[Use of levosimendan in children]. / Utilisation du levosimendan chez l'enfant.

Levosimendan is a calcium-sensitizing drug with positive inotropic properties. As an inodilator, this molecule also has a vasodilation effect. While its efficacy has been demonstrated in the adult in the context of cardiac surgery, its pediatric use is still not widespread. Many studies have shown its safety of use in children, including in the newborn. Across the world, a growing number of teams use levosimendan to treat both acute and chronic heart failure. Through a review of the literature, we describe its pharmacodynamic effects, its current applications, and its perspectives of use in children.

Antisera against catecholamines: specificity studies and physicochemical data for anti-dopamine and anti-p-tyramine antibodies.

Antibodies against dopamine and p-tyramine were raised in rabbits. The two catecholamines were conjugated to albumin by glutaraldehyde. The specificity of the antibodies was established by equilibrium dialysis competition experiments using an immunoreactive tritiated derivative synthesized by coupling dopamine or p-tyramine to N-alpha-acetyl-L-lysine N-methylamide with glutaraldehyde. Hence, these radiolabelled ligands mimicked the antigenic determinant of conjugated immunogens. A comparison of the data obtained showed the high specificity of each antiserum for its hapten coupled by glutaraldehyde. The anti-dopamine antibodies recognized dopamine-glutaraldehyde but not p-tyramine-glutaraldehyde. The opposite occurred for the anti-p-tyramine antibodies. A slight modification of the molecular structure provided the opportunity for a specific response against that molecule. But this difference was more important when related to the hapten region where the antibody affinity was maximal. The cross-reactivity was observed to be more important dopamine and p-tyramine than between dopamine and noradrenaline on the one hand and between p-tyramine and dopamine than p-tyramine and octopamine on the other hand.

Ductal closure and near-infrared spectroscopy for regional oxygenation monitoring in ductus-dependent congenital heart disease.

In ductus-dependent congenital heart disease, preserving the blood flow through the ductus arteriosus (DA) is vital before surgery. We present the cases of three full-term neonates with ductus-dependent congenital heart disease for whom near-infrared spectroscopy (NIRS) monitoring was performed. We recorded cyclical drops in regional oxygen saturation, both cerebral and renal, that corresponded to constrictions of the DA. These findings appeared either simultaneously or previous to SpO2 drops and were corrected by prostaglandin infusion. Through these cases, we assume that cyclical constrictions of ductal cells participate in the DA closure process in its early phase.

Primary structure and expression of a naturally truncated human P2X ATP receptor subunit from brain and immune system.

A novel member of the ionotropic ATP receptor gene family has been identified in human brain. This 422 amino acid long P2X receptor subunit has 62% sequence identity with rat P2X5. Several characteristic motifs of ATP-gated channels are present in its primary structure, but this P2X5-related subunit displays a single transmembrane domain. Heterologous expression of chimeric subunits containing the C-terminal domain of rat P2X5 leads to the formation of desensitizing functional ATP-gated channels in Xenopus oocytes. The developmentally regulated mRNA, found in two splicing variant forms, is expressed at high levels in brain and immune system.

Distribution of GABA-immunoreactive neurons in the basal ganglia of the squirrel monkey (Saimiri sciureus).

The distribution of GABA-immunoreactive neurons was visualized in the basal ganglia of the squirrel monkey (Saimiri sciureus), by using a highly specific antiserum raised against GABA-glutaraldehyde-lysyl-protein conjugate and revealed by the indirect peroxidase-antiperoxidase immunohistochemical method. In the dorsal striatum, GABA-immunoreactive nerve cell bodies were small to medium in size (sectional area ranging from 90 to 125 microns2), but some larger ones (500-600 microns2) were also found. These cells displayed no obvious clustering but were significantly more numerous in the caudate nucleus than in the putamen; their number was also markedly greater at caudal than at rostral striatal levels. A moderate number of evenly distributed positive axon terminals were visible in both the caudate nucleus and the putamen. In the ventral striatum, GABA-immunoreactive nerve cell bodies and axon terminals were seen in fair number within the nucleus accumbens and in the deep layers of the olfactory tubercle. Many positive terminals but no somata were found in the islands of Calleja. In the globus pallidus, virtually all nerve cell bodies were GABA-immunoreactive and the neuropil exhibited a multitude of positive terminals. In the substantia innominata, clusters of small, globular GABA-immunoreactive somata were scattered among aggregates of larger, nonimmunoreactive neurons belonging to the nucleus basalis, and the whole region showed a low to moderate number of evenly spread GABA-positive terminals. In the subthalamic nucleus, nerve cell bodies were generally surrounded by several GABA-positive terminals but were not themselves immunoreactive. The substantia nigra showed many GABA-immunoreactive somata, which predominated in the pars lateralis and diminished progressively in number along the lateromedial axis of the pars reticulata. These cells formed a rather pleomorphic group comprising round, fusiform, or polygonal elements of relatively large size (sectional area ranging from 200 to 800 microns2). In the pars compacta and ventral tegmental area, a few GABA-immunoreactive neurons of small size were dispersed among larger, unreactive neurons. In both pars lateralis and pars reticulata of the substantia nigra, the number of GABA-positive terminals was high and their distribution was rather uniform; a smaller number were visible in the pars compacta of the substantia nigra and in the ventral tegmental area. The present results demonstrate that GABA-containing neurons are widely and heterogeneously distributed in the various components of the squirrel monkey's basal ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural relationships of serotonin axon terminals in the cerebral cortex of the adult rat.

PAP immunocytochemistry with an antiserum against serotonin (5-HT)-glutaraldehyde-protein conjugate (kindly donated by M. Geffard) was used to analyze the ultrastructural relationships of 5-HT axon terminals (varicosities) in the frontal (Fr1-Fr2), parietal (Par1), and occipital (Oc1M-Oc2) cortex of adult rats. One hundred-forty-five immunostained varicosities from Fr1-Fr2 (54 from layers I-II; 91 from layer VI) and 97 each from the upper layers (I-II) of Par1 and OcM1-Oc2 were examined in groups of serial thin sections (mean number of sections in series: 3.2 to 7.3). These terminals were of comparable shape and size in the 4 cortical sectors examined, and averaged 0.66 +/- 0.2 microns in mean diameter. The proportion of varicosities engaged in synaptic contact was evaluated by linear transformation of the relationship between the frequency of observed synaptic junctions and the number of thin sections available for examination. Reliability of the sampling was evidenced by a high coefficient of correlation (r greater than 0.95) in each cortical sector. The synaptic incidence extrapolated for whole varicosities ranged from 28% (layer VI of Fr1-Fr2) to 46% (Par1), without statistically significant differences between the 4 sectors examined. The interregional mean could thus be evaluated at 38%. The synaptic 5-HT terminals always made asymmetrical junctions, which were exclusively found on dendritic spines and shafts, and appeared more frequent on spines than shafts in the deep frontal and the upper occipital cortex. In all 4 sectors, dendritic shafts and spines and other axonal varicosities were frequently encountered in the immediate microenvironment of the immunostained varicosities. It is concluded that the cortical 5-HT innervation is predominantly nonjunctional throughout the neocortex of the adult rat, which reinforces earlier views of a highly divergent afferent system with particular functional properties and perhaps capable of widespread, global and/or sustained influences in this part of the brain.

Distribution and morphological characteristics of dopamine-immunoreactive neurons in the midbrain of the squirrel monkey (Saimiri sciureus).

The distribution and morphological characteristics of dopamine (DA) neurons in the midbrain of the squirrel monkey (Saimiri sciureus) were investigated by peroxidase-antiperoxidase (PAP) immunohistochemistry with a highly specific antiserum raised against DA-glutaraldehyde-lysyl-protein conjugate (donated by M. Geffard). Four contiguous areas contained DA-immunostained nerve cell bodies: (1) the substantia nigra, pars compacta (SNc), (2) the ventral tegmental area (VTA), (3) the retrorubral area (RRA), and (4) the periaqueductal gray (PAG). The SNc composed the vast majority of DA-immunostained neurons. Most of these neurons were relatively large (mean diameters: 35 x 15 micron) and varied in shape from fusiform to polygonal, but a few smaller (16 x 10.5 micron) globular cells were dispersed among them. The caudal two-thirds of the SNc was particularly rich in DA somata. Rostrally, these DA cells formed several distinct columns impinging deeply upon the underlying pars reticulata. Large oval sectors mostly devoid of immunoreactivity were delineated by these trabeculae. The long dendritic processes of DA neurons in the SNc were generally oriented in prominent dorsoventral bundles the ventralmost portion of which arborized diffusely along the dorsal surface of the cerebral peduncle. In the VTA, the DA neurons were regrouped in a triangular zone located dorsal to the interpeduncular nucleus, medial to the substantia nigra and ventral to the oculomotor nucleus. These DA cells were of medium size (19 x 10.5 micron), globular or fusiform, and usually showed one or two thick primary dendrites oriented dorsoventrally. The DA cells in the RRA lay in continuity with the most caudal DA-containing elements of the substantia nigra but could be distinguished by their smaller size (26 x 12 micron), shorter and more profusely branched dendrites, and darker immunostaining. These DA neurons were characteristically scattered among and medial to the fibers of the medial lemniscus, and a few could be observed as far caudally as the pedunculopontine nucleus. In the PAG, DA-immunostained neurons were seen in the rostral half of the mesencephalic central gray and predominated in its ventral half. These cells were of medium size (22.5 x 10 micron) and some of them were found in proximity to the ventricular lining. At caudal levels, the DA-positive cells in the PAG did not intermingle with dorsal raphe neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective modulation of heteromeric ASIC proton-gated channels by neuropeptide FF.

Proton-gated channels of the acid-sensing ion channel (ASIC) family are candidates for mediating the fast ionotropic transduction of extracellular acidification in neurons. ASIC subunits can assemble in homomeric and heteromeric channels with specific biophysical and pharmacological properties. Using heterologous expression of ASIC subunits in Xenopus oocytes, we show here that the biphasic response of heteromeric rat and human ASIC2A+3 subtypes to low pH is selectively modulated by the neuropeptide FF (NPFF) and by the related peptide FMRFamide. We recorded both a dramatic potentiation (up to 275%) of the amplitude of acid-gated human ASIC2A+3 maximal currents and a change of desensitization kinetics in the presence of NPFF (EC(50)=2 microM) leading to a slowly inactivating phenotype. These modulatory effects were not observed with the corresponding homomeric human ASIC2A or ASIC3 receptor subtypes. Moreover, the sensitivity of ASIC2A+3 receptors to extracellular protons was increased in the presence of NPFF (DeltapH(50)=+0.5). Our data therefore suggest that the direct sensitization of heteromeric proton-gated channels by endogenous neuropeptides might play a role in the neuronal response to noxious acidosis in sensory and central pathways.

Distribution of GABA-immunoreactive neurons in the thalamus of the squirrel monkey (Saimiri sciureus).

A light microscopic study of the cellular localization of GABA in the thalamus of the squirrel monkey (Saimiri sciureus) was undertaken by means of the indirect peroxidase-antiperoxidase method using a highly purified antiserum directed against GABA-glutaraldehyde-lysyl-protein conjugate. GABA-immunoreactive cell bodies and axon terminals were visualized in all thalamic nuclei in the squirrel monkey but their relative density varied from one nucleus to the other. At the level of the anterior nuclear group, GABA-positive cells and terminals abounded in the anterodorsal nucleus but were much less numerous in the anteromedial and anteroventral nuclei. In the nuclei of the ventral group, GABA-immunoreactive cells were found to be smaller and less numerous than nonimmunoreactive neurons. In the ventral anterior nucleus, GABA-positive neuronal profiles formed typical clusters, whereas they were more uniformly distributed in the posterior nuclei of the ventral group. In the intralaminar nuclei, GABA-immunoreactive cells and terminals abounded in the dorsal portion of the paracentral and centrolateral nuclei, whereas more caudally, GABA-positive terminals pervaded the entire parafascicular nucleus. In the mediodorsal nucleus, GABA-positive cell bodies and axon terminals formed typical clusters of various sizes scattered within the lateral parvocellular portion of the nucleus, while GABA-immunoreactive neuronal profiles were less numerous and more uniformly distributed in the medial portion of this structure. In the nuclei of the posterior group, GABA-immunoreactive neuronal profiles were uniformly distributed except in the pulvinar where they abounded in the inferior and oral parts but were scarce in the medial part. In the dorsal lateral geniculate nucleus, the magnocellular layers received the most massive GABA-positive innervation and contained the largest number of GABA-immunoreactive cell bodies. In the ventral lateral geniculate nucleus, GABA-positive cells occurred only ventrolaterally while GABA-immunoreactive terminals pervaded the entire structure. In the medial geniculate nucleus, GABA-immunoreactive cell bodies and terminals abounded particularly within the ventromedial third of the structure. In the habenula, a few GABA-immunoreactive cell bodies and numerous GABA-positive terminals were scattered throughout the lateral habenular nucleus, whereas only a few GABA-immunoreactive terminals surrounded the closely packed unreactive cells in the medial habenular nucleus. In contrast to other thalamic nuclei all neurons in the reticular nucleus displayed GABA immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural features of the serotonin innervation in adult rat hippocampus: an immunocytochemical description in single and serial thin sections.

This study was aimed at characterizing the fine-structural features of the normal serotonin (5-HT) innervation in adult rat hippocampus, by means of electron microscopic immunocytochemistry with a polyclonal antiserum against 5-HT-glutaraldehyde-protein conjugate (donated by Michel Geffard, Bordeaux). Two hippocampal sectors were examined, at mid-level along the septo-temporal axis: CA3-a of Ammon's horn and crest of the dentate gyrus (DG-c). A large number of axonal varicosities (terminals) were sampled in single ultrathin sections, to achieve a statistically significant comparison of their size and of their relative frequency of synaptic specialization, junctional targets and juxtaposed elements, between the oriens and the radiatum layer of CA3-a, and the molecular and the polymorph layer of DG-c. In both CA3-a layers, the microenvironment of the immunostained terminals was also compared to that of a population of unlabeled varicosities randomly selected from the same micrographs. Moreover, 57 varicosities from the oriens and the radiatum layer of CA3-a were visualized in a long series of thin sections, allowing for their examination from end to end in 43 instances. As measured in single sections, hippocampal 5-HT varicosities were of comparable diameter (0.57 microns on the average) in the two anatomical sectors and four neuropil layers examined. As extrapolated stereologically to whole varicosities, the proportion making a synaptic membrane specialization (synaptic incidence) ranged from 18 to 33% (average of 24%), without statistically significant differences between the two sectors and four layers. The synaptic incidence determined directly from serial sections of CA3-a (18%) was nearly identical to that extrapolated from single sections (18.1% in the oriens and 19.5% in the radiatum layer). In both CA3-a and DG-c, the 5-HT varicosities showing a junctional complex were slightly larger than their non-junctional counterparts. In CA3-a, only dendritic shafts were targeted by synaptic 5-HT varicosities, whereas in DG-c there were also a few axo-spinous synapses. The microenvironment of CA3-a 5-HT varicosities differed markedly from that of randomly selected unlabeled varicosities, due to its much lower frequency of synaptic targets and higher frequency of juxtaposed axonal varicosities, at least in the radiatum layer. In all four layers examined, other axonal varicosities were indeed the most frequently encountered neuronal element in the immediate vicinity of immunostained 5-HT varicosities. Neurites and dendritic shafts were also common, but dendritic spines (4%) were relatively infrequent.(ABSTRACT TRUNCATED AT 400 WORDS)

Noradrenaline axon terminals in adult rat neocortex: an immunocytochemical analysis in serial thin sections.

Peroxidase-antiperoxidase electron microscope immunocytochemistry with an antiserum against noradrenaline-glutaraldehyde-protein conjugate was used to identify cortical noradrenaline terminals (axonal varicosities) from the upper layers of the frontal, parietal and occipital cortex in adult rat. A large number of immunostained varicosities were examined in serial thin sections, and compared with a control population of randomly chosen unlabeled terminals from the same sections. Both groups of varicosities were measured and scrutinized for the presence of a junctional complex indicative of synaptic specialization. Cellular elements juxtaposed to the membrane of both types of varicosities were also identified and counted. Noradrenaline varicosities in all three cortical regions averaged 0.65 microns in diameter. In contrast to their unlabeled counterparts, these profiles rarely showed a membrane differentiation characteristic of a synaptic contact (junctional complex). The rare junctional complexes formed by cortical noradrenaline varicosities were invariably symmetrical and almost always found on dendritic shafts. The microenvironment of noradrenaline varicosities also differed, exhibiting a greater number of apposed axonal varicosities and a smaller number of dendritic spines than that of the random population. The proportion of noradrenaline varicosities making a synaptic contact (synaptic incidence) was determined by plotting the incidence of visible junctions as a function of the number of thin sections available for examination. As extrapolated for whole varicosities after linear transformation (double reciprocal plot), this proportion was 17% or 26% depending on the stringency of the criteria used in identifying the junctional complex. The same analysis provided a figure of 98% for the control population. The present study largely confirmed our initial radioautographic characterization of the cortical noradrenaline innervation as a mostly non-junctional system, and also indicated that these varicosities are set in a particular microenvironment. These new data further support the eventuality of a diffuse release of cortical noradrenaline in the extracellular space, compatible with both its neuromodulatory role and multiplicity of actions on diverse cellular targets in the cerebral cortex. The functions assigned to the coeruleocortical noradrenaline system must therefore be viewed as the product of a widespread and ubiquitously distributed neuronal organization characterized by loose intercellular relationships. This system might be capable of selectivity and specificity of action, however, owing to the distribution of its receptors, and in view of intrinsically or extrinsically driven control mechanisms triggered by the release of its own or other transmitters and which may also involve target-initiated feedback mechanisms.

Ultrastructural immunocytochemistry of gamma-aminobutyrate in the cerebral and cerebellar cortex of the rat.

gamma-Aminobutyrate containing structures in the cerebral and cerebellar cortex of the rat were visualized by an immunocytochemical method using glutaraldehyde fixation and an antiserum developed against a gamma-aminobutyrate-glutaraldehyde-protein conjugate. Labelled elements (perikarya and cell processes) were observed to be distributed throughout the layers of the cerebral cortex in a pattern similar to that described using glutamate decarboxylase immunocytochemistry. The morphological features of many immunoreactive cell bodies were typical of stellate neurons. In the cerebellar cortex, Purkinje, basket, Golgi and stellate, cell bodies were found to be immunoreactive along with numerous labelled neuronal processes. At the ultrastructural level, the labelled processes in both areas corresponded to immunoreactive dendrites and fibres. Labelled synaptic boutons, generally of the symmetrical type, could also be seen in contact with positive or negative cell bodies and dendrites. In the cerebellum, glomeruli could be clearly identified including mossy fibres surrounded by unlabelled dendrites in contact with immunoreactive terminals. At the subcellular level in both brain regions, the areas occupied by the Golgi apparatus were never labelled, although the nuclei had varied reactions. The strong glutaraldehyde fixation that limits the diffusion of gamma-aminobutyrate limits also antibody diffusion. However, this fixation is compatible with a good morphological preservation and should enable immunocytochemistry studies to be compared to other methods such as autoradiography.

Sensory presynaptic and widespread somatodendritic immunolocalization of central ionotropic P2X ATP receptors.

Recent evidence suggests that extracellular ATP plays a neurotransmitter role in the central nervous system. Its fast ionotropic effects are exerted through a family of P2X ATP-gated channels expressed in brain and spinal cord. To determine the physiological significance of central ATP receptors, we have investigated the localization of a major neuronal P2X receptor at the cellular and subcellular levels using affinity-purified antibodies directed against the C-terminal domain of P2X4 subunit. Subunit-specific anti-P2X4 antibodies detected a single band of 57,000 +/- 3000 mol. wt in transfected HEK-293 cells and in homogenates from adult rat brain. The strongest expression of central P2X receptors was observed in the olfactory bulb, lateral septum, cerebellum and spinal cord. P2X4 immunoreactivity was also evident in widespread areas including the cerebral cortex, hippocampus, thalamus and brainstem. In all regions examined, P2X receptors were associated with perikarya and dendrites where they were concentrated at the level of afferent synaptic junctions, confirming a direct involvement of postsynaptic ATP-gated channels in fast excitatory purinergic transmission. Moreover, P2X4-containing purinoceptors were localized in axon terminals in the olfactory bulb and in the substantia gelatinosa of nucleus caudalis of the medulla and dorsal horn of the spinal cord, demonstrating an important selective presynaptic role of ATP in the modulation of neurotransmitter release in central sensory systems.

Immunocytochemical localization and circadian variations of serotonin and N-acetylserotonin in photoreceptor cells. Light and electron microscopic study in the teleost pineal organ.

Using two immunocytochemical procedures (i.e., immunofluorescence and the unlabeled peroxidase-antiperoxidase method), the localization of a serotonin(HT)-like and of a N-acetylserotonin (aHT)-like immunoreactivity in the pineal organ of the pike was studied during winter. It was shown that immunostaining was exclusively restricted to the cells of the receptor line (CRL = typical and modified photoreceptors). The intensity of the reactions varied through the light-dark cycle, HT-like immunoreactivity being high during the photophase and low during the scotophase. In contrast, aHT-like immunoreactivity was highest at the beginning of the scotophase. HT and aHT-like immunoreactivities were detected in all cell types of the pineal epithelium after administration of a monoamine oxidase inhibitor. Up to now, only HT immunoreactivity could be localized at the ultrastructural level. In a number of typical and modified photoreceptors, a HT-positive staining seemed to be confined within the hyaloplasm of the inner segment, particularly with that of the perikaryon and basal pedicle. Our previous and present results strongly suggest that indole compounds, which are involved in the regulation of various neuroendocrine processes in fish, are synthetized within the CRL. Taking into account that the CRL of the pike are also photosensitive, it appears more and more likely that they are photoneuroendocrine cells involved in mediating the effects of the photoperiod on various physiological and behavioral processes.

Identification in the rat neurotensin receptor of amino-acid residues critical for the binding of neurotensin.

In order to identify charged amino-acid residues of the cloned rat brain neurotensin (NT) receptor (NTR) that are critical for NT binding, we performed site-directed mutagenesis on the cDNA encoding this protein, followed by transient expression into mammalian COS-7 cells and in Xenopus laevis oocytes. Point substitutions of charged residues in the N-terminal part and in the 2nd and 3rd extracellular loop of the receptor either did not affect (125)I-Tyr3-NT binding or resulted in a decrease in binding affinity by a factor of 2-3. Mutations of amino acids Asp113 in the second transmembrane domain (TM) and of Arg149 or Asp150 in TM III yielded receptors that bound NT as efficiently as the native receptor. By contrast, replacement of the Asp139 residue in the 1st extracellular loop, or of Arg143 or Arg327-Arg328 residues at the top of TM III and in TM VI, respectively, completely abolished ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal sequence of the vesicular stomatitis virus glycoprotein (VSV-G), indicated that this loss of binding was not due to altered receptor expression or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocytes, Asp139-Gly- and Arg143-Gly-modified receptors remained functional in spite of a lowered response to NT whereas the Arg327-Arg328 mutant form was totally insensitive to NT at concentrations up to 10 microM. In the case of the Arg327-Arg328 mutation, the observed insensibility to NT could be the result of a drastic conformational alteration of this mutant protein. By contrast, it would appear that Asp139 and Arg143 residues located in the first extracellular loop of the receptor may be directly involved in the interaction of the receptor with neurotensin.

Antisera against small neurotransmitter-like molecules.

Antisera were produced against three types of small neurotransmitter-like molecules: indolealkylamines, catecholamines and amino acid derivatives such as GABA. The specificity of the antisera were evaluated using radioimmunological or immunoenzymatic competition tests between a radiolabelled ligand or conjugated hapten, and analog molecules from the same metabolic pathway. The antibody site was characterized by the ratios of cross-reactivity and the affinity constants. On the basis of these in vitro studies, each immune response was found to be specific for the target molecule.

Ultrastructural features of dopamine axon terminals in the anteromedial and the suprarhinal cortex of adult rat.

The ultrastructural features and synaptic relationships of dopamine (DA) axon terminals were examined in the prefrontal cortex of adult rat after immunocytochemical staining with a highly specific polyclonal antiserum directed against DA-glutaraldehyde-lysyl-protein conjugate (donated by M. Geffard). Single and serial ultrathin sections were obtained from the deep layers of the anteromedial and the suprarhinal DA fields. The DA axon terminals from both regions averaged 0.7 micron in diameter, contained a mixed population of small, round and clear synaptic vesicles associated with a few larger dense-cored or fully immunostained vesicles, and frequently exhibited synaptic contacts which were exclusively made on dendritic shafts and spines. These synapses were mostly of the symmetrical type (80%) and were more often seen on dendritic shafts than spines, particularly in the suprarhinal (89%) compared with the anteromedial cortex (62%). As estimated either by stereological extrapolation from single sections or by direct observation in serial sections, the synaptic incidence of these DA varicosities was significantly greater in the anteromedial than suprarhinal DA field. In the longest series of thin sections, a junctional complex could be observed on 93% of the DA varicosities from the anteromedial cortex but only on 56% in the suprarhinal cortex. Such an inter-regional disparity in the relational characteristics of the DA input will need to be taken into account in elucidating the role and properties of this monoamine in cerebral cortex.

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies.

The distribution of GABA-immunoreactive neurons and axonal varicosities was investigated in the hippocampal region of the rat brain by means of an indirect peroxidase immunocytochemical method with recently developed anti-GABA antibodies. The immunolabeling was found to be restricted to nervous structures: neuronal cell bodies, dendrites and axon terminals. Myelinated axons showing GABA-immunoreactivity were also observed. GABA-immunoreactive neurons were found in great number in the stratum pyramidale, the superficial part of the stratum oriens and the deep part of the stratum radiatum in the Ammon's horn. Less were found in the other regions; rare labeled cells were observed in the superficial part of the stratum radiatum and the middle part of the stratum oriens. The dentate gyrus exhibited numerous labeled cells in the granular layer, few in the hilus, rare in the molecular layer. A high density of GABA-immunoreactive terminals was found at the limit of the stratum oriens with the alveus, in the stratum pyramidale and in the stratum lacunosum. A lower density of labeled fibers was observed in the other areas. The somata and proximal dendrites of pyramidal and granular cells were encompassed by characteristic pericellular arrangements of GABA-immunoreactive varicosities. Ultrastructural observations revealed a diffuse immunoreaction product spread over the cytoplasm and the nucleus without specific relationship with the organelles, and immunoreactive aggregates in the cytoplasm. Labeled dendrites often showed enlargements displaying the immunoreaction whereas thinner segments were devoid of it. They received numerous asymmetrical synapses from unlabeled axon terminals. GABA-immunoreactive terminals were filled with small clear vesicles with immunopositive membranes and were observed in symmetrical contact with somata and dendrites.

First demonstration of highly specific and sensitive antibodies against dopamine.

Dopamine was coupled to bovine serum albumin (BSA) with glutaraldehyde, precautions were taken in order to preserve the catechol ring. After injection of this immunogen into rabbits, anti-dopamine antibodies were obtained and tested using radioimmunochemical binding studies and adsorption to catecholamine covered sepharose beads. A good correlation was found between the results of the different test systems, allowing us to visualize dopamine specifically in glutaraldehyde-fixed rat brains.

Immunorecognition of anti-serotonin antibodies by using a radiolabelled ligand.

Specificity of anti-serotonin antibodies to be assayed for immunocytochemical studies was tested in vitro by using a radiolabelled ligand mimicking the hapten structure. A good immunorecognition was found after coupling [3H]HT to a lysine with formaldehyde. Competition experiments by equilibrium dialysis were then carried out between [3H]HT-F-ALM and both conjugated and unconjugated indoles. The cross-reactivity ratios showed that the antibody recognition, though less specific than expected, was found suitable for immunocytochemical applications.