Calretinin-immunoreactive nerves in the uterus, pelvic autonomic ganglia, lumbosacral dorsal root ganglia and lumbosacral spinal cord

Nerves containing the calcium-binding protein calretinin have been reported in several organs but not in female reproductive organs and associated ganglia. This study was undertaken to determine if nerves associated with the uterus contain calretinin and the source(s) of calretinin-synthesizing nerves in the rat (are they sensory, efferent, or both?). Calretinin-immunoreactive nerves were present in the uterine horns and cervix where they were associated with arteries, uterine smooth muscle, glands, and the epithelium. Calretinin-immunoreactive terminals were apposed to neurons in the paracervical ganglia; in addition, some postganglionic neurons in this ganglion were calretinin positive. Calretinin perikarya were present in the lumbosacral dorsal root ganglia, no-dose ganglia, and lumbosacral spinal cord. Retrograde axonal tracing, utilizing Fluorogold injected into the uterus or paracervical parasympathetic ganglia, revealed calretinin-positive/Fluorogold-labeled neurons in the dorsal root and nodose ganglia. Also, capsaicin treatment substantially reduced the calretinin-positive fibers in the uterus and pelvic ganglia, thus indicating the sensory nature of these fibers. The presence of calretinin immunoreactivity identifies a subset of nerves that are involved in innervation of the pelvic viscera and have origins from lumbosacral dorsal root ganglia and vagal nodose ganglia. Though the exact function of calretinin in these nerves is not currently known, calretinin is likely to play a role in calcium regulation and their function.     

Cholinergic neurotransmission and muscarinic receptors in the enteric nervous system

There is a rich knowledge of the enteric nervous system (ENS), especially the neurochemical and neurophysiological properties of enteric neurons and how they communicate in neural circuits underlying intestinal reflexes. The major pathways of excitatory transmission within the ENS are mediated by cholinergic and tachykinergic transmission, with transmitters Acetylcholine (ACh) and Tachykinins (TK), respectively, producing excitatory potentials in post-synaptic effectors. This review focuses on the cholinergic pathways of the ENS. The cholinergic circuitry of the ENS is extensive and mediates motility (muscular) and secretory (mucosal) reflexes, in addition to intrinsic sensory and vascular reflexes. The capacity of ACh to mediate multiple physiologically significant intestinal reflexes is largely due to having multiple sites of neuronal and non-neuronal release and reception within the intestine. This review will concentrate on one of two classes of ACh receptors, Muscarinic receptors (mAChr), in particular their location and function in mediating synaptic transmission within enteric circuits underlying intestinal reflexes.     

Immunohistochemical properties and spinal connections of pelvic autonomic neurons that innervate the rat prostate gland

Autonomic innervation of the prostate gland supplies the acini, and non-vascular and vascular smooth muscle. The activity of each of these tissues is enhanced by sympathetic outflow, whereas the role of the parasympathetic nervous system in this organ is unclear. In the present study, a range of methods was applied in rats to determine the location of autonomic neurons supplying this gland, the immunohistochemical properties of these neurons, the spinal connections made with the postganglionic pathways and the distribution of various axon types within the gland. Injection of the retrograde tracer, FluoroGold, into the ventral gland visualised neurons within the major pelvic ganglion and sympathetic chain. Fluorescence immunohistochemical studies on the labelled pelvic neurons showed that most were noradrenergic (also containing neuropeptide Y, NPY), the others being non-noradrenergic and containing either vasoactive intestinal peptide (VIP) or NPY. Sympathetic dyelabelled neurons were identified by the presence of varicose nerve terminals stained for synaptophysin on their somata following lesion of sacral inputs. Parasympathetic innervation of dye-labelled neurons was identified by continued innervation after hypogastric nerve lesion. Most noradrenergic prostate-projecting neurons were sympathetic, as were many of the non-noradrenergic VIP neurons. Parasympathetic prostate-projecting neurons were largely non-noradrenergic and contained either VIP or NPY. All substances found in retrogradely labelled somata were located in axons within the prostate gland but had slightly different patterns of distribution. The studies have shown that there are a significant number of non-noradrenergic sympathetic prostate-projecting neurons, which contain VIP.     

Cholinergic stimulation of pancreatic amylase release and muscarinic receptors: effect of ionophore A23187

Dispersed rat pancreatic acini were incubated in 0.5mM calcium medium with increasing concentrations of carbamylcholine, with or without the ionophore A23187 (10(-6)M). Addition of the ionophore reduced maximal amylase release, increased the maximal effective concentration of carbamylcholine and dramatically impaired the agonist's capacity to induce enzyme secretion at low concentration. The ionophore also abolished the inhibition of secretion observed at high carbamylcholine concentrations. These effects of the ionophore on the cholinergic secretory response cannot be explained by interaction at the muscarinic receptor since neither the Bmax, the affinity of the receptor for the [3H]QNB nor the binding of carbamylcholine were affected by the ionophore. It is suggested that for the conditions studied, the ionophore can interact with the secretory process at one or several points ulterior to the initial recognition site of carbamylcholine on its receptor.     

Nonselective cation conductance activated by muscarinic and purinergic receptors in rat spiral ganglion neurons

The present studycharacterizes the ionic conductances activated by acetylcholine (ACh)and ATP, two candidate neuromodulators, in isolated spiral ganglionneurons (SGNs). Brief application (1 s) of ACh evoked in adose-dependent manner (EC₅₀ = 4.1 µM) a reversibleinward current with a long latency (average 1.3 s), at holdingpotential (V_h) = 50 mV. This current wasreversibly blocked by atropine and mimicked by muscarine. Applicationof ATP also evoked a reversible inward current atV_h = 50 mV, but the current showed twocomponents. A fast component with a short latency was largely reducedwhen N-methyl-D-glucamine (NMDG) replaced extracellular sodium, implying a P2X-like ionotropic conductance. Thesecond component had a longer latency (average 1.1 s) and waspresumably activated by metabotropic P2Y-like receptors. The secondcomponent of ATP-evoked current shared similar characteristics with theresponses evoked by ACh: the current reversed near 0 mV, displayedinward rectification, could be carried by NMDG, and was insensitive toextracellular and intracellular calcium. This ACh-/ATP-evokedconductance was reversibly inhibited by preapplication of ionomycin.These results suggest that muscarinic receptors and purinergicmetabotropic receptors activate a similar large nonselective cationconductance via a common intracellular pathway in SGNs, a candidatemechanism to regulate neuronal excitability of SGNs.

     

Cholinergic afferents to gonadotropin-releasing hormone neurons of the rat

Gonadotropin-releasing hormone-synthesizing neurons represent the final common pathway in the hypothalamic regulation of reproduction and their secretory activity is influenced by a variety of neurotransmitters and neuromodulators acting centrally in synaptic afferents to gonadotropin-releasing hormone neurons. The present study examined the anatomical relationship of cholinergic neuronal pathways and gonadotropin-releasing hormone neurons of the preoptic area. The immunocytochemical detection of choline acetyltransferase or vesicular acetylcholine transporter revealed a fine network of cholinergic fibers in this region. At the light microscopic level, the cholinergic axons formed appositions to the gonadotropin-releasing hormone immunoreactive cell bodies and dendrites. Results of electron microscopic studies confirmed the absence of glial interpositions in many of these neuronal contacts. Classical cholinergic synapses, which belonged to the asymmetric category, were only observed rarely on gonadotropin-releasing hormone neurons. The lack of synaptic density in most contacts corroborates previous observations on the cholinergic system elsewhere in the brain. Further, it suggests a dominantly non-synaptic route also in this cholinergic neuronal communication. This study provides direct neuromorphological evidence for the involvement of the cholinergic system in the afferent neuronal regulation of gonadotropin-releasing hormone neurons. The sources of cholinergic afferents and the receptorial mechanisms underlying this interaction will require further clarification.     

Effect of alcohol on the rat juxtaprostatic pelvic ganglia and gonads

In the present paper, it was investigated the aspect of the intrapelvic autonomic neuronal system (juxtaprostatic ganglion) and gonads of rats submitted to a experimental alcoholism. The animals which had water substituted for 35% sugar cane spirit for 30 d, presented testicular lesions as well as numeric and degenerative alterations in the juxtaprostatic pelvic ganglia neurons.     

Coexistence of calcitonin gene-related peptide and galanin immunoreactivity in female rat pelvic and lumbosacral dorsal root ganglia.

Coexistence of immunoreactivity for calcitonin gene-related peptide (CGRP) and galanin (GAL) was examined in varicose nerve endings in female rat pelvic paracervical ganglia (PG) and in perikarya of lumbosacral dorsal root ganglia (DRG). Varicose peptide-containing nerves were closely adjacent to somata of neurons in the PG, certain somata being virtually surrounded by immunoreactive varicosities. Some nerve endings were immunoreactive for either CGRP or GAL; in others, immunoreactivity for CGRP and GAL coexisted. Likewise, many perikarya in DRG were CGRP immunoreactive, fewer were GAL immunoreactive, and in some immunoreactivity for CGRP and GAL coexisted. The results suggest there are subpopulations of neuropeptide-containing sensory nerve endings in the PG; some contain CGRP, some contain GAL, and in some CGRP and GAL coexist. These substances contained in sensory nerve endings could have important roles in pelvic ganglionic functions.     

Neurotrophin-3 and TrkC-immunoreactive neurons in rat dorsal root ganglia correlate by distribution and morphology

Previous studies have shown that a subpopulation of large dorsal root ganglion neurons contains neurotrophin-3 (NT3)-like immunoreactivity. It is not known, however, whether these NT3 immunoreactive neurons also express the high affinity receptor for NT3, trkC. In the present study, the distribution and morphology of trkC immunoreactive neurons have been correlated with those of NT3 immunoreactive neurons in the dorsal root ganglia. Size and segmental distributions of both antigens indicate that they are present in the same group of large sensory neurons. Almost twice the number of these neurons are present in the cervical and lumbar spinal ganglia than in the thoracic. Co-localization study indicates that 94% of NT3 immunoreactive neurons express trkC. Our findings support the proposal that NT3 in these neurons is derived from their peripheral targets rather than synthesized in situ. Special issue dedicated to Dr. Hans Thoenen.     

Changes in lectin binding of lumbar dorsal root ganglia neurons and peripheral axons after sciatic and spinal nerve injury in the rat

Summary The effects of chronic lesions of rat lumbar spinal or sciatic nerves on the binding of Glycine max (soybean) agglutinin to galacto-conjugates, in small-and medium-size primary sensory neurons of the L₄ and L₅ dorsal root ganglia, were examined over a 580-day period. Spinal nerve section resulted in a marked decrease in the population of stained neurons within 7 days. However, despite some retrograde morphological changes triggered by axonal injury, the proportion of stained nerve cells was normalized 180 days postoperatively. This temporary decrease in perikaryal lectin reactivity was initially associated with a marked accumulation of stained material in the nerve, proximal and distal to the site of section, with similar accumulations also being noticeable at each level of injury in sciatic nerves subjected to double ligature. This may reflect the presence of glycocompounds linked to the autolysis of nerve fibers during the phase of retrograde dying-back and Wallerian degeneration. At later stages, stained deposits could be seen scattered along central and peripheral axonal processes of the dorsal root ganglion neurons in the vicinity of the cell body. They may indicate a disturbance in the peripheral turnover of glycoproteins in chronically-transected nerves, with piling up of neuronal products. Sciatic nerve injury caused similar but less severe effects which, except for the L₄ ganglion cells, were rapidly reversible.     

Calcium distribution and exchange in the rat uterus

The calcium content and distribution of the rat uterus were determined employing flame photometry and Ca⁴⁵ determinations. The total uterine calcium concentration was found to be 2.25 millimoles (mmoles) per kilogram wet weight, 0.45 of which was inexchangeable. The exchangeable Ca could be divided into 0.8 mmole/kg wet weight extracellular and 1.0 mmole/kg wet weight intracellular. The concentration of ionic Ca in rat serum was obtained by equilibrium dialysis as 1.5 mM or 53 % of the total serum Ca. The observed Ca distribution required that its active transport be postulated, since the membrane was shown to be permeable to Ca and the internal Ca concentration was far below its electrochemical equilibrium value. Metabolic inhibition by iodoacetate or dinitrophenol caused a net Ca uptake, but cooling to 4°C and ouabain did not. Iodoacetate did not affect the Ca⁴⁵ efflux, but did increase the influx, suggesting that active Ca transport is accomplished by an exclusion mechanism. In experiments with varied external sodium concentrations, no evidence was obtained that sodium competes with calcium for inward transport. Cellular Ca binding was measured under conditions of prolonged metabolic inhibition, which abolished both active transport and the membrane potential. The association constants obtained were compatible with intracellular Ca binding to proteins, but insufficient to account for the low level of intracellular ionic Ca believed essential for relaxation. Hence metabolically dependent intracellular Ca binding was postulated. The Ca⁴⁵ efflux was slowed down by Ca-free efflux media. The presence of Sr or EDTA could completely prevent this decrease in efflux rate, and Ba could partly prevent it. Changes in Mg and Na concentration did not affect the rate of Ca⁴⁵ efflux. A model to explain Ca exchange across smooth muscle membranes has been proposed.     

Expression of sphingosine 1-phosphate receptors in the rat dorsal root ganglia and defined single isolated sensory neurons

Previously, we demonstrated that sphingosine 1-phosphate (S1P) increased the excitability of small-diameter sensory neurons, in part, through activation of S1P receptor 1 (S1PR(1)), suggesting that other S1PRs can modulate neuronal excitability. Therefore, studies were undertaken to establish the expression profiles of S1PRs in the intact dorsal root ganglion (DRG) and in defined single isolated sensory neurons. To determine mRNA expression of S1PRs in the DRG, SYBR green quantitative PCR (qPCR) was used. To determine the expression of S1PR mRNAs in single neurons of defined diameters, a preamplification protocol utilizing Taqman primer and probes was used to enhance the sensitivity of detection. The preamplification protocol also permitted detection of mRNA for two hallmark neuronal receptor/ion channels, TRPV1 and P(2)X(3). Expression profiles of S1PR mRNA isolated from lung and brain were used as positive control tissues. In the intact DRG, the order of expression of S1PRs was S1PR(3)>R(1)≈R(2)>R(5)≈R(4). In the single neurons, the expression of S1PRs was quite variable with some neurons expressing all five subtypes, whereas some expressing only one subtype. In contrast to the DRG, S1PR(1) was the highest expressing subtype in 10 of the 18 small-, medium-, and large-diameter sensory neurons. S1PR(1) was the second highest expressor in ～50% of those remaining neurons. Overall, in the single neurons, the order of expression was S1PR(1)>R(3)≈R(5)>R(4)>R(2). The results obtained from the single defined neurons are consistent with our previous findings wherein S1PR(1) plays a prominent but not exclusive role in the enhancement of neuronal excitability.     

Effects of castration on the immunoreactivity to NGF, BDNF and their receptors in the pelvic ganglia of the male rat

Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and are members of the neurotrophin family, a family of neurotrophic factors that also includes neurotrophin (NT) 3 and NT4/5. Neurotrophins have essential roles in the survival, development and differentiation of neurons in the central and peripheral nervous systems. Neurotrophins exert their effects by binding to corresponding receptors which are formed by the tyrosine protein kinases TrkA, TrkB and TrkC, and the low affinity neurotrophic receptor (p75NTR). In the present study, using immunohistochemistry and quantitative analysis, we have investigated immunoreactivity to BDNF, NGF, TrkB, p75NTR and TrkA in the pelvic ganglia of normal and castrated rats. Neurons of the pelvic ganglia expressed both these neurotrophins and their receptors. After castration the immunoreactivity persisted. However, the number of BDNF- and p75NTR-IR cells statistically significant decreased after castration. These results suggest that castration modulates the expression of neurotrophins and their receptors in pelvic autonomic neurons.