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Physiological differences in low- (tonic like) and high-output (phasic like) synapses match many of the expected anatomical features of these terminals. However, investigation in the recruitment of synaptic vesicles from a reserve pool (RP) to a readily releasable pool (RRP) of synaptic vesicles within these types of nerve terminals has not been fully addressed. This study highlights physiological differences and differential modulation of the vesicles in a RP for maintaining synaptic output during evoked depression of the RRP. With the use of bafilomycin A1, a vacuolar ATPase blocker, recycling vesicles are blocked in refilling with transmitter. The tonic terminal is fatigue resistant due to a large RRP, whereas the phasic depresses rapidly upon continuous stimulation. These differences in rates of depression appear to be in the size and degree of utilization of the RRP of vesicles. The working model is that upon depression of the tonic terminal, serotonin (5-HT) has a large RP to act on in order to recruit vesicles to the RRP; whereas, the phasic terminal, 5-HT can recruit RP vesicles to the RRP prior to synaptic depression but not after depression. The vesicle pools are physiologically differentiated between phasic and tonic output terminals., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Calcium channels that mediate glutamate release (N-type and P/Q-type) are expressed in distinct populations of cerebrocortical nerve terminals in adult mice. mGlu7 receptors are exclusively expressed in nerve terminals containing N-type Ca(2+) channels, which are less tightly coupled to glutamate release than P/Q-type Ca(2+) channels. We recently reported that in addition to inhibit, mGlu7 receptors can also potentiate glutamate release via phosphatidyl inositol (4,5)-bisphosphate hydrolysis and activation of the non-kinase diacylglycerol binding protein Munc13-1, a protein that primes synaptic vesicles for exocytosis. Here, we assessed whether mGlu7 receptor-mediated potentiation of glutamate release is restricted to nerve terminals expressing N-type Ca(2+) channels to compensate for their weak coupling to release. In the hippocampus, mGlu7 receptors are expressed both in nerve terminals containing N-type Ca(2+) channels and in nerve terminals containing P/Q-type Ca(2+) channels. When analyzed, we observed potentiation of mGlu7 receptor mediated release in wild type hippocampal nerve terminals at physiological (1.3 mM) and low (0.1 mM) concentrations of external Ca(2+). By contrast, in nerve terminals from mice lacking the α1B subunit of N-type channels (Ca(v)2.2), in which evoked release is mediated by P/Q-type channels only, no release potentiation was observed at 1.3 mM Ca(2+). We conclude that release potentiation at 1.3 mM [Ca(2+)](e) occurs in nerve terminals expressing N-type channels, whereas that which occurs at low 0.1 mM [Ca(2+)](e) represents the release from nerve terminals containing P/Q-type Ca(2+) channels. Although, mGlu7 receptor mediated potentiation is independent of Ca(2+) channel activity, as it was induced by the Ca(2+) ionophore ionomycin, release potentiation is influenced by the Ca(2+) channel type and/or the associated release machinery., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pH(cyto)) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pH(cyto) shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pH(cyto). Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤ 4 s) trains of action potentials but did buffer slow (~60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca(2+) increase upon stimulation, and partial inhibition of the plasma membrane Ca(2+)-ATPase, a Ca(2+)/H(+) exchanger, attenuated pH(cyto) shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (~0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pH(cyto) shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pH(cyto) shifts cannot be dismissed as artifacts of ex vivo preparations.

Palisade endings are nerve specializations found in the extraocular muscles (EOMs) of mammals, including primates. They have long been postulated to be proprioceptors. It was recently demonstrated that palisade endings are cholinergic and that in monkeys they originate from the EOM motor nuclei. Nevertheless, there is considerable difference of opinion concerning the nature of palisade ending function. Palisade endings in EOMs were examined in cats to test whether they display motor or sensory characteristics. We injected an anterograde tracer into the oculomotor or abducens nuclei and combined tracer visualization with immunohistochemistry and α-bungarotoxin staining. Employing immunohistochemistry, we performed molecular analyses of palisade endings and trigeminal ganglia to determine whether cat palisade endings are a cholinergic trigeminal projection. We confirmed that palisade endings are cholinergic and showed, for the first time, that they, like extraocular motoneurons, are also immunoreactive for calcitonin gene-related peptide. Following tracer injection into the EOM nuclei, we observed tracer-positive palisade endings that exhibited choline acetyl transferase immunoreactivity. The tracer-positive nerve fibers supplying palisade endings also established motor terminals along the muscle fibers, as demonstrated by α-bungarotoxin. Neither the trigeminal ganglion nor the ophthalmic branch of the trigeminal nerve contained cholinergic elements. This study confirms that palisade endings originate in the EOM motor nuclei and further indicates that they are extensions of the axons supplying the muscle fiber related to the palisade. The present work excludes the possibility that they receive cholinergic trigeminal projections. These findings call into doubt the proposed proprioceptive function of palisade endings.

VGLUT3-expressing unmyelinated low-threshold mechanoreceptors (C-LTMRs) are proposed to mediate pleasant touch and/or pain, but the molecular programs controlling C-LTMR development are unknown. Here, we performed genetic fate mapping, showing that VGLUT3 lineage sensory neurons are divided into two groups, based on transient or persistent VGLUT3 expression. VGLUT3-transient neurons are large- or medium-diameter myelinated mechanoreceptors that form the Merkel cell-neurite complex. VGLUT3-persistent neurons are small-diameter unmyelinated neurons that are further divided into two subtypes: (1) tyrosine hydroxylase (TH)-positive C-LTMRs that form the longitudinal lanceolate endings around hairs, and (2) TH-negative neurons that form epidermal-free nerve endings. We then found that VGLUT3-persistent neurons express the runt domain transcription factor Runx1. Analyses of mice with a conditional knock-out of Runx1 in VGLUT3 lineage neurons demonstrate that Runx1 is pivotal to the development of VGLUT3-persistent neurons, such as the expression of VGLUT3 and TH and the formation of the longitudinal lanceolate endings. Furthermore, Runx1 is required to establish mechanosensitivity in C-LTMRs, by controlling the expression of the mechanically gated ion channel Piezo2. Surprisingly, both acute and chronic mechanical pain was largely unaffected in these Runx1 mutants. These findings appear to argue against the recently proposed role of VGLUT3 in C-LTMRs in mediating mechanical hypersensitivity induced by nerve injury or inflammation. Thus, our studies provide new insight into the genetic program controlling C-LTMR development and call for a revisit for the physiological functions of C-LTMRs.

Parvalbumin (PV)-producing fast-spiking neurons are well known to generate gamma oscillation by mutual chemical and electrical connections in the neocortex. Although it was clearly demonstrated that PV neurons form a dense gap junction network with each other not only at the proximal sites but also at the distal dendrites, comprehensive quantitative data on the chemical connections are still lacking. To elucidate the connectivity, we investigated inhibitory inputs to PV neurons in the somatosensory cortex, using the transgenic mice in which the dendrites and cell bodies of PV neurons were clearly visualized. We first examined GABAergic inputs to PV neurons by labeling postsynaptic and presynaptic sites with the immunoreactivities for gephyrin and vesicular GABA transporter. The density of GABAergic inputs was highest on the cell bodies, and almost linearly decreased to the distal dendrites. We then investigated inhibitory inputs from three distinct subgroups of GABAergic interneurons by visualizing the axon terminals immunopositive for PV, somatostatin (SOM), or vasoactive intestinal polypeptide (VIP). PV and SOM inputs were frequently located on the dendrites with the ratio of 2.5:1, but much less on the cell bodies. By contrast, VIP inputs clearly preferred the cell bodies to the dendrites. Consequently, the dendritic and somatic compartments of PV neurons received ∼60 and 62% of inhibitory inputs from PV and VIP neurons, respectively. This compartmental organization of inhibitory inputs suggests that PV neurons, together with gap junctions, constitute mutual connections at the dendrites, and that their activities are negatively controlled by the somatic inputs of VIP neurons.

Spinocerebellar tract neurons are inhibited by various sources of input via pathways activated by descending tracts as well as peripheral afferents. Inhibition may be used to modulate transmission of excitatory information forwarded to the cerebellum. However it may also provide information on the degree of inhibition of motoneurons and on the operation of inhibitory premotor neurons. Our aim was to extend previous comparisons of morphological substrates of excitation of spinocerebellar neurons to inhibitory input. Contacts formed by inhibitory axon terminals were characterised as either GABAergic, glycinergic or both GABAergic/glycinergic by using antibodies against vesicular GABA transporter, glutamic acid decarboxylase and gephyrin. Quantitative analysis revealed the presence of much higher proportions of inhibitory contacts when compared with excitatory contacts on spinal border (SB) neurons. However similar proportions of inhibitory and excitatory contacts were associated with ventral spinocerebellar tract (VSCT) and dorsal spinocerebellar tract neurons located in Clarke's column (ccDSCT) and the dorsal horn (dhDSCT). In all of the cells, the majority of inhibitory terminals were glycinergic. The density of contacts was higher on somata and proximal versus distal dendrites of SB and VSCT neurons but more evenly distributed in ccDSCT and dhDSCT neurons. Variations in the density and distribution of inhibitory contacts found in this study may reflect differences in information on inhibitory processes forwarded by subtypes of spinocerebellar tract neurons to the cerebellum., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mixed (electrical and chemical) synaptic contacts on the Mauthner cells, known as Club endings, constitute a valuable model for the study of vertebrate electrical transmission. While electrical synapses are still perceived by many as passive intercellular channels that lack modifiability, a wealth of experimental evidence shows that gap junctions at Club endings are subject to dynamic regulatory control by two independent activity-dependent mechanisms that lead to potentiation of electrical transmission. One of those mechanisms relies on activation of NMDA receptors and postsynaptic CaMKII. A second mechanism relies on mGluR activation and endocannabinoid production and is indirectly mediated via the release of dopamine from nearby varicosities, which in turn leads to potentiation of the synaptic response via a PKA-mediated postsynaptic mechanism. We review here these two forms of potentiation and their signaling mechanisms, which include the activation of two kinases with well-established roles as regulators of synaptic strength, as well as the functional implications of these two forms of potentiation. Special Issue entitled Electrical Synapses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mammalian peripheral cold thermoreceptors respond to cooling of their sensory endings with an increase in firing rate and modification of their discharge pattern. We recently showed that cultured trigeminal cold-sensitive (CS) neurons express a prominent hyperpolarization-activated current (I(h)), mainly carried by HCN1 channels, supporting subthreshold resonance in the soma without participating in the response to acute cooling. However, peripheral pharmacological blockade of I(h), or characterization of HCN1(-/-) mice, reveals a deficit in acute cold detection. Here we investigated the role of I(h) in CS nerve endings, where cold sensory transduction actually takes place. Corneal CS nerve endings in mice show a rhythmic spiking activity at neutral skin temperature that switches to bursting mode when the temperature is lowered. I(h) blockers ZD7288 and ivabradine alter firing patterns of CS nerve endings, lengthening interspike intervals and inducing bursts at neutral skin temperature. We characterized the CS nerve endings from HCN1(-/-) mouse corneas and found that they behave similar to wild type, although with a lower slope in the firing frequency vs. temperature relationship, thus explaining the deficit in cold perception of HCN1(-/-) mice. The firing pattern of nerve endings from HCN1(-/-) mice was also affected by ZD7288, which we attribute to the presence of HCN2 channels in the place of HCN1. Mathematical modeling shows that the firing phenotype of CS nerve endings from HCN1(-/-) mice can be reproduced by replacing HCN1 channels with the slower HCN2 channels rather than by abolishing I(h). We propose that I(h) carried by HCN1 channels helps tune the frequency of the oscillation and the length of bursts underlying regular spiking in cold thermoreceptors, having important implications for neural coding of cold sensation.

This article describes current views on motor and sensory control of extraocular muscles (EOMs) based on anatomical data. The special morphology of EOMs, including their motor innervation, is described in comparison to classical skeletal limb and trunk muscles. The presence of proprioceptive organs is reviewed with emphasis on the palisade endings (PEs), which are unique to EOMs, but the function of which is still debated. In consideration of the current new anatomical data about the location of cell bodies of PEs, a hypothesis on the function of PEs in EOMs and the multiply innervated muscle fibres they are attached to is put forward.

Nonvisual pineal and retinal photoreceptors are synchronizing circadian and circannual periodicity to the environmental light periods in the function of various organs. Melatonin of the pineal organ is secreted at night and represents an important factor of this periodic regulation. Night illumination suppressing melatonin secretion may result in pathological events like breast and colorectal cancer. Experimental works demonstrated the role of autonomic nerves in the pineal melatonin secretion. It was supposed that mammalian pineals have lost their photoreceptor capacity that is present in submammalians, and sympathetic fibers would mediate light information from the retina to regulate melatonin secretion. Retinal afferentation may reach the organ by central nerve fibers via the pineal habenulae as well. In our earlier works we have found that the pineal organ developing from lobular evaginations of the epithalamus differs from peripheral endocrine glands and is composed of a retina-like central nervous tissue that is comprised of cone-like pinealocytes, secondary pineal neurons and glial cells. Their autonomic nerves in submammalians as well as in mammalian animals do not terminate on pineal cells, rather, they run in the meningeal septa among pineal lobules and form vasomotor nerve endings. Concerning the adult human pineal there are no detailed fine structural data about the termination of autonomic fibers, therefore, in the present work we investigated the ultrastructure of the human pineal peripheral autonomic nerve fibers. It was found, that similarly to other parts of the brain, autonomic nerves do not enter the human pineal nervous tissue itself but separated by glial limiting membranes take their course in the meningeal septa of the organ and terminate on vessels by vasomotor endings. We suppose that these autonomic vasomotor nerves serve the regulation of the pineal blood supply according to the circadian and circannual changes of the metabolic activity of the organ and support by this effect the secretion of pineal neurohormones including melatonin., (© 2012 The Authors APMIS © 2012 APMIS.)

We examined abdominal vagal afferents (n = 33) and the distributions of their intraganglionic laminar endings (IGLEs) in the duodenum. Rats (male, Sprague-Dawley) received a partial subdiaphragmatic vagotomy that spared a single branch. Wheat germ agglutinin-horseradish peroxidase (0.5-1.0 μl) was injected into the nodose ganglion ipsilateral to the vagotomized side. We observed that the hepatic branch does not project to the stomach, that the accessory celiac and celiac branches course along the celiac artery and innervate the intestines, and that the left nodose afferents innervate predominantly the duodenum. The hepatic branch innervates the duodenum via the "hepatoduodenal" subbranch and has the densest IGLE distribution in both the dorsoventral and the rostrocaudal extensions of the first 4-cm segment. Both gastric branches have two subbranches that innervate the duodenum; the "lesser curvature" subbranches follow the lesser curvature artery and may join the "hepatoduodenal" subbranch, whereas the "pyloric" subbranches run through the antrum and pylorus to reach the proximal duodenum. Moreover, the subbranches of ventral and dorsal gastric branches innervate more in the ventral and dorsal parts of the duodenum, respectively, and have more IGLEs in the rostral region than in the caudal. A posteriori comparisons indicate that, in the first-centimeter segment, the ventral gastric branch has significantly more IGLEs, whereas, in the third- and fourth-centimeter segments, the hepatic branch has more IGLEs. The finding that three different vagal branches innervate the duodenum with different densities of afferent endings might indicate a viscerotopic receptive field that coordinates digestive functions in feeding., (Copyright © 2011 Wiley Periodicals, Inc.)

Purpose: To determine the effects of end-to-side nerve repair performed only with fibrin glue containing nerve growth in rats., Methods: Seventy two Wistar rats were divided into six equal groups: group A was not submitted to nerve section; group B was submitted to nerve fibular section only. The others groups had the nerve fibular sectioned and then repaired in the lateral surface of an intact tibial nerve, with different procedures: group C: ETS with sutures; group D: ETS with sutures and NGF; group E: ETS with FG only; group F: ETS with FG containing NGF. The motor function was accompanied and the tibial muscle mass, the number and diameter of muscular fibers and regenerated axons were measured., Results: All the analyzed variables did not show any differences among the four operated groups (p>0.05), which were statistically superior to group B (p<0.05), but inferior to group A (p>0.05)., Conclusion: The end-to-side nerve repair presented the same recovery pattern, independent from the repair used, showing that the addition of nerve growth factor in fibrin glue was not enough for the results potentiating.

The rodents are an excellent model for understanding the development and plasticity of the visual system. In this study, we explored the feasibility of Mn-enhanced MRI (MEMRI) and diffusion tensor imaging (DTI) at 7 T for in vivo and longitudinal assessments of the retinal and callosal pathways in normal neonatal rodent brains and after early postnatal visual impairments. Along the retinal pathways, unilateral intravitreal Mn2+ injection resulted in Mn2+ uptake and transport in normal neonatal visual brains at postnatal days (P) 1, 5 and 10 with faster Mn2+ clearance than the adult brains at P60. The reorganization of retinocollicular projections was also detected by significant Mn2+ enhancement by 2%-10% in the ipsilateral superior colliculus (SC) of normal neonatal rats, normal adult mice and adult rats after neonatal monocular enucleation (ME) but not in normal adult rats or adult rats after monocular deprivation (MD). DTI showed a significantly higher fractional anisotropy (FA) by 21% in the optic nerve projected from the remaining eye of ME rats compared to normal rats at 6 weeks old, likely as a result of the retention of axons from the ipsilaterally uncrossed retinal ganglion cells, whereas the anterior and posterior retinal pathways projected from the enucleated or deprived eyes possessed lower FA after neonatal binocular enucleation (BE), ME and MD by 22%-56%, 18%-46% and 11%-15% respectively compared to normal rats, indicative of neurodegeneration or immaturity of white matter tracts. Along the visual callosal pathways, intracortical Mn2+ injection to the visual cortex of BE rats enhanced a larger projection volume by about 74% in the V1/V2 transition zone of the contralateral hemisphere compared to normal rats, without apparent DTI parametric changes in the splenium of corpus callosum. This suggested an adaptive change in interhemispheric connections and spatial specificity in the visual cortex upon early blindness. The results of this study may help determine the mechanisms of axonal uptake and transport, microstructural reorganization and functional activities in the living visual brains during development, diseases, plasticity and early interventions in a global and longitudinal setting., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hairs are known as a sensory apparatus for touch. Their follicles are innervated predominantly by palisade endings composed of longitudinal and circumferential lanceolate endings. However, little is known as to how their original primary neurons make up a part of the ending. In this study, innervation of the palisade endings was investigated in the auricular skin of thy1-YFP transgenic mouse. Major observations were 1) Only a small portion of PGP9.5-immunopositive axons showed YFP-positivity, 2) All of thy1-YFP-positive sensory axons were thick and myelinated, 3) Individual thy1-YFP-positive trunk axons innervated 4-54 hair follicles, 4) Most palisade endings had a gap of lanceolate ending arrangement, 5) PGP9.5-immunopositive 10-32 longitudinal lanceolate endings were closely arranged. Only a part of them were thy1-YFP-positive axons that originated from 1-3 afferents, and 6) Single nerve bundles of the dermal nerve network included both bidirectional afferents. Palisade endings innervated by multiple sensory neurons might be highly sensitive to hair movement.

Afferent signals from the stomach play an important role in inhibition of food intake during a meal. The gastric hormone ghrelin can influence gastric satiety signalling by altering the sensitivity of gastric vagal afferents. Changes in diet, including food restriction and high fat diet (HFD) alter satiety signalling. We hypothesised that the function of gastric vagal afferent endings are affected by both a period of food restriction and a high fat diet, and that the inhibitory effect of ghrelin on vagal afferents is influenced by the different feeding conditions. We found that both fasting and HFD reduced the responses of gastric vagal tension receptors to distension, but not responses of mucosal receptors to mucosal contact. We traced vagal afferents anterogradely to their terminals in the mucosa where we found they were in close apposition to ghrelin-containing cells. Ghrelin receptor mRNA was expressed in vagal afferent cell bodies of the nodose ganglia, and increased in response to caloric restriction, but decreased in HFD mice. In control mice, ghrelin decreased the sensitivity of tension but not mucosal receptors. After caloric restriction or high fat diet, ghrelin inhibited mucosal receptors, and the inhibition of mechanosensitive tension receptors was enhanced. Therefore, both caloric restriction and HFD decrease mechanosensory vagal afferent signals, and augment the inhibitory effect of ghrelin on vagal afferents, but different mechanisms mediate the short- and longer-term changes.

We established the effect of ATP, which is released together with acetylcholine (ACh), on the non-quantal ACh release (NQR) in rat diaphragm endplates and checked what kind of purine receptors are involved. NQR was estimated by the amplitude of endplate hyperpolarization (the H-effect) following the blockade of postsynaptic nicotinic receptors and cholinesterase. 100 μM ATP reduced the H-effect to 66% of the control. The action of ATP remained unchanged after the inhibition of ionotropic P2X receptors by Evans blue and PPADS, but disappeared after the application of the broad spectrum P2 receptor antagonist suramin, metabotropic P2Y receptor blocker reactive blue 2 and U73122, an inhibitor of phospholipase C. P2Y-mediated regulation is not coupled to presynaptic voltage-dependent Ca(2+) channels. During the simultaneous application of ATP and glutamate (which is another ACh cotransmitter reducing non-quantal release), the additive depressant effect led to a disappearance of the H-effect. This can be explained by the independence of the action of ATP and glutamate. Unlike the effects of purines on the spontaneous quantal secretion of ACh, its non-quantal release is regulated via P2Y receptors coupled to G(q/11) and PLC. ATP thus regulates the neuromuscular synapse by two different pathways., (Copyright © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Epidermal growth factor (EGF) and neuregulin-1 (NRG) belong to the ErbB ligand family and both exert neurotrophic actions on midbrain dopamine neurons. According to the immune inflammatory hypothesis for schizophrenia, we have established rodent models for this illness by exposing their neonates to these cytokines. At post-pubertal stage, these animals develop various neurobehavioral abnormalities such as prepulse inhibition (PPI) and social interaction deficits. In this review, we introduce neurochemical features of the EGF-treated rats and NRG-treated mice, which exhibit persistent increases in tyrosine hydroxylase levels and dopamine release in the globus pallidus and prelimbic cortex (medial prefrontal cortex), respectively. Local blockade of the hyperdopaminergic state in EGF-treated rats ameliorates their behavioral deficits. These findings suggest that development of the midbrain dopamine system is vulnerable to circulating cytokines at perinatal and/or prenatal stages and potentially influences schizophrenia risk or neuropathology. The dopamine hypothesis for schizophrenia is re-evaluated with the obtained results as well as with published literatures in this review.

Vertebrate neuromuscular junctions are highly stable synapses, retaining the morphology they achieve in early postnatal development throughout most of life. However, these synapses undergo dramatic change during aging. The acetylcholine receptors (AChRs) change from smooth gutters into fragmented islands, and the nerve terminals change similarly to be varicosities apposed to these islands. These changes have been attributed to a slow deterioration in mechanisms maintaining the synapse. We have used repeated, vital imaging to investigate how these changes occur in the sternomastoid muscle of aging mice. We have found, contrary to expectation, that individual junctions change infrequently, but change, when it occurs, is sudden and dramatic. The change mimics that reported previously for cases in which muscle fibers are deliberately damaged: most of the AChRs present disappear rapidly and are replaced by a new set of receptors that become fragmented. The fiber segment underneath the synapse has centrally located nuclei, showing that this segment has undergone necrosis, quickly regenerated, and been reinnervated with an altered synapse. We show that necrotic events are common in aged muscle and have likely been missed previously as a cause of the alterations in aging because central nuclei are a transient phenomenon and the necrotic events at the junction infrequent. However, the changes are permanent and accumulate over time. Interventions to reduce the neuromuscular changes during aging should likely focus on making muscle fibers resistant to injury.

4-Aminopyridine (4-AP) is a convulsing agent that in vivo preferentially releases Glu, the most important excitatory amino acid neurotransmitter in the brain. Here the ionic dependence of 4-AP-induced Glu release and the effects of several of the most common antiepileptic drugs (AEDs) and of the new potential AED, vinpocetine on 4-AP-induced Glu release were characterized in hippocampus isolated nerve endings pre-loaded with labelled Glu ([3H]Glu). 4-AP-induced [3H]Glu release was composed by a tetrodotoxin (TTX) sensitive and external Ca2+ dependent fraction and a TTX insensitive fraction that was sensitive to the excitatory amino acid transporter inhibitor, TBOA. The AEDs: carbamazepine, phenytoin, lamotrigine and oxcarbazepine at the highest dose tested only reduced [3H]Glu release to 4-AP between 50-60%, and topiramate was ineffective. Vinpocetine at a much lower concentration than the above AEDs, abolished [3H]Glu release to 4-AP. We conclude that the decrease in [3H]Glu release linked to the direct blockade of presynaptic Na+ channels, may importantly contribute to the anticonvulsant actions of all the drugs tested here (except topiramate); and that the significantly greater vinpocetine effect in magnitude and potency on [3H]Glu release when excitability is exacerbated like during seizures, may involve the increase additionally exerted by vinpocetine in some K+ channels permeability., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Release of conventional neurotransmitters is mainly controlled by calcium (Ca²⁺) influx via high-voltage-activated (HVA), Ca(v)2, channels ("N-, P/Q-, or R-types") that are opened by action potentials. Regulation of transmission by subthreshold depolarizations does occur, but there is little evidence that low-voltage-activated, Ca(v)3 ("T-type"), channels take part. GABA release from cortical perisomatic-targeting interneurons affects numerous physiological processes, and yet its underlying control mechanisms are not fully understood. We investigated whether T-type Ca²⁺ channels are involved in regulating GABA transmission from these cells in rat hippocampal CA1 using a combination of whole-cell voltage-clamp, multiple-fluorescence confocal microscopy, dual-immunolabeling electron-microscopy, and optogenetic methods. We show that Ca(v)3.1, T-type Ca²⁺ channels can be activated by α3β4 nicotinic acetylcholine receptors (nAChRs) that are located on the synaptic regions of the GABAergic perisomatic-targeting interneuronal axons, including the parvalbumin-expressing cells. Asynchronous, quantal GABA release can be triggered by Ca²⁺ influx through presynaptic T-type Ca²⁺ channels, augmented by Ca²⁺ from internal stores, following focal microiontophoretic activation of the α3β4 nAChRs. The resulting GABA release can inhibit pyramidal cells. The T-type Ca²⁺ channel-dependent mechanism is not dependent on, or accompanied by, HVA channel Ca²⁺ influx, and is insensitive to agonists of cannabinoid, μ-opioid, or GABA(B) receptors. It may therefore operate in parallel with the normal HVA-dependent processes. The results reveal new aspects of the regulation of GABA transmission and contribute to a deeper understanding of ACh and nicotine actions in CNS.

Muscle fibers degenerate and regenerate in response to contractile damage, during aging, and in various muscle diseases that weaken the fibers. It is known that degeneration and regeneration of the segment of the postsynaptic fiber produces dramatic alterations in the neuromuscular junction (NMJ) that forms on the regenerated fiber, but the mechanisms here are incompletely understood. We have used a laser microbeam to damage the postsynaptic fibers at individual NMJs in the sternomastoid muscle of living young adult mice and then followed the synapses vitally over time using fluorescent proteins expressed in motor neurons and glial cells and staining of postsynaptic acetylcholine receptors. We find, in contrast to previous reports, that the mouse nerve terminal retains contact with the synaptic basal lamina marked by cholinesterase staining even in the absence of the target, showing that this terminal does not require a continuous supply of target-derived molecules for its maintenance. Thus, remodeling of the nerve terminal during the period of target absence does not explain the subsequent changes in the new NMJ. Rather, we see that the synapse becomes altered as the new fiber segment regenerates. Mechanisms for remodeling the synapse include failure of the regenerating muscle fiber to contact the old basal lamina and nerve terminal, growth of the nerve terminal and its glia toward the regenerating fiber, and remodeling of the initial contact as the nerve terminal becomes varicose.

Palisade endings (PEs), which are unique to the eye muscles, are associated with multiply innervated muscle fibers. They lie at the myotendinous junctions and form a cap around the muscle fiber tip. They are found in all animals investigated so far, but their function is not known. Recently, we demonstrated that cell bodies of PEs and tendon organs lie around the periphery of the oculomotor nucleus in the C- and S-groups. A morphological analysis of these peripheral neurons revealed the existence of different populations within the C-group. We propose that a small group of round or spindle-shaped cells gives rise to PEs, and another group of multipolar neurons provide the multiple motor endings. If PEs have a sensory function, then their cell body location close to motor neurons would be in an ideal location to control tension in extraocular muscles; in the case of the C-group, its proximity to the preganglionic neurons of the Edinger-Westphal nucleus would permit its participation in the near response. Despite their unusual properties, PEs may have a sensory function., (© 2011 New York Academy of Sciences.)

There is abundant evidence suggesting the relevance of glutamate to depression and antidepressant mechanisms. Curcumin, a major active compound of Curcuma longa, has been reported to have the biological function of antidepressant. The aim of the present study was to investigate the effect of curcumin on endogenous glutamate release in nerve terminals of rat prefrontal cortex and the underlying mechanisms. The results showed that curcumin inhibited the release of glutamate that was evoked by exposing synaptosomes to the K(+) channel blocker 4-aminopyridine (4-AP). This phenomenon was blocked by the chelating the extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyl-oxyaspartate (DL-TBOA). Further experiments demonstrated that curcumin decreased depolarization-induced increase in [Ca(2+)](C), whereas it did not alter the resting membrane potential or 4-AP-mediated depolarization. Furthermore, the inhibitory effect of curcumin on evoked glutamate release was prevented by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release or Na(+)/Ca(2+) exchange. These results suggest that curcumin inhibits evoked glutamate release from rat prefrontocortical synaptosomes by the suppression of presynaptic Ca(v)2.2 and Ca(v)2.1 channels. Additionally, we also found that the inhibitory effect of curcumin on 4-AP-evoked glutamate release was completely abolished by the clinically effective antidepressant fluoxetine. This suggests that curcumin and fluoxetine use a common intracellular mechanism to inhibit glutamate release from rat prefrontal cortex nerve terminals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We observed endocytosis in real time in stimulated frog motor nerve terminals by imaging the growth of large membrane infoldings labeled with a low concentration of FM dye. The spatial and temporal information made available by these experiments allowed us to image several new aspects of this synaptic vesicle recycling pathway. Membrane infoldings appeared near synaptic vesicle clusters and grew rapidly during long-duration, high-frequency stimulation. In some cases, we observed large, elongated infoldings growing laterally into the terminal. We used these observations to calculate infolding growth rates. A decrease in stimulation frequency caused a decrease in growth rates, but the overall length of these structures was unaffected by frequency changes. Attempts to wash the dye from these infoldings after stimulation were unsuccessful, demonstrating that the fluorescent structures had been endocytosed. We also used this technique to trigger and image infoldings during repeated, short trains. We found that membrane uptake occurred repeatedly at individual endocytosis sites, but only during a portion of the total number of trains delivered to the terminal. Finally, we showed that phosphatidylinositol 3-kinase, but not actin, was involved in this endocytosis pathway. The ability to monitor many individual bulk endocytosis sites in real time should allow for new types of endocytosis measurements and could reveal novel and unexpected mechanisms for coordinating membrane recovery during synaptic activity.

Chronic unproductive coughing and dyspnea are symptoms that severely diminish the quality of life in a substantial proportion of the population. There are presently few if any drugs that effectively treat these symptoms. Rational drug targets for cough and dyspnea have emerged over the recent years based on developments in our understanding of the innervation of the respiratory tract. These drug targets can be subcategorized into those that target the vagal afferent nerve endings, and those that target neural activity within the CNS. This review focuses on targets presumed to be in the peripheral terminals of afferent nerves within the airways. Conceptually, the activity of peripheral afferent nerves involved with unwanted urge-to-cough or dyspnea sensations can be inhibited by limiting the intensity of the stimulus, inhibiting the amplitude of the stimulus-induced generator potential, or inhibiting the transduction between the generator potential and action potential discharge and conduction. These mechanisms reveal many therapeutic strategies for anti-tussive and anti-dyspnea drug development with peripheral sites of action., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Neural competition among multiple inputs can affect the refinement and maintenance of terminal fields in sensory systems. In the rat gustatory system, the chorda tympani, greater superficial petrosal, and glossopharyngeal nerves have distinct but overlapping terminal fields in the first central relay, the nucleus of the solitary tract. This overlap is largest at early postnatal ages followed by a significant refinement and pruning of the fields over a 3 week period, suggesting that competitive mechanisms underlie the pruning. Here, we manipulated the putative competitive interactions among the three nerves by sectioning the greater superficial petrosal and glossopharyngeal nerves at postnatal day 15 (P15), P25, or at adulthood, while leaving the chorda tympani nerve intact. The terminal field of the chorda tympani nerve was assessed 35 d following nerve sections, a period before the sectioned nerves functionally regenerated. Regardless of the age when the nerves were cut, the chorda tympani nerve terminal field expanded to a volume four times larger than sham controls. Terminal field density measurements revealed that the expanded terminal field was similar to P15 control rats. Thus, it appears that the chorda tympani nerve terminal field defaults to its early postnatal field size and shape when the nerves with overlapping fields are cut, and this anatomical plasticity is retained into adulthood. These findings not only demonstrate the dramatic and lifelong plasticity in the central gustatory system, but also suggest that corresponding changes in functional and taste-related behaviors will accompany injury-induced changes in brainstem circuits.

Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS. Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled, large-diameter DR axons in thy1-YFPH mice elongated through a DR crush site, but not a transection site, and grew along the root at >1.5 mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastructural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.

Dentinal hypersensitivity is a very common clinical finding that can cause considerable concern for the patient. Clinicians must understand the various etiological factors, their complexities, and numerous treatment options available. This article reviews the etiology, management, and prevention of dentinal hypersensitivity.

Aim: The present study was aimed at investigating the effect of trans-6-(4-chlorobutyl)-5-hydroxy-4-(phenylthio)-1-tosyl-5,6-dihydropyridine-2(1H)-one (HTDP-2), a novel synthetic compound, on the release of endogenous glutamate in rat cerebrocortical nerve terminals (synaptosomes) and exploring the possible mechanism., Methods: The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by an on-line enzyme-coupled fluorimetric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca²⁺ indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca²⁺ concentrations ([Ca²⁺](c))., Results: HTDP-2 inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by HTDP-2 was prevented by the chelating intraterminal Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyloxyaspartate. HTDP-2 did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in [Ca²⁺](c). Furthermore, the inhibitory effect of HTDP-2 on the evoked glutamate release was abolished by the N-, and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP37157., Conclusion: Based on these results, we suggest that, in rat cerebrocortical nerve terminals, HTDP-2 decreases voltage-dependent Ca²⁺ channel activity and, in so doing, inhibits the evoked glutamate release., (Copyright © 2011 S. Karger AG, Basel.)

The relationship between bone and nervous system has been considered based on clinical observations such as Reflex Sympathetic Dystrophy (RSD) or ectopic bone formation associated with spinal cord injury. Sympathtic nervous tone has been reported to control both bone formation and bone resorption. Unloading induces bone loss due to an increase in bone resorption and decrease in bone formation. Both of these two arms are shown to be influenced by sympathetic tone. In addition, cannabinoid receptor has been observed to be involved in regulation of bone mass. Psychiatric diseases such as depression has also been suggested to linked to the alteration in the levels of bone mass. These observations together point to importance of the relationship between bone mass and nervous system.

Renin, the rate-limiting enzyme in the activation of the renin-angiotensin system (RAS), is synthesized and stored in cardiac mast cells. In ischemia/reperfusion, cardiac sensory nerves release neuropeptides such as substance P that, by degranulating mast cells, might promote renin release, thus activating a local RAS and ultimately inducing cardiac dysfunction. We tested this hypothesis in whole hearts ex vivo, in cardiac nerve terminals in vitro, and in cultured mast cells. We found that substance P-containing nerves are juxtaposed to renin-containing cardiac mast cells. Chemical stimulation of these nerves elicited substance P release that was accompanied by renin release, with the latter being preventable by mast cell stabilization or blockade of substance P receptors. Substance P caused degranulation of mast cells in culture and elicited renin release, and both of these were prevented by substance P receptor blockade. Ischemia/reperfusion in ex vivo hearts caused the release of substance P, which was associated with an increase in renin and norepinephrine overflow and with sustained reperfusion arrhythmias; substance P receptor blockade prevented these changes. Substance P, norepinephrine, and renin were also released by acetaldehyde, a known product of ischemia/reperfusion, from cardiac synaptosomes and cultured mast cells, respectively. Collectively, our findings indicate that an important link exists in the heart between sensory nerves and renin-containing mast cells; substance P released from sensory nerves plays a significant role in the release of mast cell renin in ischemia/reperfusion and in the activation of a local cardiac RAS. This culminates in angiotensin production, norepinephrine release, and arrhythmic cardiac dysfunction.

We have previously described a rat model for the contribution of neuroplastic changes in nociceptors to the transition from acute to chronic pain. In this model a prior injury activates protein kinase C epsilon (PKCepsilon), inducing a chronic state characterized by marked prolongation of the hyperalgesia induced by inflammatory cytokines, prototypically prostaglandin E(2) (PGE(2)), referred to as hyperalgesic priming. In this study we evaluated the population of nociceptors involved in priming, by lesioning isolectin B4-positive (IB4(+)) nociceptors with intrathecal administration of a selective neurotoxin, IB4-saporin. To confirm that the remaining, TrkA(+)/IB4(-), nociceptors are still functional, we evaluated if nerve growth factor (NGF) induced hyperalgesia. While pretreatment with IB4-saporin eliminated the acute mechanical hyperalgesia induced by glia-derived neurotrophic factor (GDNF), NGF and PsiepsilonRACK, a highly selective activator of PKCepsilon, induced robust hyperalgesia. After injection of NGF, GDNF or PsiepsilonRACK, at a time at which hyperalgesia induced by PGE(2) is markedly prolonged (hyperalgesic priming) in control rats, in IB4-saporin-pretreated rats PGE(2) failed to produce this prolonged hyperalgesia. Thus, while PKCepsilon is present in most dorsal root ganglion neurons, where it can contribute to acute mechanical hyperalgesia, priming is restricted to IB4(+)-nociceptors, including those that are TrkA(+). While PKCepsilon activation can induce acute hyperalgesia in the IB4(+) population, it fails to induce priming. We suggest that hyperalgesic priming occurs only in IB4(+) nociceptors, and that in the peripheral terminals of nociceptors separate intracellular pools of PKCepsilon mediate nociceptor sensitization and the induction of hyperalgesic priming., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Important physiological functions of neurotrophins (NTs) in airways and lungs are the early development, differentiation and maintenance of peripheral sensory neurons. The main pulmonary sensory innervation is of vagal origin, with several nerve fibre populations that selectively contact complex morphologically well-characterized receptor end-organs, called neuroepithelial bodies (NEBs). NEBs in mouse lungs are innervated by at least two separate myelinated vagal sensory nerve fibre populations, of which the neurochemical coding is suggestive of a mechanosensory function. Since neurotrophin-4 (NT-4) has been especially described to be important for the maintenance of mechanosensory nerve terminals, the present study aimed at investigating the NT-4 dependency of the two myelinated vagal sensory nerve fibre populations innervating mouse pulmonary NEBs. Multiple immunostaining in 21-day-old and adult mouse lungs revealed the expression of the NT-4 receptor TrkB on the two different myelinated vagal sensory nerve fibre populations, i.e., the vesicular glutamate transporter/calbindin-positive and the P2X2/3-positive fibres, which selectively contact pulmonary NEBs. Examination of the effect of the lack of NT-4 on these NEB-related nerve fibre populations, by comparing adult NT-4-/- and wild-type mice, revealed that in NT-4-/- mice the percentage of NEBs contacted by P2X2/3+ is reduced by 75%, while the VGLUT+/CB+ population seemed to be unaffected. This study demonstrated that although mouse pulmonary NEBs are contacted by two distinct TrkB expressing populations of vagal myelinated afferents, only one is distinctly reduced in NT-4 deficient mice, suggesting the involvement of NTs. In view of the growing evidence for the involvement of NTs in neuronal plasticity associated with airway diseases, pulmonary NEBs innervated by NT-sensitive vagal afferents may play a significant role.

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activity-dependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high- and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

The contributions of P- and R-type Ca2+ channels on glycinergic nerve endings (boutons) projecting to the rat spinal sacral commissural nucleus (SDCN) neurons are not understood. Thus, we investigated the functional role of P- and R-type Ca2+ channels by measuring the inhibitory postsynaptic currents (eIPSCs) evoked from individual nerve endings (boutons) by focal electrical stimulation. The current amplitude and failure rate (Rf) of glycinergic eIPSCs varied directly with changes in [Ca2+](o). Low concentration of omega-Aga IVA (P-type selective antagonist) suppressed eIPSCs as much as high concentration (both P- and Q-type selective) indicating little contribution of Q-type Ca2+ channels. Antagonism of R-type Ca2+ channels with SNX-482 and Ni2+ greatly decreased the current amplitude and increased failure rate (Rf) of glycinergic eIPSCs. Overall, our results suggest that the dominant control of glycine release depends on Ca2+ entry through P- and R-type Ca2+ channels that ubiquitously populate spinal glycine release sites., (2010 Elsevier Ltd. All rights reserved.)

Primary afferent neurotransmission is the fundamental first step in the central processing of sensory stimuli and is controlled by pre- and postsynaptic inhibitory mechanisms. Presynaptic inhibition (PSI) is probably the more powerful form of inhibitory control in all primary afferent fibers. A major mechanism producing afferent PSI is via a channel-mediated depolarization of their intraspinal terminals, which can be recorded extracellularly as a dorsal root potential (DRP). Based on measures of DRP latency it has been inferred that this primary afferent depolarization (PAD) of low-threshold afferents is mediated by minimally trisynaptic pathways with pharmacologically identified GABAergic interneurons forming last-order axo-axonic synapses onto afferent terminals. There is still no "squeaky clean" evidence of this organization. This paper describes recent and historical work that supports the existence of PAD occurring by more direct pathways and with a complex pharmacology that questions the proprietary role of GABA and GABA(A) receptors in this process. Cholinergic transmission in particular may contribute significantly to PAD, including via direct release from primary afferents.

Polyethylene glycol (PEG) has been widely used as a solvent among other applications. An ideal solvent is one that does not interfere with an in vitro biological system, unless it is a bioactive agent. Herein, a facilitatory neurotransmission effect was exhibited by PEG (20 microM) in mammalian (67 +/- 12.5%, n = 4) and avian (74 +/- 6.8%, n = 6) neuromuscular preparations. In curarized preparations, PEG did not reverse the neurotransmission blockade induced by D-tubocurarine (D-Tc, 5.8 microM, n = 6) as promoted by neostigmine (12 microM, n = 4). A possible presynaptic action of PEG was ruled out, because quantal acetylcholine (ACh) content was similar to the control Tyrode-incubated mammalian preparation. PEG showed improved sarcolemmal sensitivity, both under direct (sarcolemma) and indirect stimulation (motor axon), because it was able to release calcium from the sarcoplasmic reticulum, even when 30 microM dantrolene (n = 5) was previously applied. Neurotransmission decreased at a higher PEG concentration (100 microM, n = -6) in the depolarized membrane, but it did not alter normal muscle fiber morphology. In addition, it partially recovered twitch tension amplitude (55 +/- 5.7%) after washing the preparations. More than a simple solvent, we suggest that PEG 400 is able to act on the sarcolemmal membrane, probably at the triad level, which is in line with its well-known ability as drug carrier.

Visceral afferents play a key role in neural circuits underlying the physiological function of visceral organs. They are responsible for the detection and transmission of a variety of visceral sensations (e.g. satiety, urge, discomfort and pain) from the viscera to the central nervous system. A comprehensive account of the different functional types of visceral sensory neurons would be invaluable in understanding how sensory dysfunction occurs and how it might be diagnosed and treated. Our aim was to explore the morphology of different nerve endings of visceral afferents within the gastrointestinal tract and urinary bladder and how the morphology of these nerve endings may relate to their functional properties. Morphological studies of mechanosensitive endings of visceral afferents to the gut and bladder correlated with physiological recordings have added a new dimension to our ability to distinguish different functional classes of visceral afferents., (Copyright 2009 Elsevier B.V. All rights reserved.)

Recent progress in understanding visceral afferents, some of it reviewed in the present issue, serves to underscore how little is known about the aging of the visceral afferents in the gastrointestinal (GI) tract. In spite of the clinical importance of the issue-with age, GI function often becomes severely compromised-only a few initial observations on age-related structural changes of visceral afferents are available. Primary afferent cell bodies in both the nodose ganglia and dorsal root ganglia lose Nissl material and accumulate lipofucsin, inclusions, aggregates, and tangles. Additionally, in changes that we focus on in the present review, vagal visceral afferent terminals in both the muscle wall and the mucosa of the GI tract exhibit age-related structural changes. In aged animals, both of the vagal terminal types examined, namely intraganglionic laminar endings and villus afferents, exhibit dystrophic or regressive morphological changes. These neuropathies are associated with age-related changes in the structural integrity of the target organs of the affected afferents, suggesting that local changes in trophic environment may give rise to the aging of GI innervation. Given the clinical relevance of GI tract aging, a more complete understanding both of how aging alters the innervation of the gut and of how such changes might be mitigated should be made research priorities., (Copyright 2009 Elsevier B.V. All rights reserved.)

Most intercellular glutamate signaling in the nervous system occurs at synapses. Some intercellular glutamate signaling occurs outside synapses, however, and even outside the nervous system where high ambient extracellular glutamate might be expected to preclude the effectiveness of glutamate as an intercellular signal. Here, I briefly review the types of intercellular glutamate signaling in the nervous system and beyond, with emphasis on the diversity of signaling mechanisms and fundamental unanswered questions.

Enteric co-innervation of striated muscle fibers in the esophagus occurs in several mammalian species including humans. However, the functional significance is still unknown. Phylogenetic data may be instrumental in gaining further insight. We examined the bat Glossophaga soricina and the shrew Suncus murinus as representatives for phylogenetically old mammals. As ruminants the antelope Tragelaphus imberbis, the he-goat Capra falconeri and the sheep Ovis aries were selected. As non-mammals the clawed frog Xenopus laevis as representative for the taxon amphibian and the rainbow trout Oncorhynchus mykiss as representative for the taxon fish were included. Histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase and acetylcholinesterase as well as immunofluorescence for vasoactive intestinal peptide and alpha-bungarotoxin were used to demonstrate enteric nerve fibers and motor endplates, respectively. Motor endplates were associated with enteric nerve fibers in all species investigated, although the rates of co-innervation varied from approximately 10 to 20% in shrew, antelope, he-goat, frog and fish, approximately 40% in bat to nearly 90% in sheep. These results demonstrate that enteric co-innervation, in spite of varying co-innervation rates, is conserved through vertebrate evolution, and underline the significance of this newly discovered innervation component.

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca(2+)-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca(2+) entry and enhanced nerve-terminal growth. The impaired Ca(2+) entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca(2+) channels, or that it regulates the PI4Kbeta pathway as described in other organisms. To determine whether Frq interacts with PI4Kbeta with consequent effects on Ca(2+) channels, we first characterized a PI4Kbeta-null mutant and found that PI4Kbeta was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kbeta-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kbeta. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the alpha(1)-subunit of voltage-gated Ca(2+) channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca(2+) entry through a functional interaction with the alpha(1) voltage-gated Ca(2+)-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.

The aim of this study was to examine the importance of the vesicle-associated synapsin I and II phosphoproteins for the accumulation of neurotransmitters in central cholinergic as compared to central glutamatergic and GABAergic nerve terminals. In brain homogenate samples from mice devoid of synapsin I and II, the levels of vesicular transporters for glutamate (VGLUT1-2) and GABA (VGAT) were decreased by 35-40% in striatum and cortex, while no change was apparent for the vesicular acetylcholine transporter (VAChT). The severe decrease in the levels of amino acid vesicular transporters caused only minor changes in the concentrations of the respective neurotransmitters in homogenates of the three selected brain areas from synapsin I- and II-deficient mice. However, when measured in a crude vesicular fraction, the concentrations of glutamate and GABA were decreased by 48-60% in synapsin-deficient mice, with a similar decrease in the levels of VGLUT1, VGLUT2 and VGAT. In comparison, the concentration of acetylcholine and the level of VAChT were not significantly different from wild-type in the vesicular fraction. No changes were seen in the activity of specific enzymes involved in the synthesis of acetylcholine, glutamate or GABA, however, immunoblotting indicated a decrease in the protein level of glutamic acid decarboxylase, isoform 65 (GAD(65)). In conclusion, the results indicate that neurotransmitter regulation in central cholinergic synapses may be less dependent on synapsin I and II compared to the marked alterations seen in the glutamatergic and GABAergic synapses.

The aim of this paper is to review diagnostic techniques which are useful and applicable in the evaluation of constipation. Besides widely-spread available techniques such as taking a specific and structured history and performing a clinical examination sophisticated diagnostic tools such as colon transit time, anal manometry, defecography endorectal ultrasonography as well as electrophysiological investigations such as anal sphincter electromyography and pudendal nerve terminal motor latency measurement are illustrated.

We describe a new method of investigation of the integrative action of fusimotor inputs in mammalian muscle spindles by stimulation of multiple fusimotor axons using independent pseudorandom pulse trains, each of low mean rate with pseudorandomly distributed stimulus intervals. Technically it was feasible only because of the development of (1) a novel, highly efficient approach to functional isolation of fusimotor efferents in ventral-root filaments, which we have called the isodyne strategy; (2) a real-time, microprocessor-based stimulus artefact cancellation device (SACAD); and (3) a highly adjustable, multi-branch stimulation electrode array. The general approach of using multiple, independent, pseudorandom stimulation of several input channels has wider applications in controlled-activation paradigms.

Experiments on frog neuromuscular synapses using extracellular microelectrode recording of endplate currents (EPC) and nerve ending (NE) responses were performed to study the mechanisms of facilitation of quantum secretion of acetylcholine on replacement of extracellular Ca ions with Sr ions. Solutions with a Ca ion concentration of 0.5 mM (calcium solutions) or a Sr ion concentration of 1 mM (strontium solutions) were used; the basal levels of neurotransmitter secretion (in conditions of low-frequency stimulation) were essentially identical. In calcium solutions, the drop in EPC facilitation on paired-pulse stimulation as the interimpulse interval was increased from 5 to 500 msec was described by the sum of three exponential components - the early, the first, and the second. In strontium solutions, facilitation was decreased as compared with the level in calcium solutions predominantly because of decreases in the early and first components. At the same time, EPC facilitation in conditions of rhythmic stimulation (10 or 50 impulses/sec) in strontium solution was significantly increased as compared with the level in calcium solutions. In strontium solutions in conditions of high-frequency stimulation at 50 impulses/sec, there was also a marked decrease in the amplitude of the third phase of the NE response, reflecting NE potassium currents. These data lead to the conclusion that the facilitation sites underlying the first and early components had lower affinities for Sr ions than for Ca ions. Increases in facilitation in strontium solutions in conditions of high-frequency rhythmic activity resulted from two mechanisms: more marked widening of the NE action potential and an increase in the divalent cation influx current due to weak activation of the Ca2+-dependent potassium current in the presence of Sr ions, as well as the slow dynamics of the removal of Sr ions from the NE axoplasm as compared with that in the presence of Ca ions.