Segregation of met-enkephalin from vesicular acetylcholine transporter and choline acetyltransferase in sympathetic preganglionic varicosities mostly lacking synaptophysin and synaptotagmin.

Sympathetic preganglionic neurons (SPN) coexpress the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase and different peptides in their cell bodies, but can express them independently in separate varicosities, indicating that SPN segregate transmitters to different synapses. Consequently, there are populations of preganglionic varicosities (peptidergic and noncholinergic) that store peptides but not ACh. We studied in the cell bodies and axon processes of the rat SPN the expression and the proportional coexpression of the vesicular ACh transporter-like immunoreactivity (VAChT), a specific marker of cholinergic synaptic vesicles or ChAT-like immunoreactivity (ChAT), and the peptide methionine enkephalin-like immunoreactivity (mENK), and confirmed the presence of a population of SPN peptidergic, noncholinergic varicosities. We characterized these varicosities by exploring the occurrence of synaptophysin-like immunoreactivity (Syn), a marker of small clear vesicles, and synaptotagmin-like immunoreactivity (Syt), a preferential marker of large dense core vesicles. We found that (i) VAChT and mENK, like ChAT-mENK, were coexpressed in only 59% of the mENK-containing varicosities, although they colocalized in the SPN cell bodies; and (ii) almost 60% of the population of mENK-containing varicosities did not express Syn or Syt, and over 80% of the mENK-containing varicosities negative for VAChT also lacked Syn. These data prove that SPN segregate mENK from VAChT and ChAT, and show that most of the subset of mENKergic varicosities negative for VAChT also does not express Syn, suggesting the presence of a different vesicular pattern in these sympathetic preganglionic varicosities.

Choline acetyl transferase and neuropeptide immunoreactivities are colocalized in somata, but preferentially localized in distinct axon fibers and boutons of cat sympathetic preganglionic neurons.

Cholinergic sympathetic preganglionic neurons (SPN) coexpress the biosynthetic enzyme for acetylcholine, choline acetyl-transferase (ChAT), and neuropeptides such as enkephalin (ENK) in their cell bodies. However, it is not clear whether they also coexpress ChAT and neuropeptides in axon fibers and boutons. To explore coexpression of ChAT and neuropeptides in somata and axon processes of SPN, we investigated, using immunohistochemistry, retrograde labeling, confocal analysis, and tridimensional reconstruction, whether ChAT and the peptides neurotensin, methionine-ENK, somatostatin, calcitonin gene-related peptide, and vasoactive intestinal peptide colocalize in somata, axons fibers, and boutons of cat SPN. Practically, complete colocalization for these peptides and ChAT was observed in SPN somata. Conversely, in most instances we observed independent localization of immunoreactivity (IR) for ChAT and the peptides in axon fibers and boutons. The minor colocalization between ChAT- and peptide-IR in preganglionic fibers could correspond to a sequential axonal transport of ChAT and peptides, since we observed coexistence of these transmitters after blocking axonal transport. Contrary to Dale's principle, our results suggest that SPN can synthesize ChAT and peptides in their cell bodies and route them to distinct axon boutons or terminals in sympathetic ganglia. Presence of axon boutons containing either ChAT or neuropeptides lead us to suggest a new neurochemical pattern of cotransmission in sympathetic ganglia based on the concurrent release of transmitters and cotransmitters from distinct presynaptic boutons, rather than in the corelease of these mediators from the same axon process. The possibility that cellular segregation could be transient and depend on functional requirements is considered.

Occurrence, co-occurrence and topographic distribution of choline acetyl transferase, Met-enkephalin and neurotensin in the stellate ganglion of the cat.

The presence of the classical ganglionic transmitter acetylcholine (ACh), its occurrence and possible co-occurrence with the neuromodulator peptides methionine enkephalin (Met-ENK) and neurotensin (NT), as well as the possible coexistence of these peptides in the preganglionic axon terminals of the cat stellate ganglia were investigated with light and confocal microscopy using immunofluorescence. Choline acetyltransferase (ChAT), Met-ENK, and NT immunoreactivity was detected with light microscopy in axon terminals near tyrosine hydroxylase (TH) immunoreactive (IR) cells. Cell bodies immunopositive for ChAT or Met-ENK were also detected and were TH-negative or TH-positive. Denervation by sectioning preganglionic axons produced two effects: the almost complete elimination of IR fibers and an increase in the number of ChATIR and Met-ENKIR cell bodies, together with the appearance of NTIR cell bodies. Preganglionic ChATIR fibers and boutons form a dense network throughout the entire ganglion, with a homogeneous regional distribution. ChAT, Met-ENK, and NT are essentially stored in different nerve endings, although a low level of co-occurrence was detected. NTIR and Met-ENKIR networks of boutons were observed to have independent and somewhat complementary regional distributions. Further analysis with simultaneous triple labeling for NT, Met-ENK, and TH, and confocal microscopy showed fibers and boutons containing Met-ENK or NT reached distinct neurons separately, or both converge onto the same cells. This finding suggests that modulation (the facilitation-inhibition balance) of ganglionic transmission is achieved mainly by the selective and complementary innervation of boutons containing NT (facilitation) and Met-ENK (inhibition) and only rarely by terminals which coexpress both peptides.

Release-depletion and receptor-mediated neuronal internalization of endogenous neurotensin in the stellate ganglion of the cat.

The release and depletion of neurotensin in sympathetic preganglionic axon terminals and internalization in principal ganglion cells were investigated in the cat stellate ganglion by means of combined immunohistochemical staining, image analysis and confocal microscopy. Neurotensin stored in preganglionic boutons was released by 40 or 5 Hz electrical stimulation of preganglionic nerves, being depleted to 7.4 and 19.2% of control levels by continuous stimulation lasting 20 or 160 min (both stimuli delivered 48,000 pulses). Once released, neurotensin was internalized by the principal ganglion cells as evidenced by a ring of bright spot-like granules in the perinuclear region indicating the sites of intracellular neurotensin accumulation. Neurotensin internalization was time-dependent, thus, different content was found when the time between the end of stimulation and start of perfusion was varied. The onset of neurotensin internalization appeared in the first minutes, intracellular accumulation was evident at 20 min, maximal internalization occurred at 120 min and, 24 h later internalized neurotensin content had faded. Internalization was partially blocked by the nonpeptide neurotensin antagonist SR48692. These data provide evidence of presynaptic neurotensin release and depletion by electrical stimulation with varied frequencies. They also provide evidence for in situ receptor-mediated internalization of endogenously released neurotensin, raising the possibility that internalization may represent, in addition to some kind of turnover dynamics, an important part of the mechanisms of neuropeptide signaling.

Immunoreactive prolactin forms colocalize with vasopressin in neurons of the hypothalamic paraventricular and supraoptic nuclei.

The prolactin (PRL) gene is expressed in the hypothalamo-neurohypophyseal system as revealed by the detection of the PRL mRNA and of PRL-like immunoreactive and biologically active proteins in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and in the neurohypophysis. We have investigated the distribution of cells containing PRL-like molecules in the PVN and SON by immunocytochemistry with a specific antiserum directed against the 16-kD N-terminal fragment of PRL. PRL-positive cells were found to be concentrated throughout the ventral SON and in the lateroposterior region of the PVN. The cellular distribution of PRL-immunoreactive cells resembled more closely that of vasopressin (VP) than that of oxytocin magnocellular neurons. Moreover, double immunofluorescence labelling, followed by confocal microscopy, indicated the coexistence of PRL- and VP-related antigens within the same neurons of the PVN and SON. Pre-embedding immunoperoxidase on the ultrastructural level showed a PRL-like product in granular-type particles within the neural soma and projections in the SON and PVN. These findings are consistent with the expression and secretion of PRL-like molecules by vasopressinergic neurons of the hypothalamo-neurohypophyseal system.

Topographic relationship of neurotensin-containing axon terminals with cardiac and noncardiac principal ganglion cells in the stellate ganglia of the cat.

The pattern of association between neurotensin (NT)-immunoreactive (NTIR) preganglionic nerve terminals and cardiac and noncardiac neurons in the stellate ganglion of the cat is analyzed, based on the finding of an excitatory modulation effect of exogenous NT on cardiac functions. For this purpose, NT-containing terminals were labeled by immunohistochemistry, and ganglion cells were detected by retrograde labeling of cardiac and vertebral nerves to identify cardiac and noncardiac neurons. To determine a possible regional localization of NTIR terminals and ganglion cells, the ganglia were divided into four areas: caudal, dorsomedial, cranial, and ventromedial, related to the two major afferent nerves (thoracic white rami 3 [T3WR] and 2 [T2WR]) and the two efferent nerves (vertebral and cardiac). NTIR terminals were widespread in the complete ganglion tissue; they covered practically all the regions explored, although two clusters of high concentration of NTIR terminals were detected in the cranial and caudal areas. By retrograde labelling it was found that cardiac cells were arranged around the exit of the cardiac nerve and that the vertebral neurons were extended from the exit of the vertebral nerve to the entrance of T3WR. The finding of association of NTIR terminals with cardiac neurons may account for the cardioregulatory effect of NT; however, since the presence of NTIR terminals close to the noncardiac neurons is notorious, other regulatory functions of NT must be considered.

[Hereditary lysosomal diseases in Mexico. III. Laboratory diagnosis of sphingolipidosis]. / Enfermedades hereditarias lisosomales en México. III. Diagnóstico de laboratorio para esfingolipidosis.

Enzymatic determinations of the levels of lysosomal enzymes in serum or leukocytes samples have been carried out for the diagnosis of 7 sphingolipidosis. This methodology has allowed us to study 49 homozygotes and 33 close relatives at risk for the carrier state of a particular sphingolipidosis. So far we have diagnosed: 21 Gaucher's disease patients, 17 metachromatic leukodistrophy, 4 Niemann-Pick, 4 GM2 gangliosidosis, 2 Fabry and one GM1 gangliosidosis. Limitations in the performance and interpretation of the levels of the defective enzyme in heterozygotes, homozygotes and those variants not detected with the assays described are discussed.

[Hereditary lysosomal diseases in Mexico. II. Laboratory diagnosis of mucopolysaccharidosis and mucolipidosis]. / Enfermedades hereditarias lisosomales en México. II. Diagnóstico de laboratorio de mucopolisacaridosis y mucolipidosis.

Methods suitable for the diagnosis of the mucopolysaccharidoses and mucolipidosis using urine, serum and leucocytes are presented. The methodology includes a screening technique for the mucopolysaccharidoses, a determination of glycosaminoglycans excreted in urine, serum and leucocyte enzymatic determinations for deficient lysosomal enzymes in lysosomal storage diseases. Their use is validated in the diagnosis of 19 patient with mucopolysaccharidoses and 5 with mucolipidoses, and to establish a carrier state in 10 close relatives.

[Hereditary lysosomal diseases. I. Initial results of a diagnostic program in Mexico]. / Enfermedades hereditarias lisosomales. I. Resultados iniciales del programa para su diagnóstico en México.

Results obtained by the Lysosome Storage Disease Diagnostic Program are described as well as the criteria used for the selection of patients to be studied. This program was started in 1983 and is sponsored by the National Reference Center for the Detection and Diagnosis of Inborn Errors of Metabolism in Mexico City. Laboratory tests include chemical determinations of urinary glycosaminoglycans and enzymatic assays of 15 lysosomal enzymes that allow the identification of 25 of the 35 lysosomal storage diseases known. A total of 259 patients with clinical phenotypes suggesting a lysosomal storage disease, and 47 individuals at risk for the carrier state were studied. The disease diagnosed were 35 patients with mucopolysaccharidoses, 27 with sphingolipidoses, 5 with mucolipidoses and 7 with glycogenoses; the most common lysosomal storage disease was Gaucher followed in decreasing frequency by Morquio, Hunter and glycogenoses Ia. The carrier state was confirmed in 29 of close relatives, one of them confirmed prenatally.