Initial axon growth rate from embryonic sensory neurons is correlated with birth date.

Axon growth rate from different populations of sensory neurons is correlated with the distance they have to grow to reach their targets in development: neurons with more distant targets extend axons at intrinsically faster rates. With growth of the embryo, later-born neurons within each population have further to extend their axons to reach their targets than early-born neurons. Here we examined whether the axon growth rate is related to birth date by studying the axon growth from neurons that differentiate in vitro from precursor cells isolated throughout the period of neurogenesis. We first showed that neurons that differentiated in vitro from different precursor cell populations exhibited differences in axon growth rate related to in vivo target distance. We then examined the axon growth rate from neurons that differentiate from the same precursor population at different stages throughout the period of neurogenesis. We studied the epibranchial placode precursors that give rise to nodose ganglion neurons in the chicken embryo. We observed a highly significant, threefold difference in axon growth rate from neurons that differentiate from precursor cells cultured early and late during the period of neurogenesis. Our findings suggest that intrinsic differences in axon growth rate are correlated with the neuronal birth date.

Evidence of vagus nerve sprouting to innervate the urinary bladder and clitoris in a canine model of lower motoneuron lesioned bladder.

AIMS: Complete spinal cord injury does not block perceptual responses or inferior solitary nucleus activation after genital self-stimulation, even though the vagus is not thought to innervate pelvic structures. We tested if vagus nerve endings sprout after bladder decentralization to innervate genitourinary structures in canines with decentralized bladders. METHODS: Four reinnervation surgeries were performed in female hounds: bilateral genitofemoral nerve transfer to pelvic nerve with vesicostomy (GNF-V) or without (GFN-NV); and left femoral nerve transfer (FNT-V and FNT-NV). After 8 months, retrograde dyes were injected into genitourinary structures. Three weeks later, at euthanasia, reinnervation was evaluated as increased detrusor pressure induced by functional electrical stimulation (FES). Controls included un-operated, sham-operated, and decentralized animals. RESULTS: Increased detrusor pressure was seen in 8/12 GFNT-V, 4/5 GFNT-NV, 5/5 FNT-V, and 4/5 FNT-NV animals after FES, but not decentralized controls. Lumbar cord segments contained cells labeled from the bladder in all nerve transfer animals with FES-induced increased detrusor pressure. Nodose ganglia cells labeled from the bladder were observed in 5/7 nerve transfer animals (1/2 GNT-NV; 4/5 FNT-V), and from the clitoris were in 6/7 nerve transfer animals (2/2 GFNT-NV; 4/5 FNT-V). Dorsal motor nucleus vagus cells labeled from the bladder were observed in 3/5 nerve transfer animals (1/2 GFNT-NV; 2/3 FNT-V), and from the clitoris in 4/5 nerve transfer animals (1/2 GFNT-NV; 3/3 FNT-V). Controls lacked this labeling. CONCLUSIONS: Evidence of vagal nerve sprouting to the bladder and clitoris was observed in canines with lower motoneuron lesioned bladders. Neurourol. Urodynam. 36:91-97, 2017. © 2015 Wiley Periodicals, Inc.

Endomorphin-2 is released from newborn rat primary sensory neurons in a frequency- and calcium-dependent manner.

Recent evidence indicates that endomorphins, endogenous mu-opioid receptor (MOR) agonists, modulate synaptic transmission in both somatic and visceral sensory pathways. Here we show that endomorphin-2 (END-2) is expressed in newborn rat dorsal root ganglion (DRG) and nodose-petrosal ganglion complex (NPG) neurons, and rarely co-localizes with brain-derived neurotrophic factor (BDNF). In order to examine activity-dependent release of END-2 from neurons, we established a model using dispersed cultures of DRG and NPG cells activated by patterned electrical field stimulation. To detect release of END-2, we developed a novel rapid capture enzyme-linked immunosorbent assay (ELISA), in which END-2 capture antibody was added to neuronal cultures shortly before their electrical stimulation. The conventional assay was effective at reliably detecting END-2 only when the cells were stimulated in the presence of CTAP, a MOR-selective antagonist. This suggests that the strength of the novel assay is related primarily to rapid capture of released END-2 before it binds to endogenous MORs. Using the rapid capture ELISA, we found that stimulation protocols known to induce plastic changes at sensory synapses were highly effective at releasing END-2. Removal of extracellular calcium or blocking voltage-activated calcium channels significantly reduced the release. Together, our data provide the first evidence that END-2 is expressed by newborn DRG neurons of all sizes found in this age group, and can be released from these, as well as from NPG neurons, in an activity-dependent manner. These results point to END-2 as a likely mediator of activity-dependent plasticity in sensory pathways.

Cellular subtype distribution and developmental regulation of TRPC channel members in the mouse dorsal root ganglion.

Transient receptor potential (TRP) channels play essential roles in sensory physiology and their expression in different classes of sensory neurons reflect distinct receptive properties of these neurons. While expression of the TRPV, TRPA, and to a certain degree TRPM classes of channels has been studied in sensory neurons, little is known about the expression and regulation of TRPC channels. In this study we examined the regulation of all TRPC members (TRPC1-C7) throughout embryonic and postnatal development of the dorsal root ganglion (DRG) and nodose ganglion (NG). In adult mice, mRNAs for all channels were present in the DRG, with TRPC1, 3, and 6 being the most abundant, TRPC2, C4, and C5 at lower levels, and TRPC7 at very low levels. While TRPC2 mRNAs were downregulated from high levels at embryonic (E) day 12 and E14 until adult, TRPC4, C5, and C7 expressions increased from E12 to peak levels at E18. TRPC1, C3, and C6, the most abundant TRPC channel mRNAs, increased progressively from E12 to adult. Expression and regulation of TRPC channels mRNAs in the NG were unexpectedly similar to the DRG. TRPC1 and C2 was expressed in the neurofilament-200 (NF-200)-positive large size subclass of neurons, while TRPC3 mRNAs expression, which stained up to 35% of DRG neurons, was almost exclusively present in nonpeptidergic isolectin B4 (IB4)-positive small size neurons that were largely TRPV1-negative. Our results suggest important roles of the TRPC family of channels in sensory physiology of both nociceptive as well as nonnociceptive classes of neurons.

Afferent innervation of the trachea during postnatal development.

Retrograde axonal transport of horseradish peroxidase was used in this study to determine the location and basic morphological parameters of neurons innervating the trachea in newborn, 10-, 20-, 30-day-old and 2-month-old kittens. Labeled neurons were detected in all animals in the nodose ganglion of the vagus nerve and in the spinal ganglia (C1-C7 and T1-T6 after injection of tracer into the cervical trachea, C5-C7 and T1-T8 with injection into the thoracic part of the trachea) from both sides. The content of vagal and spinal afferent neurons innervating the cervical part of trachea declined during development. The number of spinal afferent neurons with connections to the thoracic trachea did not change but the quantity of cells in nodose ganglion supplying the thoracic trachea increased from the moment of birth till 10 and 20 days and decreased later in postnatal development. In newborn, 10-day-old and 20-day-old animals, the largest number of afferent cells was connected with the cervical part of the trachea in comparison with the thoracic one, whereas in 2-month-old kittens the relation was opposite. We suggest that afferent innervation of the trachea is not morphologically complete at the moment of birth and does not become mature until the second month of life.

Ontogeny of retrograde labeled chemoafferent neurons in the newborn rat nodose-petrosal ganglion complex: an ex vivo preparation.

Hypoxic chemosensitivity of the peripheral arterial chemoreceptors in the carotid body is developmentally regulated. Essential neural elements of the chemotransducing unit in the carotid body consist of the Type I cell that depolarizes and releases neurotransmitters in response to hypoxemia and the chemoafferent fibers which form synapses with Type I cells, contain postsynaptic receptors and have cell bodies in the petrosal ganglion. While many properties of the Type I cells have been characterized during postnatal development, less is known about the effect of development on the number and properties of the chemoafferents since localization of the cell bodies of chemoafferents are intermingled with the cell bodies of other sensory neurons that innervate the upper airway. Here, we describe a novel ex vivo preparation that we have developed to retrogradely label cell bodies of chemoafferents in the petrosal ganglion with rhodamine dextran. With this technique, in newborn rats, we show that there is a three-fold increase in retrogradely labeled neurons in the nodose-petrosal ganglion complex from postnatal day (PND) 3-7 with a three-fold decrease by PND 14 (P < 0.001, ANOVA). Furthermore, greater than 85% of these retrogradely labeled neurons co-express TH mRNA in all age groups. This novel ex vivo technique circumvents many of the technical difficulties encountered with retrogradely labeling chemoafferents in small newborn animals in vivo, and provides a method to identify and characterize essential neural components of the chemotranductive unit of the peripheral arterial chemoreceptors.

Patent ductus arteriosus remodels cardiac sensory neuronal chemotransduction.

We sought to determine the capacity of neonatal ventricular sensory nerve endings (neurites) to transduce the cardiac milieu in the presence of cardiovascular pathology. The spontaneous activity generated by nodose ganglion cardiac afferent neurons was identified in situ using extracellular recording techniques in two groups of piglets approximately 2 weeks old: (i). controls that underwent sham operations (n=19 piglets) 2 weeks earlier and (ii). a pathological model of patent ductus arteriosus stented open for about 2 weeks (n=16 piglets). The capacity of ventricular sensory neurites associated with nodose ganglion afferent neurons to transduce local mechanical (including alterations in right or left ventricular volumes) or chemical stimuli was studied in both groups. The average conduction velocity of afferent axons associated with identified neuronal somata was estimated to be 1.5+/-0.6 or 2.9+/-1.3 m s(-1). Ventricular afferent neurons transduced mechanical stimuli similarly in both groups. In control animals, ventricular afferent neurons transduced the following chemicals: the sodium channel modifier veratridine (delta 23+/-7 impulses min(-1)), the P(1)-purinoceptor agonist adenosine (Delta 24+/-8 impulses min(-1)), and the beta-adrenoceptor agonist isoproterenol (delta 18+/-7 impulses min(-1)). On the other hand, patent ductus arteriosus cardiac afferent neurons did not transduce these chemicals. It is concluded that neonatal cardiac afferent neuronal chemosensory-as opposed to mechanosensory-transduction remodels in the presence of a patent ductus arteriosus. The reduced capacity of neonatal cardiac afferent neurons to transduce chemicals in the presence of a patent ductus arteriosus should be taken into account when considering neonatal cardiovascular control in such a state.

Vagal intraganglionic laminar endings and intramuscular arrays mature at different rates in pre-weanling rat stomach.

To assess whether vagal afferents in the gastrointestinal (GI) tract mature postnatally and differentiate at different rates, potentially reflecting the changing functional requirements of weaning and independence, the vagal afferent innervation of the stomach was inventoried in pre-weanling and adult rats. Wheatgerm agglutinin-horseradish peroxidase was injected into the nodose ganglia of 9-day-old and adult rats, and after tracer transport, the animals were sacrificed. Their stomachs were prepared as wholemounts and processed with tetramethylbenzidine. Inventories were obtained with a counting grid that was systematically positioned throughout the wholemounts by the use of a sampling template that was adjusted for stomach size and shape. Densities in the gastric antrum, corpus, and forestomach were determined for (1) afferent bundles, (2) individual fibers separated from the bundles and presumably located near their targets, (3) differentiated intraganglionic laminar endings (IGLEs) associated with myenteric ganglia, and (4) differentiated intramuscular arrays (IMAs) situated within the smooth muscle layers. In pre-weanling rats, which were 10 days old at perfusion, the distributions of vagal bundles and fibers were similar to those of adults, suggesting that the basic vagal architecture develops early. Differentiated IGLEs were also distributed in a mature pattern in 10 day olds, whereas few IMAs had yet been distributed and differentiated in the forestomach of the pre-weanlings. The authors hypothesize that these different developmental patterns for the two types of vagal afferents are consistent with their putative functional roles as, respectively, mechanoreceptors (IGLEs) that coordinate rhythmic motor function needed early for the digestion of milk and stretch receptors (IMAs) needed later as the GI tract natures for the transition to solid food at weaning.

Gastrointestinal projection maps of the vagus nerve are specified permanently in the perinatal period.

The vagal innervation of the proximal gastrointestinal (GI) tract is lateralized. To determine whether this pattern is specified as early as the perinatal period, neonatal rat pups were given unilateral cervical vagotomies. Separate groups received (1) transections below the left nodose ganglion, (2) left cervical resections that included removal of the nodose ganglion, or (3) sham surgeries. At 4 months of age, each animal's vagal afferent projections from the unoperated side were mapped by injecting the nodose with WGA-HRP, preparing the stomach as wholemounts, and processing the tissue with tetramethyl benzidine. The two types of vagal afferent endings in GI smooth muscle, namely intraganglionic laminar endings and intramuscular arrays, were surveyed separately, and their regional distributions were mapped. Changes in the nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus (DMNX) were assessed with cell counts and area measurements. Neonatal loss of the vagus innervating one side of the GI tract, with or without ganglionectomy, did not cause the unoperated vagus to sprout to the denervated side. In addition, removal of the projections to the one side of the target organ did not produce a reorganization of the projection maps of the unoperated vagus within its normal or ipsilateral wall of the GI tract. Although the regional patterns of the unoperated ipsilateral vagus were not affected, the packing densities of both types of afferents supplied by this trunk were moderately reduced. The DMNX of the vagotomized side displayed extensive (approximately 83%) neuronal loss; the DMNX on the unoperated side as well as the NST on both sides exhibited limited (approximately 20--25%) losses. The lack of a peripheral projection field reorganization -- except for a moderate down-regulation -- after complete unilateral denervation suggests that both the laterality and the afferent terminal phenotypes (or target tissues) of the vagus in the proximal GI tract are specified by postnatal day one in the rat. The present results, taken together with other observations, also suggest that three different combinations of signals orchestrate the commitments of vagal afferents respectively to (1) the side of the organ, (2) the region within the organ wall, and (3) the accessory and innervated tissues that complex with the fully differentiated ending.

Transduction capabilities of neonatal ventricular afferent neurons in vivo.

We sought to determine the capacity of ventricular sensory nerve endings (neurites) associated with neonatal nodose ganglion afferent neurons to transduce mechanical and chemical stimuli in situ. Spontaneous activity generated by 17 nodose ganglion cardiac afferent neurons was identified in 8 anesthetized neonatal pigs (10-21 days old) using extracellular recording recording techniques. The activity generated by afferent neurons was studied when their ventricular sensory neurites were exposed to local mechanical or chemical stimuli, following systemic administration of specific chemicals or during brief periods of apnea. Gentle mechanical distortion of their ventricular sensory fields enhanced the activity generated by 6 spontaneously active afferent neurons, while suppressing the activity generated by another 3 neurons. Afferent neuronal activity was either enhanced or suppressed when the following chemicals were applied to identified ventricular epicardial sensory fields: the sodium channel modifier veratridine (92% of tested neurons); the P1-purinoceptor agonist adenosine (92%); the neuropeptides angiotensin II (100%), bradykinin (90%) and substance P (90%); and the nitric oxide donor S-nitroso-N-acetylpenicillamine (100%). Epicardial application of isoproternol or nicotine induced modest neuronal responses. Cardiac afferent neurons were also affected when these chemicals were administered systemically. Apnea of 60-100 s duration modified (enhanced, n = 2; suppressed, n = 5) the activity generated by most identified afferent neurons. The estimated average conduction velocity of afferent axons associated with these neurons was 1.0 +/- 0.2 m/s. It is concluded that neonatal nodose ganglion cardiac afferent neurons respond to many of the chemicals known to modify adult cardiac afferent neurons. That cardiac afferent neurons are capable of sensing the mechanical and chemical milieu of the neonatal heart should be taken into account when considering altered neonatal cardiovascular status such as occurs during apnea.

Reciprocal developmental changes in the roles of Bcl-w and Bcl-x(L) in regulating sensory neuron survival.

We have compared the roles of two anti-apoptotic members of the Bcl2 family, Bcl-w and Bcl-x(L), in regulating the survival of sensory neurons during development. We used microinjection to introduce expression plasmids containing Bcl-w and Bcl-x(L) cDNAs in the sense and antisense orientations into the nuclei of BDNF-dependent nodose neurons and NGF-dependent trigeminal neurons at stages during and after the period of naturally occurring neuronal death. Whilst overexpression of either protein promoted neuronal survival in the absence of neurotrophins and microinjection of antisense constructs reduced neuronal survival in the presence of neurotrophins, the magnitude of these effects changed with age. Whereas Bcl-w overexpression became more effective in promoting neuronal survival with age, Bcl-x(L) overexpression became less effective, and whereas antisense Bcl-w became much more effective in killing neurotrophin-supplemented neurons with age, antisense Bcl-x(L) became much less effective in killing these neurons. There was a marked increased in Bcl-w mRNA and Bcl-w immunoreactive neurons and a decrease in Bcl-x(L) mRNA and Bcl-x(L) immunoreactive neurons in the trigeminal and nodose ganglia over this period of development. Our results demonstrate that both Bcl-w and Bcl-x(L )play an important anti-apoptotic role in regulating the survival of NGF- and BDNF-dependent neurons, and that reciprocal changes occur in the relative importance of these proteins with age. Whereas Bcl-x(L) plays a more important role during the period of naturally occurring neuronal death, Bcl-w plays a more important role at later stages.

Axonal outgrowth from adult mouse nodose ganglia in vitro is stimulated by neurotrophin-4 in a Trk receptor and mitogen-activated protein kinase-dependent way.

The actions of neurotrophic factors on sensory neurons of the adult nodose ganglion were studied in vitro. The ganglia were explanted in an extracellular matrix-based gel that permitted observation of the growing axons. Neurotrophin-4 (NT-4) was a very efficient stimulator of outgrowth of axons from the nodose ganglion and had almost doubled the outgrowth length when this was analyzed after 2 days in culture. Brain-derived neurotrophic factor also stimulated outgrowth, but to a lesser degree, whereas NT-3 gave only weak stimulatory tendencies. Nerve growth factor and glial cell line-derived neurotrophic factor both lacked stimulatory effects. NT-4 is known to act via TrkB receptors, and the presence of these on growing nodose neurons was demonstrated immunohistochemically. In line with a Trk-mediated growth effect, the NT-4 stimulation was abolished by K252a, a selective inhibitor of neurotrophin receptor-associated tyrosine kinase activity. K252a had no effect on the unstimulated preparation. NT-4 treatment led to activation of the mitogen-activated protein kinase and inhibition of the latter pathway by PD98059 significantly reduced the NT-4 stimulated outgrowth, whereas the drug had no effect on the unstimulated growth. In conclusion, the data suggest that NT-4 can serve as a powerful growth factor for neurons of adult nodose ganglia and that the growth stimulation involves TrkB- and mitogen-activated protein kinase.

Cytokines promote the survival of mouse cranial sensory neurones at different developmental stages.

To investigate when the neurotrophic cytokines ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin-M (OSM), interleukin-6 (IL-6) and cardiotrophin-1 (CT-1) act on developing sensory neurones and whether they co-operate with neurotrophins in regulating neuronal survival, we studied the in vitro trophic effects of these factors on two well-characterized populations of cranial sensory neurones at closely staged intervals throughout embryonic development. The cutaneous sensory neurones of the trigeminal ganglion, which show an early, transient survival response to BDNF and NT3 before becoming NGF-dependent, were supported by CNTF, LIF, OSM and CT-1 during the late fetal period, several days after the neurones become NGF-dependent. At this stage of development, these cytokines promoted the survival of a subset of NGF-responsive neurones. The enteroceptive neurones of the nodose ganglion, which retain dependence on BDNF throughout fetal development, were supported throughout their development by CNTF, LIF, OSM and CT-1, and displayed an additional survival response to IL-6 in the late fetal period. These findings indicate that populations of sensory neurones display different developmental patterns of cytokine responsiveness and show that embryonic trigeminal neurones pass through several phases of differing neurotrophic factor survival requirements.

Postnatal neuron death in the nodose ganglia of the rat.

Developmental neuron death is well described in sensory and sympathetic ganglia derived from the neural crest. In this study, nodose ganglia were removed from 2 litters of postnatal rats (male and female; 1, 3, 5, 9, and 14 days old) in order to determine whether postnatal neuron degeneration occurs in the nodose ganglia, which is derived from ectodermal placode. The ganglia were embedded in paraffin, sectioned and stained with methylene blue. Neuronal nuclei were counted at a magnification of 100 and diameters of nodose nuclei were traced at each age. There was a significant (p < 0.001) increase in the nuclear diameter of the nodose neurons of male and female rats from birth to postnatal day 14. In male rats, this difference was most marked between postnatal day 5 and postnatal day 14. The results of the neuron counts for both male and female rats indicated a gradual, significant (p < 0.001) decrease in the neuron population from day 1 to 14. For the males a 57.2% decline was observed, while the females displayed a 49.2% decline. The numbers of neurons in male and female ganglia showed no consistent differences. The data for neuron counts suggest that developmental neuron death occurs in postnatal rats with a gradual decrease in number of nodose neurons. However, since our findings show no evidence of degenerating nodose neurons, we are unable to rule out the possibility of migration from the developing ganglion.

Factors affecting the expression of acetylcholine receptors on rat sensory neurones in culture.

Sensory neurones from nodose ganglia of new-born rats were grown in dissociated tissue culture either with or without satellite cells. When cultured without satellite cells, most neurones developed sensitivity to acetylcholine (ACh); time-course experiments indicated that the neurones acquire their sensitivity during the second to third week in culture. Most neurones co-cultured with satellite cells did not develop ACh sensitivity. Delayed removal of satellite cells 8-12 days after plating resulted in few neurones acquiring ACh sensitivity. Delayed addition of satellite cells to neuronal cultures that were initially grown without satellite cells had no effect on the number of ACh-sensitive neurones. The potential to develop ACh sensitivity in culture without satellite cells decreases with the age of the neurones at the time of culturing; few neurones from 2-week-old animals developed sensitivity to ACh when cultured without satellite cells. These results indicate that there is some influence from satellite cells that prevents nodose neurones from developing ACh sensitivity in culture and suggests that this influence may also operate in vivo.

The role of extracellular signals in the differentiation of cholinergic neurons from the CNS and PNS in culture.

Rat skeletal muscle cells release in culture a macromolecule which stimulates by 25-100 fold the development of choline acetyltransferase (CAT) in cultures of new-born rat sympathetic neurons. This "cholinergic factor" impaired the development of three norepinephrine synthesizing enzymes and of acetylcholinesterase (AChE) in these cultures. The 16S form of AChE failed to develop in cultures grown with the factor, but amounted to 30-40% in 3-week old cultures grown in its absence. Using the development of CAT activity in sympathetic neuron cultures as an assay, the cholinergic factor has been partially purified in 6 steps, and its hydrodynamic parameters determined. The effects of this factor on sympathetic neurotransmitter choice were qualitatively reproduced by 1-10 mM Na butyrate. The cholinergic factor increased CAT activity and decreased AChE in neuron cultures from new-born rat nodose ganglia. The factor also stimulated CAT activity in rat embryo (E14) spinal cord cultures, but stimulated the development of AChE in these cultures.

Influences on the expression of acetylcholine receptors on rat nodose neurones in cell culture.

1. Nodose neurones dissociated from new-born rats were grown in culture in the absence or presence of cells from neonatal skeletal muscle or heart. 2. In cultures devoid of non-neuronal cells cholinergic interactions between the neurones were common. In the presence of non-neuronal cells such interactions were rare. 3. A decrease in the proportion of neurones responsive to ACh was primarily responsible for the reduced incidence of synaptic interactions. Non-neuronal cells influenced the expression of ACh receptors in developing nodose neurones in culture. 4. Most neurones appeared susceptible to the non-neuronal influence during the first week in culture. 5. Many nodose ganglion neurones, whether grown in the presence or absence of non-neuronal cells, were sensitive to gamma-aminobutyric acid and serotonin but were insensitive to glutamate, glycine and L-epinephrine.