Oxytocin innervation of spinal preganglionic neurons projecting to the superior cervical ganglion in the rat

The paraventricular nucleus of the hypothalamus is a major integrative nucleus for relaying information from the suprachiasmatic nucleus to the autonomic system. The precise pathway by which this information can influence autonomic functions, such as melatonin synthesis in the pineal gland, is not clear. In the present study, we used a retrograde tracer injected in the superior cervical ganglion to identify spinal preganglionic neurons. One of the main neurotransmitters present in descending projections of the paraventricular nucleus of the hypothalamus, oxytocin, was detected with immunocytochemistry to visualise possible contacts with the neurons located in the intermediolateral column of the spinal cord and projecting to the superior cervical ganglion. Although many appositions could be seen at the light-microscopic level, this abundance could not be confirmed at the electron-microscopic level. The implications of these observations for the overall timing message received by the spinal preganglionic neurons are discussed.     

Selective stimulation of jugular ganglion afferent neurons in guinea pig airways by hypertonic saline

We evaluated the ability of hyperosmolar stimuli to activate afferent nerves in the guinea pig trachea and main bronchi and investigated the neural pathways involved. By using electrophysiological techniques, studies in vitro examined the effect of hyperosmolar solutions of sodium chloride (hypertonic saline) on guinea pig airway afferent nerve endings arising from either vagal nodose or jugular ganglia. The data reveal a differential sensitivity of airway afferent neurons to activation with hypertonic saline. Afferent fibers (both A delta and C fibers) with cell bodies located in jugular ganglia were much more sensitive to stimulation with hypertonic saline, compared with afferent neurons with cell bodies located in nodose ganglia. Additional studies in vivo demonstrated that inhalation of aerosols of hypertonic saline induced plasma extravasation in guinea pig trachea that was mediated via tachykinin NK1 receptors. Identification of a differential sensitivity of guinea pig airway afferent nerves to hypertonic saline leads to the speculation that airway responses to hyperosmolar stimuli may result from activation of afferent neurons originating predominantly from the jugular ganglion.     

Reduced levels of acetylcholine receptor expression in chick ciliary ganglion neurons developing in the absence of innervation

Chick ciliary ganglion neurons receive innervation from a single source, the accessory oculomotor nucleus (AON), and nicotinic ACh receptors (AChRs) mediate chemical synaptic transmission through the ganglion. Previous experiments examining the developmental expression of AChRs in embryonic chick ciliary ganglion neurons in situ have shown that AChR levels increase substantially in the neurons at the time of innervation. Prior to synapse formation, few AChRs are detected in the neurons. In the present experiments, the role of presynaptic inputs in inducing an increase in AChRs was established by examining AChR levels in ciliary ganglion neurons that have been deprived of innervation by surgical ablation of the AON prior to synapse formation. AChR levels were dramatically reduced in neurons of input-deprived ganglia as compared to control innervated neurons at all developmental stages examined from embryonic day (ED) 5 to ED 12 as determined by indirect immunocytochemical labeling of frozen ganglion sections with the anti-AChR monoclonal antibody mAb 35, and light microscopy. In contrast, neuronal somata of input-deprived and control ganglia had equivalent levels of immunolabeling for three other components, a transmembrane glycoprotein of synaptic vesicles, SV2, and two microtubule-associated proteins, MAP 1B and MAP 2, from ED 5 up to ED 10. The results demonstrate that presynaptic inputs specifically increase the levels of AChR expression in developing neurons. In addition, changes in the levels of immunolabeling for AChRs, SV2, MAP 1B, and MAP 2 in neuronal somata after ED 10 demonstrate that other major developmental events also influence the levels of these components in neurons. Declines in the intensity of AChR, SV2, MAP 1B, and MAP 2 immunolabeling within a subset of neuronal somata in both operated and control ganglia at ED 10 and 12 coincide with the period of neuronal cell death. Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences AChR levels in developing neurons in situ.     

Evidence for the existence of substance P autoreceptor in the membrane of rat dorsal root ganglion neurons

Substance P, a putative peptide neurotransmitter contained in primary sensory neurons, is suggested to play a major role in nociceptive transmission. In the present study, the existence of substance P autoreceptor in dorsal root ganglion neurons was identified with a method we developed recently and substance P-activated inward current in the dorsal root ganglion neurons and its ionic mechanism were also explored preliminarily. The majority of the cells examined (68/76, 89.5%) were sensitive to external application of substance P (0.01-10 microM) with a concentration-dependent inward current. This current was found to result from the opening of nonselective ion channel, preferring the Na+ channel. The substance P-activated current can be suppressed by Cd2+ (0.05 microM), which suggested Ca2+ may also be involved. Soon after the neurons had been identified to be endowed with substance P receptor with whole-cell patch-clamp technique, 17 cells were chosen for immunocytochemical staining to detect substance P-immunoreactivity. Seven neurons which were classified into small and intermediate size were found to reveal substance P-immunoreactivity. Using this method we have identified the existence of substance P autoreceptor in rat DRG neurons.     

Chemosensitivity of nociceptive, mechanosensitive afferent nerve fibres in the guinea-pig ureter

The mechanosensitivity and chemosensitivity of afferent fibres were investigated in an in vitro preparation of the guinea-pig ureter. Electrophysiological recordings were obtained from 5 U-1 (low mechanical threshold, contraction-sensitive) and 74 U-2 units (high threshold). U-2 units had significant higher levels of spontaneous activity, lower conduction velocities, higher mechanical thresholds (U-1: 7 mmHg; U-2: 39 mmHg), less pronounced phasic responses and longer latencies in the response to distensions than the U-1 units. For chemical stimulation, guinea-pig urine (> 800 mosmol/L), bradykinin and capsaicin were applied intraluminally. The responses of U-1 units mainly corresponded to the contractions induced by the chemical stimulation. The vast majority of the U-2 units were excited by urine, bradykinin (threshold: 0.1-1 microM) and capsaicin (threshold: 0.03-0.3 microM). The responses to urine could be mimicked by high concentrations of potassium ions (> 200 mM), but not by an equiosmolar solution of NaCl, urea and mannitol. Chemical stimulation could also result in a transient sensitization of the U-2 units to mechanical stimuli. In the anaesthetized guinea-pig, pseudo-affective responses could be evoked by ureteric distension (threshold: 30-60 mmHg) and serosal application of capsaicin. Intraluminal application of urine in vivo did not evoke any reactions, suggesting that the responses of the U-2 units to urine might be due to an impaired barrier function of the urothelium in vitro. The data are in agreement with the hypothesis that U-2 units are visceral polymodal nociceptors. Since the U-1 units were also able to encode at least noxious mechanical stimuli, their involvement in visceral nociception cannot be excluded.     

Lidocaine toxicity in primary afferent neurons from the rat

Evidence from both clinical studies and animal models suggests that the local anesthetic, lidocaine, is neurotoxic. However, the mechanism of lidocaine-induced toxicity is unknown. To test the hypothesis that toxicity results from a direct action of lidocaine on sensory neurons we performed in vitro histological, electrophysiological and fluorometrical experiments on isolated dorsal root ganglion (DRG) neurons from the adult rat. We observed lidocaine-induced neuronal death after a 4-min exposure of DRG neurons to lidocaine concentrations as low as 30 mM. Consistent with an excitotoxic mechanism of neurotoxicity, lidocaine depolarized DRG neurons at concentrations that induced cell death (EC50 = 14 mM). This depolarization occurred even though voltage-gated sodium currents and action potentials were blocked effectively at much lower concentrations. (EC50 values for lidocaine-induced block of tetrodotoxin-sensitive and -resistant voltage-gated sodium currents were 41 and 101 microM, respectively.) At concentrations similar to those that induced neurotoxicity and depolarization, lidocaine also induced an increase in the concentration of intracellular Ca++ ions ([Ca++]i; EC50 = 21 mM) via Ca++ influx through the plasma membrane as well as release of Ca++ from intracellular stores. Finally, lidocaine-induced neurotoxicity was attenuated significantly when lidocaine was applied in the presence of nominally Ca(++)-free bath solution to DRG neurons preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Our results indicate: 1) that lidocaine is neurotoxic to sensory neurons; 2) that toxicity results from a direct action on sensory neurons; and 3) that a lidocaine-induced increase in intracellular Ca++ is a mechanism of lidocaine-induced neuronal toxicity.     

Neurochemical classification of myenteric neurons in the guinea-pig ileum

A strategy has been developed to identify and quantify the different neurochemical populations of myenteric neurons in the guinea-pig ileum using double-labelling fluorescence immunohistochemistry of whole-mount preparations. First, six histochemical markers were used to identify exclusive, non-overlapping populations of nerve cell bodies. They included immunoreactivity for the calcium binding proteins calbindin and calretinin, the neuropeptides vasoactive intestinal polypeptide, substance P and somatostatin, and the amine, 5-hydroxytryptamine. The sizes of these populations of neurons were established directly or indirectly in double-labelling experiments using a marker for all nerve cell bodies. Each of these exclusive populations was further subdivided into classes by other markers, including immunoreactivity for enkephalins and neurofilament protein triplet. The size of each class was then established directly or by calculation. These distinct, neurochemically-identified classes were related to other published work on the histochemistry, electrophysiology and retrograde labelling of enteric neurons and to the simple Dogiel morphological classification. A classification scheme, consistent with previous studies, is proposed. It includes 14 distinct classes of myenteric neurons and accounts for nearly all neurons in the myenteric plexus of the guinea-pig ileum.     

Paradoxical facilitation of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by isoprenaline

1. Previous studies have provided evidence that activation of beta-adrenoceptors on cholinergic nerve terminals can inhibit neurotransmission in the airways. However, in most cases, this conclusion has been based on indirect evidence obtained from mechanical experiments where changes in airways smooth muscle tone were measured. 2. We have assessed whether modulation of cholinergic neurotransmission by beta-adrenoceptor agonists is due to a pre- or post-junctional action by investigating the effect of isoprenaline on contractile responses evoked by exogenous acetylcholine (ACh) and electrical field stimulation (EFS; 4 Hz, 40 V, 0.5 ms pulse width every 15 s), and on EFS-induced ACh release from cholinergic nerves innervating guinea-pig and human trachea. Furthermore, the subtype of beta-adrenoceptor which modulates neurotransmission and the potential role of cyclic AMP in this response were evaluated. 3. In guinea-pig trachea, isoprenaline (1 nM-1 microM) inhibited the contractile response evoked by exogenous ACh (1 microM) to a similar extent to that evoked by EFS (EC50 = 19.9 and 23 nM, respectively). 4. In epithelium-denuded guinea-pig strips treated with indomethacin (10 microM), isoprenaline significantly enhanced EFS-induced ACh release from cholinergic nerve terminals (by 36% at 0.3 microM). This effect was blocked by propranolol and ICI 118, 551 (each 0.1 microM). In contrast, isoprenaline failed to affect EFS-induced ACh release from parasympathetic nerves innervating human trachea. 5. To evaluate the role of cyclic AMP in the beta-adrenoceptor-induced facilitation of cholinergic neurotransmission, the effects of various cyclic AMP elevating drugs on ACh release were studied. Forskolin (10 microM) significantly augmented (by 17%) EFS-induced ACh release, an effect which was not reproduced by 1,9-dideoxyforskolin (10 microM) which does not activate adenylyl cyclase. Similarly, the cyclic AMP analogue, 8-bromo-cyclic AMP (1 mM) and cholera toxin (1 microgram ml-1) facilitated ACh output by 22 and 47% respectively, whereas prostaglandin E2 (PGE2, 0.1 nM-1 microM) inhibited this response (by 67% at 1 microM). 6. Zardaverine (10 microM), a dual inhibitor of the phosphodiesterase (PDE)3 and PDE4 isoenzyme families, did not affect EFS-induced ACh release and failed to facilitate the actions of either isoprenaline or PGE2. Similarly, neither SK&F 94120 (10 microM) nor rolipram (10 microM), selective inhibitors of PDE3 and PDE4 respectively, significantly affected the release of ACh in response to EFS. 7. The result of this study suggests that isoprenaline facilitates cholinergic neurotransmission in guinea-pig, but not human, trachea by activation of pre-junctional beta 2-adrenoceptors, an effect that may be mediated via activation of the cyclic AMP/cyclic AMP-dependent protein kinase cascade. Furthermore, the data presented herein illustrate the need to undertake direct measurements of neurotransmitter release when examining the effect of agents purported to act pre-junctionally.     

Distribution of myenteric NO neurons along the guinea-pig esophagus

This review surveys the most important and promising contributions of agricultural biotechnology to the development of sustainable, environment-friendly agriculture. It deals with the recent achievements of genetic technology for the development of new transgenic microbial, plant and animal products. It also deals with the newest developments and perspectives of microbial intervention in agricultural practices, such as biofertilizers, biocontrol agents, and various microbiological products used in modern agriculture. The review surveys the outlook for a waste-free, environment-friendly sustainable agricultural practice, including waste management, recycling and bioremediation technologies. The review lists the most important marketable agrobiotechnological products, and their present and projected sales volume.     

Ascending afferent regulation of rat midbrain dopamine neurons

Standard, extracellular single-unit recording techniques were used to examine the electrophysiological and pharmacological responsiveness of midbrain dopamine (DA) neurons to selected, ascending afferent inputs. Sciatic nerve stimulation-induced inhibition of nigrostriatal DA (NSDA) neurons was blocked by both PCPA (5-HT synthesis inhibitor) and 5,7-DHT (5-HT neurotoxin), suggesting mediation by a serotonergic (5-HT) system. Direct stimulation of the dorsal raphe (which utilizes 5-HT as a neurotransmitter and inhibits slowly firing NSDA neurons) inhibited all mesoaccumbens DA (MADA) neurons tested. Paradoxically, DPAT, a 5-HT1A agonist which inhibits 5-HT cell firing, enhanced sciatic nerve stimulation-induced inhibition of NSDA neurons. MADA neurons were not inhibited by sciatic nerve stimulation and, therefore, could not be tested in this paradigm. In contrast to the dorsal raphe, electrical stimulation of the pedunculopontine tegmental nucleus preferentially excited slowly firing NSDA and MADA neurons. Thus, both excitatory and inhibitory ascending afferents influence the activity of midbrain DA neurons, and intact 5-HT systems are necessary for sciatic nerve stimulation to alter DA cell activity. However, the role that 5-HT plays in mediating peripheral sensory input remains unclear.     

Neurokinin A in rat renal afferent neurons and in nerve fibres within smooth muscle and epithelium of rat and guinea-pig renal pelvis

Neurokinin A-like immunoreactivity of dorsal root ganglion neurons innervating the kidney were studied with retrograde tracing of FluoroGold dye applied to the cut renal nerves. The proportions and sizes of renal afferent neurons with neurokinin A-like immunoreactivity were quantified in T9-L2 dorsal root ganglia from five rats. Of 240 renal afferent neuronal somata examined, 26 +/- 3% (S.E.M.) showed neurokinin A-like immunoreactivity. Compared with the overall size distribution of renal afferent neurons, those staining for neurokinin A were mostly small-sized neurons with a few medium-sized neurons. All somata with neurokinin A-like immunoreactivity were neurofilament-poor as judged by labelling with an anti-neurofilament antibody, RT97, and it is therefore likely that they had unmyelinated fibres. To examine the sites to which the renal afferent fibres with neurokinin A might project, sections of rat and guinea-pig kidney and upper ureter were examined. Fibres with neurokinin A-like immunoreactivity were found beneath and within the transitional epithelium lining the inner surface of the pelvis, and within the smooth muscle layer beneath the transitional epithelium. Epithelial innervation was found only in regions with underlying smooth muscle and loose connective tissue, and not in sites where the epithelium was closely applied to the renal parenchyma. The network of fibres was most dense towards the pelvo-uretic junction. Fibres with neurokinin A-like immunoreactivity were not seen beneath or within the cuboidal/columnar epithelium covering the papilla within the renal pelvis. Furthermore, only very few fibres with neurokinin A were observed penetrating the transitional epithelium of the upper ureter in both rat and guinea-pig. The distribution of fibres labelled with antibodies to substance P and calcitonin gene-related peptide in the renal pelvis was similar to that for fibres with neurokinin A-like immuno-reactivity, although a few fibres penetrated further into the fornices than fibres with neurokinin-A-like immunoreactivity. Thus, many afferent fibres in the renal pelvis may contain neurokinin A as well as substance P and calcitonin gene-related peptide. These fibres may be the source of the neurokinin A, substance P and calcitonin gene-related peptide which can be released by topical capsaicin treatment. In addition they may be the mechano- and chemo-receptive fibres in the renal pelvis that are known to play important roles in renal haemodynamics. The intra-epithelial position of some of these fibres in the epithelial layer suggests a possible chemosensory or osmosensory role.     

Vestibular ganglion neurons survive the loss of their cerebellar targets

Neuronal survival during mammalian development crucially depends on target-derived neurotrophic factors. Target loss removes this trophic support and leads in most cases to the transsynaptic retrograde degeneration of the respective afferents. Primary vestibular afferents (PVA) originating from bipolar neurons in the vestibular ganglion (VG) are the first mossy fibers that enter the cerebellum, but little is known about the survival requirements of VG neurons. In the present study the influence of the differential granule cell (GC) target loss on the survival of VG neurons was studied quantitatively using unbiased stereological methods in the cerebellar mutants Purkinje cell degeneration (pcd/pcd), Lurcher (Lc/+), and Weaver (wv/wv). Neither the secondary GC loss in the Purkinje cell deficient mutants pcd/pcd and Lc/+, nor the primary loss of GCs in wv/wv produced any significant reduction in the total number of bipolar neurons in the VG compared to controls. So, PVA neurons are highly resistant to cerebellar target deprivation and survive in the absence of cerebellar granule and Purkinje cells, regardless of whether the target loss occurs before (in wv/wv), during (in Lc/+) or after (in pcd/pcd) the mossy fiber-granule cell synaptogenesis.     

Analysis of spontaneous activity of auditory neurones in the spiral ganglion of the guinea-pig cochlea

1. Spontaneous activity of single afferent neurones in the cochlea of the guinea-pig was studied by measurement of mean rates and computation of interspike interval histograms. 2. The shapes of interval histograms agreed with those found in cat cochlear nerve fibres. Modes of the histograms were less than 7 msec for all cells with mean rates ranging from 20 to 150 spikes/sec. 3. The distribution of mean rates showed higher maximum rates than those found in cat. Cells from animals with abnormally high thresholds showed lower mean rates than those from sensitive animals. 4. No correlation was found between mean spontaneous rate and sensitivity to acoustic stimulation. No discrete populations of cells could be discerned on any of the criteria used. 5. The simultaneous effects of hypoxia on cell thresholds, mean spontaneous rate and scala media potentials are reported. These and other results are discussed in relation to possible sites of generation of spontaneous activity in primary auditory afferents.     

Enkephalinergic innervation of GABAergic neurons in the dorsal raphe nucleus of the rat

The preembedding double immunoreaction method was used to study interrelations of enkephalinergic and GABAergic neuronal elements in the dorsal raphe nucleus of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the peroxidase-antiperoxidase method and silver-gold intensified, which showed strongly and was specific. The GABA-like immunoreactive neurons were immunoreacted by the peroxidase-antiperoxidase method only. GABA-like neural somata were postsynaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. The enkephalin-like immunoreactive axon terminals were also found to synapse GABA-like immunoreactive dendrites. The GABA-like immunoreactive neuronal elements were also found to receive synapses from other non-immunoreactive as well as GABA-like immunoreactive axon terminals. Almost all of the synapses appeared to be asymmetrical. Possible functional activity of interactions among the enkephalinergic, GABAergic, and serotonergic neuronal elements in the dorsal raphe nucleus are discussed.     

Influence of the subesophageal ganglion on the electrical activity of neurons of the metathoracic ganglion of the cockroach Periplaneta americana

Studies have been made of non-specific effect of suboesophageal gnaglion on excitability of the segmental centers in the cockroach P. americana. It was shown that the electrical stimulation of the ganglion increases the frequency of the spontaneous activity and decreases the threshold of the evoked reactions in neurons of the metathoracic ganglion. The level of activation depends on the intensity of stimulation. Application of GABA (0.1 M) to the suboesophageal ganglion decreases the frequency of the background activity in the segmental neurons, this effect being presumably due to activation of the inhibitory structures. The descending influences from the suboesophageal ganglion may spread to centers of the metathoracic ganglion along monosynaptic pathways.     

Transneuronal labeling of CNS neurons involved in the innervation of the adrenal gland

In the light of the observation obtained with the application of the viral-tract tracing method, the CNS connection of the adrenal gland, with focusing on the central nervous connection of the adrenal cortex is reviewed.     

Characterization of neurons of the nodose ganglion having constant impulse activity

The present studies were undertaken to characterize selenium distribution in egg white. Ion-exchange chromatography fast protein liquid chromatography (FPLC) and flow injection atomic (absorption) spectrometry (FIAS) were used to separate egg white proteins and to determine the selenium content of different fractions. After purification, nine different proteins were identified with sodium dodecyl sulphate-polyacrylamide gel electrophoresis and 56% of the total selenium content was found to be associated with ovalbumin-1 and -2 (+/- 500 ng/g), which is the main protein in egg white. Flavoprotein was determined to be the richest selenium-containing protein (1800 ng/g). The selenium content of the other proteins (lysozyme, conalbumin, globulins and ovomucoid) ranged from 359 to 1094 ng/g.