 1972; Sternberger, 1979; Pickel, 1981) has superseded, to some extent, the autoradiographic method based on the uptake of radioactive exogenous substances. Thus, major strides have been made in the localization of dopamine, gamma amino butyric acid and acetylcholine through the use of antibodies to their corresponding synthesizing enzymes (tyrosine hudroxylase, glutamic acid decarboxylase and choline aeetyltransferase), and more directly with antibodies to certain peptides such as the enkephalins and substance P (Ribak et a., 1979; Pickel et al., 1980, 1981; Kimura et al., 1980; Cuello et al., 1981, 1982; DiFiglia et al., 1982a; Somogyi et al., 1982; Bolam et al., 1983).

A new case for a presynaptic role of dendrites: An immunocytochemical study of the N. Raphé Dorsalis

The distribution of serotonin (5-HT) was determined by the application of the prembedding peroxidase-anti-peroxidase (PAP) technique in vibratome and ultrathin sections of the brain stem. The antiserum stained the neuronal groups B1 to B9. Somata, dendrites and axons of multipolar and bipolar neurons were recognized in the usual locations. The most commonly found profiles in the area of the n.raphe dorsalis were dendrites. The search for axon terminals was unsuccesful. The labeled dendrites appear in synaptic contact with unlabeled endings containing round or pleomorphic vesicles, and occasionally some large dense core vesicles. Contacts between two labeled dendrites or processes were not found. Occasionally a dendrodendritic junction between a 5-HT labeled dendrite and an unlabeled dendrite has been found. There are areas of the dendritic membrane free of synaptic junctions and free of glial insulation. Results are discussed in relation with the previously proposed presynaptic role of the dendrites in the neuronal circuitry of then. raphé dorsalis.

Serotonin innervation of enkephalin containing neurones in the rat spinal trigeminal nucleus

Light and electron microscopic immunocytochemistry was used to examine the serotonin (5-HT) innervation of the rat spinal trigeminal subnucleus caudalis. 5-HT-immunoreactive fibres form a dense plexus in lamina I and outer lamina II and synapse with the cell bodies and proximal dendrites of local neurones. Light microscopic double labelling revealed that the 5-HT axons contact enkephalin immunoreactive neurones in both laminae. The 5-fHT electron microscopic results indicate that at least some of these contacts are likely to be synapses. 5-HT axons are therefore in a position to exert a direct action on enkephalinergic interneurones and this may contribute to the analgesic actions of the 5-HT system.

Immunocytochemical study of S-100 positive glial cells in the brainstem and spinal cord of the rat embryo

A light and electronmicroscopic immunocytochemical study of the glial cells in the brainstem and spinal cord of the 18th day rat embryo was performed using an anti‐S‐100 protein antiserum. Only the radial glia and the free immature glial cells are S‐100 immunoreactive. Neurons are devoid of S‐100 immunoreactivity. The radial glia form two paramedial plates and a great number of lateral plates, uniformly spaced along the ventral portion of the brainstem from the mesencephalon to the medulla. The S‐100 protein was also detected in the perivascular membranes and glial limitans. Embryonic glia adopt a highly organized spatial pattern in the brainstem that could set the structural basis for an organized assembly of the developing nervous tissue. The use of the S‐100 protein as a glial marker in the embryonic rat brain proved to be of great value. Antibodies to S‐100 protein allow the demonstration of immature glial cells and a highly organized spatial pattern in the brainstem and spinal cord of the rat embryo.