Molecular and cellular events in alcohol-induced muscle disease.

Alcohol consumption induces a dose-dependent noxious effect on skeletal muscle, leading to progressive functional and structural damage of myocytes, with concomitant reductions in lean body mass. Nearly half of high-dose chronic alcohol consumers develop alcoholic skeletal myopathy. The pathogenic mechanisms that lie between alcohol intake and loss of muscle tissue involve multiple pathways, making the elucidation of the disease somewhat difficult. This review discusses the recent advances in basic and clinical research on the molecular and cellular events involved in the development of alcohol-induced muscle disease. The main areas of recent research interest on this field are as follows: (i) molecular mechanisms in alcohol exposed muscle in the rat model; (ii) gene expression changes in alcohol exposed muscle; (iii) the role of trace elements and oxidative stress in alcoholic myopathy; and (iv) the role of apoptosis and preapoptotic pathways in alcoholic myopathy. These aforementioned areas are crucial in understanding the pathogenesis of this disease. For example, there is overwhelming evidence that both chronic alcohol ingestion and acute alcohol intoxication impair the rate of protein synthesis of myofibrillar proteins, in particular, under both postabsorptive and postprandial conditions. Perturbations in gene expression are contributory factors to the development of alcoholic myopathy, as ethanol-induced alterations are detected in over 400 genes and the protein profile (i.e., the proteome) of muscle is also affected. There is supportive evidence that oxidative damage is involved in the pathogenesis of alcoholic myopathy. Increased lipid peroxidation is related to muscle fibre atrophy, and reduced serum levels of some antioxidants may be related to loss of muscle mass and muscle strength. Finally, ethanol induces skeletal muscle apoptosis and increases both pro- and antiapoptotic regulatory mechanisms.

Tetrahydrobiopterin stimulates L-DOPA release from striatal tissue.

In the present study we have analyzed the effect of tetrahydrobiopterin (BH4) essential cofactor for tyrosine hydroxylase and nitric oxide synthase, on the 3,4-dihydroxyphenylalanine (L-DOPA) release from in vitro incubated striatal tissue. dl-6-methyl-5,6,7,8 tetrahydropterine (6-MPH4)-stimulated L-DOPA release in a concentration-dependent manner in the range from 25 to 100 microM. At these concentrations 6-MPH4 did not have any effect on dopamine release. Presence of Nomega-Nitro-L-arginine methyl ester (L-NAME, 200 microM), a nitric oxide synthase inhibitor, but not of alpha-methyl-rho-tyrosine (alpha-MPT, 100 microM), a tyrosine hydroxylase inhibitor, blocked L-DOPA release induced by 6-MPH4 (200 microM). Also, the addition to the incubation medium of melatonin (MEL, 300 microM), which is a scavenger of NO and other free radicals, blocked the L-DOPA release induced by 6-MPH4 (200 microM) but this effect did not occur with the addition of the peroxynitrite scavenger uric acid (UA, 300 microM). Sodium nitroprusside (SNP, 100 muM), a NO generator and l-DOPA releaser as previously reported, potentiated the L-DOPA releasing effect of 6-MPH4 (200 microM) which was also blocked by melatonin. In summary 6-MPH4 stimulates L-DOPA release from striatal fragments incubated in vitro by a mechanism which involves NO or other free radicals derived from NO but not peroxynitrite.

Electrophysiological and morphological evidence for a GABAergic nigrostriatal pathway.

The electrophysiological and neurochemical characteristics of the nondopaminergic nigrostriatal (NO-DA) cells and their functional response to the degeneration of dopaminergic nigrostriatal (DA) cells were studied. Three different criteria were used to identify NO-DA cells: (1) antidromic response to striatal stimulation with an electrophysiological behavior (firing rate, interspike interval variability, and conduction velocity) different from that of DA cells; (2) retrograde labeling after striatal injection of HRP but showing immunonegativity for DA cell markers (tyrosine hydroxylase, calretinin, calbindin-D28k, and cholecystokinin); and (3) resistance to neurotoxic effect of 6-hydroxydomine (6-OHDA). Our results showed that under normal conditions, 5-8% of nigrostriatal neurons are immunoreactive for GABA, glutamic acid decarboxylase, and parvalbumin, markers of GABAergic neurons, a percentage that reached 81-84% after 6-OHDA injection. Electrophysiologically, NO-DA cells showed a behavior similar to that found in other nigral GABAergic (nigrothalamic) cells. In addition, the 6-OHDA degeneration of DA cells induced a modification of their electrophysiological pattern similar to that found in GABAergic nigrothalamic neurons. Taken together, the present data indicate the existence of a small GABAergic nigrostriatal pathway and suggest their involvement in the pathophysiology of Parkinson's disease.

Alcoholic myopathy: lack of effect of zinc supplementation.

A chronic form of myopathy has been described in alcoholics, characterized by atrophy of type II fibers, due both to reduced protein synthesis and increased protein breakdown. Increased production of reactive oxygen species could probably play a role in increased protein breakdown. In addition, treatment with zinc might be beneficial, since it acts as a cofactor of several enzymes involved in the synthesis of proteins and antioxidants as copper-zinc-superoxidedismutase (SOD) and selenium dependent glutathione peroxidase (GPX). Based on these facts, we analyze the relative and combined effects of ethanol, protein malnutrition and treatment with zinc, 227 mg/l in form of zinc sulphate, on muscle changes in 8 groups of adult Sprague-Dawley rats fed following the Lieber-de Carli model during 5 weeks. We also study the association between muscle histological changes and the activity of GPX, SOD and lipid peroxidation products (MDA), with hormones such as IGF-1, and with trace elements involved in antioxidant systems and/or in lipid peroxidation, such as selenium, copper, zinc, and iron. We found type IIa and IIb fiber atrophy in the alcoholic animals, especially in the low-protein fed ones. This effect was mainly due to protein deficiency. Zinc played no role at all. Muscle iron increased in ethanol, low protein fed rats, either with or without zinc, and was directly related with muscle MDA levels, which in turn were related with muscle atrophy, as was also found for serum IGF-1 levels. Ethanol was the main responsible for all these changes, although protein undernutrition also played an independent role in MDA levels. A positive interaction between ethanol and protein deficiency on serum IGF-1 was also detected. These results suggest that both protein deficiency and ethanol contribute to muscle atrophy observed in alcoholized rats; this atrophy is associated with increased lipid peroxidation and muscle iron overload. Treatment with zinc sulphate confers no benefit.

Developmental changes in layer I of the human neocortex during prenatal life: a DiI-tracing and AChE and NADPH-d histochemistry study.

The development of fetal layer I (marginal zone, MZ) was studied in the human neocortex by using DiI tracing and AChE and NADPH-d histochemistry, and examining the Nissl-stained material of the Yakovlev Collection. We describe the sequential maturation of the Cajal-Retzius (CR) cells and the granule cells of the subpial granular layer (SGL), and the close relationship between both. The first CR cells appear in the primordial plexiform layer, at 6 gestational weeks (GW). After the formation of the cortical plate, they settle under the pial surface. At 13 GW, the SGL begins to form around the CR cells. The horizontal members of a polymorphic population of CR cells begin to mature at 13 GW, and the intermediate and vertical forms differentiate at 16 and 18 GW, respectively. All CR cells project into an axonal plexus in the lower third of the MZ. From 18 GW, CR cells and SGL become segregated, and the polymorphic CR cells lie now below the SGL, with which they remain connected by ascending processes. Granule cells invade the lower MZ contacting somata and processes of CR cells. The somata of vertical CR cells elongate until 23/24 GW when they show degenerative signs. After 24 GW, all polymorphic CR cells die. Granule cells degenerate after 24 GW; the SGL disappears at 28/30 GW. A population of persisting CR cells, morphologically different from the transient polymorphic forms, appears in a subpial position and survives in small numbers throughout life. Small non-CR neurons differentiate first in the lower half of layer I, thereafter also in the upper half. Histochemically, all CR cells are AChE-positive; they contain NADPH-d only transiently at 20 GW. We propose that CR cells and SGL provide a transient innervation network for the developing cortical plate at a time when the definitive fiber systems of the molecular layer are not yet established.

Compartmental organization and chemical profile of dopaminergic and GABAergic neurons in the substantia nigra of the rat.

The substantia nigra (SN) is a midbrain center composed of dopaminergic (DA-) and gamma aminobutyric acid (GABA)ergic (GABA-) neurons. In this study, we investigated the topographical relationship between both cell populations and their chemical profile by using single and double immunostaining for tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), cholecystokinin (CCK), calretinin (CR), calbindin (CB), parvalbumin (PV), and nitric oxide synthase (NOS). Our results showed that DA-cells are arranged in two bands, one rostrodorsal that corresponds to the SN pars compacta (SNC), and another caudoventral that corresponds to the SN pars reticulata (SNR) and emits cell bridges that make contact with the rostrodorsal one. In the SNR, GABA-cells are arranged in dorsoventrally elongated clusters that occupy DA-cell free regions. According to cytoarchitectural, topographical, and chemical criteria, we identified ten different cell groups: five dopaminergic ones, and five GABAergic ones. Within DA-cells, we found a cell group in the dorsomedial portion of the SNC which contains CCK, CR, and CB (dmSNC); DA-cells in the SN pars lateralis (SNL) which also contain CCK, CR and CB; DA-cells in the rostral half of the SNC containing CCK and CR (rSNC); DA-cells in the SNR and the caudal half of the SNC which only express CR (cSNC-SNR), and a DA-cell group in the lateral part of the SNC that contains none of the markers studied (lSNC). Within GABA-cells, we distinguished: large GABA-cells in the SNL that contain PV; large GABA-cells in the rostrolateral part of the SNR containing PV and NOS (rlSNR), small GABA-cells in the caudomedial part of the SNR containing PV (cmSNR), and two groups of small GABA-cells in the rostromedial portion of the SNR, one of them containing CR (rmcSNR), and the other containing NOS (rmnSNR). These data suggest that over a compartmental and complementary organization, DA- and GABA-nigral cells form a mosaic of neurochemically different subnuclei which probably differ in their physiological and pharmacological properties and vulnerability to aggression.

Nitric oxide synthase and growth-associated protein are coexpressed in primary sensory neurons after peripheral injury.

A possible role for nitric oxide in growth and regeneration of dorsal root ganglion (DRG) afferents has been explored in lesion experiments by comparing immunocytochemistry for nitric oxide synthase (NOS) with that for the growth-associated phosphoprotein 43 (GAP-43). Sciatic nerve ligature induced a progressive increase in the number of small DRG cell profiles immunopositive for NOS between 2 days and 4 weeks of survival. In the proximal stump of the ligature, NOS-immunopositive fibers began to appear 2 days after injury and their growth cones were especially evident after 7 days. NOS-immunopositive fibers appeared past (i.e., distal to) the ligature at 14 days of survival and extended for at least 6 mm in either direction 4 weeks after the lesion. Dorsal root ligature alone at L4-L5 did not result in expression of NOS in DRG neurons or in the appearance of NOS-immunopositive fibers. In rats with dorsal root ligature and nerve ligature, the results were similar to those with nerve ligature only. DRG cell profiles immunopositive for GAP-43 kept increasing from 2 days to 4 weeks after sciatic nerve ligature and included small neurons initially and large neurons subsequently. Numerous axons became GAP-43 immunopositive on both sides of the ligature from 2 days after injury. In double-labeled material, about 80% of DRG cell profiles immunopositive for NOS were also immunopositive for GAP-43. The two antigens co-occurred in peripheral nerve axons proximal to the ligature starting at about 7 days and distal to it at about 2 weeks after ligature. Thus, in response to nerve lesion, nitric oxide may not only provide an injury signal to the central nervous system but may also contribute to the growth and regeneration of injured axons.

Pyramidal and nonpyramidal callosal cells in the striate cortex of the adult rat.

The aim of this study has been to determine the neuronal types (pyramidal and nonpyramidal) within the rat's visual cortex, which project through the corpus callosum. To this end, the morphology and laminar distribution of callosal cells have been investigated by combining Diamidino Yellow retrograde tracing with intracellular injection of Lucifer Yellow in slightly fixed tissue slices. The visual callosal projection arises from pyramidal cells of diverse morphology in layers II to VIb, as well as from several modified pyramids located mainly in layers II, IV (star pyramids) and VIb (horizontal or inverted pyramids and related forms of spiny stellate cells). Our results indicate that in rats, as in other mammals, several types of nonpyramidal neurons also contribute to the contralateral projection. Bitufted cells in layers II-III and V were found to project contralaterally. Moreover, a spine-free layer V cell and a sparsely spiny multipolar neuron of layer IV were also labeled. In both stellate cells, partial axonal labeling reveals that these callosal cells display a local axonal arborization. Finally, our results of retrograde transport with Diamidino Yellow and with another sensitive retrograde tracer, the beta subunit of the cholera toxin, demonstrate for the first time that the two main neuronal types of layer I participate in the callosal projection. In layer I, several small horizontal cells of the inner half of layer I and a large subpial cell displaying long radiating dendrites were also injected. The latter cell may correspond to the Cajal-Retzius cell of the adult rat. In spite of the important differences in the organization of the visual system between rodents and cats, the callosal projection in both mammals is composed of a large variety of pyramidal cells and several nonpyramidal neurons. This high morphological diversity suggests that the callosal projection is much more physiologically complex than the extracortical efferents of the visual cortex, resembling other cortico-cortical connections. The roles that the different callosal cells may play in the processing of visual information are discussed in relation to the known functions of the corpus callosum.

The deep mesencephalic nucleus as an output center of basal ganglia: morphological and electrophysiological similarities with the substantia nigra.

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region between the superior colliculus, the substantia nigra compacta, the periaqueductal gray, and the medial geniculate body. Although some data suggest that it is involved in nociception and visceral control, its functions remain unclear. In the present study, by using morphological (combination of anterograde and retrograde tracers with immunocytochemistry and in situ hibrydization) and electrophysiological (firing activity and transynaptic response to striatal stimulation) methods, we show that a subpopulation of DMN cells shares many morphological and electrophysiological characteristics with those of the substantia nigra reticulata (SNR). These similarities include the following: 1) firing rate, firing pattern, and conduction velocity; 2) expression of GAD65, GAD67, and PV; 3) excitatory and inhibitory inputs from the striatum; and 4) projections to the ventral thalamus, superior colliculus, and pedunculopontine tegmental nucleus. Some differences were also found. In comparison with SN, DMN cells and striatal afferents are more sparsely distributed and they show conspicuous contralateral projections to the thalamus and superior colliculus. This suggests that, similarly to the SNR, the DMN acts as an output center of basal ganglia and probably facilitates the inter-hemispheric regulation of these centers.

Aggregations of granule cells in the basal forebrain (islands of Calleja): Golgi and cytoarchitectonic study in different mammals, including man.

The granule cell islands in the olfactory tubercle (islands of Calleja) and the insula magna of Calleja are present in all species examined in this study: cat, rat, mouse, rabbit, hedgehog, monkey, man, and dolphin, displaying the same basic morphology. They appear as rather undifferentiated neurons with a poorly developed dendritic tree and a short unramified axon that does not leave the island. The larger islands and the insula magna are associated with medium-sized neurons often lying in cell-sparse core regions; they probably represent the efferent component of the islands. The distribution of granule cell islands in the olfactory tubercle varies from species to species: in the cat, they are restricted to the superficial cap regions; in the hedgehog and rabbit, they lie in cap regions and in the deep polymorph layer. In the rat, they are confined mainly to the deep polymorph layer, whereas in the mouse they extend through the three layers. In most species, the lateral islands form part of the cap regions, and they may receive fibers from the lateral olfactory tract. However, the consistent relationship between dwarf cells in the cap regions and granule cells seems to be a merely topographical one. The variable location of granule cell islands indicates that they are not related to specific cell types or cell groups in the olfactory tubercle, except to the large neurons in the hilus zones, which send their dendrites into the islands. Another close and constant relationship exists between granule islands and fibers of the medial forebrain bundle. The medial islands and the insula magna are the largest and most constant aggregations of granule cells. They are present even in the dolphin, which lacks lateral islands. Medial islands and insula magna are continuous in the hedgehog and the newborn kitten and seem to belong to a medial system of granule cells that is independent from the olfactory tubercle and from olfactory fibers. Aggregations of granule cells occur also outside the olfactory tubercle and the insula magna: in the hedgehog and the rabbit, clusters lie scattered in the n. accumbens. Distribution of granule cells outside the olfactory tubercle is related to ontogenetic development: in newborn kittens, granule cells extend from the subependymal layer of the lateral ventricle, where they probably originate, to the medioventral border of the hemisphere, and also distribute throughout the n. accumbens and the ventral pallidum. Thus, the granule cell territory is initially wider, and the original distribution is maintained in some species.(ABSTRACT TRUNCATED AT 400 WORDS)

Sources of GABAergic input to the inferior colliculus of the rat.

We have studied the GABAergic projections to the inferior colliculus (IC) of the rat by combining the retrograde transport of horseradish peroxidase (HRP) and immunohistochemistry for gamma-amino butyric acid (GABA). Medium-sized (0.06-0.14 microliter) HRP injections were made in the ventral part of the central nucleus (CNIC), in the dorsal part of the CNIC, in the dorsal cortex (DCIC), and in the external cortex (ECIC) of the IC. Single HRP-labeled and double (HRP-GABA)-labeled neurons were systematically counted in all brainstem auditory nuclei. Our results revealed that the IC receives GABAergic afferent connections from ipsi- and contralateral brainstem auditory nuclei. Most of the contralateral GABAergic input originates in the IC and the dorsal nucleus of the lateral lemniscus (DNLL). The dorsal region of the IC (DCIC and dorsal part of the CNIC) receives connections mostly from its homonimous contralateral region, and the ventral region from the contralateral DNLL. The commissural GABAergic projections originate in a morphologically heterogeneous neuronal population that includes small to medium-sized round and fusiform neurons as well as large and giant neurons. Quantitatively, the ipsilateral ventral nucleus of the lateral lemniscus is the most important source of GABAergic input to the CNIC. In the superior olivary complex, a smaller number of neurons, which lie mainly in the periolivary nuclei, display double labeling. In the contralateral cochlear nuclei, only a few of the retrogradely labeled neurons were GABA immunoreactive. These findings give us more information about the role of GABA in the auditory system, indicating that inhibitory inputs from different ipsi- and contralateral, mono- and binaural auditory brainstem centers converge in the IC.

Effects of ethylcholine mustard azirinium ion (AF64A) on the choline acetyltransferase and nitric oxide synthase activities in mesopontine cholinergic neurons of the rat.

The choline analogue, ethylcholine mustard azirinium ion (AF64A), has been proposed as a selective neurotoxin that produces degeneration of central cholinergic neurons. However, the mechanisms of action and the specificity or non-specificity of this toxin are still undefined. In this study, we have investigated the effects of AF64A, in comparison with kainic acid, on cholinergic neurons of the mesopontine formation (pedunculopontine and laterodorsal tegmental nuclei), a neuronal population also expressing nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide. We used choline acetyltransferase immunohistochemistry as a marker of acetylcholine activity, and nitric oxide synthase immunohistochemistry and NADPH-diaphorase histochemistry as markers of nitric oxide synthase activity. Our results show that the injection of low doses of AF64A produces: (1) an area of cavitation in the injection site of pedunculopontine tegmental nucleus (local non-specific effect), and (2) a transient decrease in choline acetyltransferase immunoreactivity in choline acetyltransferase-nitric oxide synthase neurons in both the ipsilateral laterodorsal tegmental nucleus and the perilesional area of the pedunculopontine tegmental nucleus, while their morphology and nitric oxide synthase immunoreactivity remain unaltered (post-diffusion specific effect). These findings indicate that the loss of choline-related enzymatic activity is not necessarily associated with degeneration of cholinergic neurons, and that the recovery of choline acetyltransferase immunoreactivity may arise from neurons whose activity is diminished during the first postinjection weeks. Taking into account that AF64A is a suitable tool to develop a reversible model of neurological disorders related to cholinergic deficit, further efforts should be directed toward elimination of its local non-specific effect.

Development of neuronal types and laminar organization in the central nucleus of the inferior colliculus in the cat.

The development of neuronal morphology and laminar organization in the central nucleus of the inferior colliculus has been studied with the different Golgi methods in kittens and cats of 1 day-2 years of age. The different Golgi methods used allowed us to selectively visualize the axonal or dendritic component of the fibrodendritic laminae. The characteristic lamination of the central nucleus defined by the fiber system of the lateral lemniscus is already present at birth. The axonal component of the laminae is constituted by parallel condensations of varicose terminals, myelinated axons, and preterminal fibers, oriented from ventrolateral to dorsomedial. The laminae are smaller in the dorsolateral edge of the nucleus. Neurons are classified mainly on the basis of their dendritic trees and the axonal ramification patterns. Three main types are distinguished: spinous disk-shaped neurons, aspinous to sparsely spinous disk-shaped neurons, and large or giant multipolar neurons. Our results suggest that the basic structures of the central nucleus--neuronal types and lamination of the lemniscal fibers--are already established at birth. The different neuronal types can be distinguished from the first days of life according to the ramification pattern of dendritic and axonal arbors. The characteristics of the different cell types, such as the density and distribution of dendritic spines, and the presence of varicose dendritic branchlets, are recognizable from the second week. At the end of the first month, neurons display an adult-like morphology, although the density of dendritic spines is higher than in the adult. Our morphological data can be related to the development of response properties in the inferior colliculus.

The spiny stellate neurons in layer IV of the human auditory cortex. A Golgi study.

The spiny stellate neurons have been studied by the Golgi method in the auditory koniocortex and parakoniocortex of man. Spiny stellate cells are a consistent though not very common component of layer IV. They are not confined to specific sublayers but occur at all depths of layer IV, and also in layer IIIc. Spiny stellate cells in the auditory areas show a great variety of their dendritic arborization pattern. The presence of all intermediate forms between small pyramidal cells--which constitute the dominant cell type in layer IV and which display an extraordinary heteromorphism--and spiny stellate cells shows the close kinship between both neuronal types. The morphology and distribution of spines along the dendrites of spiny stellate neurons are similar to those of the small pyramidal cells of the same layer. The axons, which were impregnated only in their proximal portions, mostly descend, giving rise to recurrent ascending collaterals, but initially ascending axons do also occur. Spiny stellate neurons are present in the different cytoarchitectonic areas examined, and thus they are not confined to the auditory koniocortex.

Effect of intracerebroventricular injection of lipopolysaccharide on the tuberoinfundibular dopaminergic system of the rat.

Numerous studies indicate that monoaminergic systems are sensitive to both peripheral and central inflammatory stimuli, and in particular dopaminergic neurons in the nigrostriatal system degenerate after local injection of lipopolysaccharide (LPS). However, data about the response of other dopaminergic groups to local inflammation are very sparse. In this study, we have examined the effect of i.c.v. injection of LPS on the tuberoinfundibular dopaminergic (TIDA) system by using biochemical and morphological parameters. Our results show that 6 h after i.c.v. injection of LPS, in parallel to a transient and intense immunoreaction to interleukin-1beta in arcuate microglial cells, there is a decrease in tyrosine hydroxylase (TH) activity in the median eminence and in the number of TH- and TH mRNA-positive cells in the arcuate nucleus, and at 12 h, an increase of prolactin levels in serum. Posterior changes were found in the TH mRNA labeling pattern, mostly in the ventrolateral region of the arcuate nucleus, but they were not accompanied by any changes in TH activity and immunoreactivity and TH-cell count. This suggests that the TIDA system is functionally susceptible to local inflammation, but the effects are transient and do not induce neurodegeneration.

Response of GABAergic cells in the deep mesencephalic nucleus to dopaminergic cell degeneration: an electrophysiological and in situ hybridization study.

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region located between the substantia nigra compacta and the superior colliculus. It contains GABAergic cells that share striatal afferents, thalamic and collicular efferents, as well as neurochemical and electrophysiological similarities, with those of the substantia nigra reticulata. In the present paper we used electrophysiological (firing rate and firing pattern) and morphological (densitometric analysis of in situ hybridization histochemical labeling for glutamic acid decarboxylase (GAD)65 and GAD67 mRNA) techniques, to study the response of DMN GABAergic cells to the degeneration of nigral dopaminergic cells. Our results showed that unilateral dopaminergic cell loss (after injection of 6-hydroxydopamine in the medial forebrain bundle) induces a bilateral and symmetrical increase in both firing rate and GAD67 mRNA levels and a decrease in GAD65 mRNA levels. These findings support the involvement of DMN GABAergic cells in the basal ganglia modifications that follow dopaminergic cell loss, also suggesting its participation in the pathophysiology of Parkinson's disease. The symmetry of effects, together with its recently reported bilateral projections to the thalamus and superior colliculus, suggest that unlike substantia nigra reticulata, DMN is involved in the interhemispheric regulation of basal ganglia, probably keeping their functional symmetry even after asymmetric lesions.

Gender differences and the effect of different endocrine situations on the NOS expression pattern in the anterior pituitary gland.

The presence of neuronal nitric oxide synthase (nNOS) in two populations of pituitary cells, gonadotrophs (LH) and folliculostellate (FS) cells, suggests that pituitary nitric oxide (NO) is involved in the control of hormone secretion. We have used single and double immunostaining and quantitative procedures to investigate possible gender-related differences in the nNOS expression pattern in the anterior pituitary lobe and its possible alterations in different endocrine situations. Our results reveal a sexual dimorphism in the pattern of nNOS expression. In males, nNOS is mainly found in FS cells, whereas only a few LH cells express nNOS. Conversely, in females, nNOS is mainly found in LH cells. After gonadectomy, paralleling an increase in LH cell size and serum luteinizing hormone (LH) levels, there is nNOS upregulation in LH cells and nNOS downregulation in FS cells. After testoterone replacement, LH cells become nNOS-immunonegative again. In lactating rats, LH cells overexpress nNOS, but LH cell size and serum LH levels are low. This suggests that, depending on its cellular source, pituitary NO can exert either an inhibitory or a stimulatory effect on hormone secretion. When released from FS cells, NO exerts a paracrine inhibitory effect, and when released from gonadotrophs it exerts an autocrine or paracrine stimulatory effect on LH or prolactin secretion, respectively.

Histochemical and immunohistochemical detection of neurons that produce nitric oxide: effect of different fixative parameters and immunoreactivity against non-neuronal NOS antisera.

This study focused on two points concerning the histochemical and immunohistochemical detection of neurons that produce nitric oxide (NO): (a) the effect of fixation and other methodological parameters on the staining pattern of both NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, and (b) the possibility that neurons display immunoreactivity against NOS antisera obtained from non-neuronal sources. Frontal sections of rat brains, fixed with 4% paraformaldehyde according to different protocols, were processed for single and double labeling using NADPH-d histochemistry and neuronal (nNOS), macrophagic (macNOS), and endothelial (eNOS) NOS immunohistochemistry. Our results show that variations in the fixative schedule, even within standard parameters, produce qualitative and quantitative changes in NADPH-d labeling. The effect of fixative on weakly stained neurons is different from that on heavily stained neurons. In subfixed brains, a large number of NOS-positive neurons lose their NADPH-d activity, whereas NOS immunolabeling remains unaltered. This finding may be particularly interesting in morphological studies that compare NADPH-d activity under experimental conditions that can affect brain perfusion. On the other hand, many cortical and subcortical neurons show macNOS immunoreactivity, most of it colocalized with nNOS.

NADPH-d activity in the islands of Calleja: a regulatory system of blood flow to the ventral striatum/pallidum?

We have used dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry to study the anatomical relationships between the islands of Calleja (ICs), ventral striatum (VS) and ventral pallidum (VP), and the perforating branches of the anterior communicating and anterior cerebral arteries traversing the olfactory tubercle. The granule cells of the ICs are intensely positive for NADPH-d, a marker for neuronal nitric oxide synthetase (NOS), and closely surround all arterioles perfusing the VP and most of the arterioles supplying the VS. In contrast, they are not related to the arteries destined for the dorsal striatum. On the ground of the vasodilatory properties of the nitric oxide, we propose that the ICs may play a role in the regulation of blood flow to specific centres of the limbic forebrain.

Transient NADPH-diaphorase activity in motor nuclei of the foetal human brain stem.

NADPH-d activity is a marker for neurones which synthesize nitric oxide (NO). In the present paper we study the NADPH-d activity in the human brain stem between 16 and 24 gestational weeks (GW). The adult distribution of NADPH-d containing neurones is established at 24 GW. Between 19 and 21 GW, neurones of the facial, hypoglossal and ambiguus nuclei show transient NADPH-d positivity. Therefore, in the human brain stem there are two chronological patterns of neuronal nitric oxide synthase (NOS) expression, one that appears during prenatal development and persists during the whole life, and another one, present only at a precise foetal moment. The transient NADPH-d activity in motor nuclei suggests that NO is involved in early regulatory processes of the human brain development.