Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons.

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.

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.

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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.

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Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

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.