Neurofilament immunoreactivity in developing rat autonomic and sensory ganglia

Immunoreactivity to neurofilament (NF) antiserum appears early in the development of both the central and peripheral nervous systems of the rat fetus. In 10 somite embryos, positive cell bodies are present in the ventromedial part of anterior rhombencephalic and mesencephalic neural tube. From there the appearance of immunoreactivity spreads cranially to the prosencephalic anlage before closure of the anterior neuropore and caudally following the sequence of neural tube closure. Immunoreactivity increases rapidly in axon bundles of central and peripheral systems, but in immature cell bodies of sensory ganglia the NF material only forms a ring around the nucleus. At 16 days of gestation, some cell bodies are progressively loaded with NF-immunoreactive material as a thick perinuclear network first and then in more excentrically located aggregates. This category of neurons is mainly observed in the distal part of the trigeminal ganglion, in petrous and nodose ganglia and in cervical dorsal root ganglia. In adult ganglia large cell bodies and some small ones present high NF immunoreactivity. In autonomic cell bodies (in superior cervical ganglion and in parasympathetic cranial ganglia) the immunoreactive material only forms a perinuclear ring slowly transformed into a loose perinuciear meshwork at the end of gestation. Intensely reactive nerve fibers are observed in cranial sensory as well as in sympathetic and parasympathetic ganglia and nerves. No positive cell bodies and only a few NF-immunoreactive nerves are observed in the carotid bodies. The NF immunoreactivity is better visualized on sections of fresh frozen material, treated with acetone, than in fixed specimens.These results are compared to previous observations reported for other species and for developing dorsal root ganglia. This immunostaining may be used to detect differentiation of peripheral sensory and autonomic neurons under experimental conditions. The uneven distribution of NF immunoreactivity in sensory neurons from stage 16 days of gestation as specific for precise subpopulations of neurons is discussed.     

Prostacyclin enhances the evoked-release of substance P and calcitonin gene-related peptide from rat sensory neurons

Prostacyclin (PGI₂) is a potent prostanoid producing various symptoms of inflammation, including an increased sensitivity to noxious stimulation. One component of these PGI₂-mediated actions may involve activation or sensitization of sensory neurons to enhance release of neuroactive peptides. We, therefore, examined whether PGI₂ and carra prostacyclin (CPGI₂), a stable analog of PGI₂, could alter the resting and evoked release of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) from embryonic rat sensory neurons grown in culture. Treating isolated sensory neurons with CPGI₂ (10–1000 nM) for 30 min caused a 3-fold increase in the resting release of both peptides. One nM CPGI₂, a concentration that did not alter the resting release, significantly enhanced neuropeptide release evoked by capsaicin, 100 nM bradykinin, or 40 mM KCl. Similarly, 10 nM PGI₂ did not alter resting release, but augmented capsaicin-stimulated release of SP and CGRP 2–3 fold. In contrast, prostaglandin F_2α was ineffective in altering either resting or capsaicin-evoked peptide release. Our results demonstrate that low concentrations of PGI₂ sensitize sensory neurons to other stimuli, whereas higher concentrations evoke release directly. This PGI₂-induced augmentation of neuropeptide release may be one mechanism contributing to neurogenic inflammation.     

Development of substance P-immunoreactive neurons in cranial sensory ganglia of the rat

Substance P-like immunoreactivity has been observed in fetal and adult cranial sensory ganglia. It first appears at day 16 of gestation in sensory neurons of trigeminal, superior-jugular, petrous and nodose ganglia, as well as in the autonomic myenteric plexus, and at day 17 in cervical dorsal root ganglion cells. Substance P immunoreactivity can be visualized much earlier (day 12) in the central nervous system. The ganglionic immunoreactivity subsequently increases during fetal life but drops at birth. The reactive material is first diffuse, then slowly becomes granular, and is mostly concentrated in coarse perinuclear inclusions in adult sensory neurons. Most substance P-positive neurons in trigeminal and superior-jugular ganglia are small, but medium-sized and large positive neurons are also observed in the trigeminal, petrous and nodose ganglia.Our observations give a precise picture of the development of substance P immunoreactivity in sensory neurons and are in general agreement with previous reports on some fetal and adult rat sensory ganglia. They indicate that in the rat, maturation of peripheral substance P-containing sensory neurons is slower than that of central substance P neurons or equivalent sensory neurons in other species. The examination of fetal material allows the observation of numerous immunoreactive sensory neurons which cannot be visualized after birth. We hypothesize a possible different embryonic origin (neural crest or placodal) for small nociceptive and larger substance P-containing neurons in rat cranial sensory ganglia.     

Muscarinic receptors modulate intracellular calcium level in chick sensory neurons

In the present work we have studied the variation of intracellular calcium levels induced by muscarinic agonists in chick dorsal root ganglia neurons. Muscarinic agonists such as muscarine and oxotremorine cause an increase of intracellular calcium levels in fura-2AM-loaded DRG neurons of E18 chick embryos. This increase was abolished following treatment with 1 microM atropine but not by 1 microM mecamylamine, indicating that the observed intracellular calcium increase, was dependent on muscarinic receptor activation. Stimulation in absence of external calcium or pre-incubation of the DRG cultures with thapsigargin or Mn(2+) demonstrated that [Ca(2+)](i) increase is mainly due to its release from intracellular stores. The use of selective antagonists of muscarinic receptor subtypes also indicated that M(1) and to a lesser extent M(3) receptor subtypes are responsible for the observed intracellular calcium mobilization. Finally pre-treatment of DRG cultures with pertussis toxin showed that the variation of [Ca(2+)](i) levels was dependent on PTX-insensitive G-protein. Moreover muscarinic agonists induce in DRG also the increase of IPs level, suggesting that the variations of intracellular calcium levels may be due at least in part to the activation of the phosphoinositide transduction pathway. In conclusion the reported observations demonstrate the activity of muscarinic receptors in sensory neurons, suggesting a functional role for acetylcholine in sensory transduction.     

The life span of ganglionic glia in murine sensory ganglia estimated by uptake of bromodeoxyuridine

Studies of ganglionic glia turnover in the sensory nervous system have implications for understanding nervous system maintenance and repair. These glial cells of the sensory ganglia in the peripheral nervous system (PNS) comprise satellite cells (SCs) and, to a lesser extent, Schwann cells. SCs proliferate in response to trauma such as axotomy; however, the half-life of these glial cells under normal circumstances has not been estimated. To estimate the half-life of sensory ganglionic glial cells, we employed the DNA precursor analog 5-bromo-2'-deoxyuridine (BrdU) to measure the rate of turnover of these cells. BrdU was administered to inbred C57BL6 and outbred Swiss white mice via their drinking water. BrdU incorporation into ganglionic glia in the PNS was estimated by immunofluorescent staining of nervous tissue sections, and the fraction of ganglionic glial cells that acquired BrdU label was measured as a function of time. Mathematical modeling of the rate of uptake of BrdU into murine ganglionic glia enables calculation of the half-life of these cells. The kinetics of BrdU uptake is linear, consistent with ganglionic glia being a homogenous population. The value of the proliferation rate (p) plus death rate (d) derived from the slope of BrdU uptake as a function of time is approximately 2.4 x 10(-3) cells per day. Assuming that p = d (because ganglionic glial numbers are in equilibrium and they are assumed to neither emigrate from, or immigrate into, sensory ganglia), then the daily death rate is d = 1.2 x 10(-3) cells/day, which implies a half-life for ganglionic glia of about 600 days. Thus murine ganglionic glia in the untraumatized state appear to behave as a homogenous, slowly replicating population.     

Adult dorsal root ganglia sensory neurons express the early neuronal fate marker doublecortin

It has been widely accepted that doublecortin (DCX) may represent a neuronal fate marker transiently expressed by immature neurons during development of the central and peripheral nervous tissue and in neurogenic areas of the adult brain. Previous work described the presence of DCX in the developing dorsal root ganglia (DRG), structures of the peripheral nervous system originating from the neural crest, but no information is available on its expression in adulthood. To this purpose, we have performed an immunohistochemical and biochemical analysis for DCX expression in DRG from adult male mice and rats. To our surprise, we demonstrated that the majority of DRG neurons do express DCX, both in somata and in fibers. DCX(+) cells have been characterized morphologically and phenotypically with well-established markers of DRG neuronal subpopulations. A large number of DCX(+) cells belong to the small and medium-sized nociceptive neurons. Additionally, DCX immunoreactivity is present in the spinal cord dorsal horns, the projection area of DRG neurons. The novel and unexpected localization for DCX protein opens up new, interesting vistas on the functional role of this protein in mature neurons and in particular in sensory neurons.     

Structure of neurons and ganglia of the guinea pig gallbladder: light and electron microscopic studies.

This study was undertaken to examine the morphological features of cells within ganglia of the guinea pig gallbladder, and to examine the ultrastructure of the ganglionated plexus. Gallbladder neurons are large, with a relatively simple form, having only one or two major processes. Neurobiotin often filled axons to their varicose arbors on smooth muscle in close proximity to the interganglionic connectives. With the exception of connective tissue clefts that sometimes penetrated into them, ganglia were devoid of intercellular spaces, capillaries, or connective tissue elements such as collagen and basal laminae. However, ganglia were surrounded by a single, continuous basal lamina that was enclosed within a fibroblast and collagen capsule. Within ganglia, neurons were insulated by the processes of cells that resembled the astrocyte-like glia of enteric ganglia. Although few classical synapses were observed, numerous sites of direct apposition were identified between vesicle-rich profiles and processes of gallbladder neurons. Direct appositions between vesicle-rich profiles and the ganglion-limiting basal laminae were also observed. Vesiculated profiles contained small clear vesicles and large dense-core vesicles. Within interganglionic connectives, axons were unmyelinated and were isolated from one another by processes of glia that resembled Schwann cells. As was seen in the ganglia, direct appositions between vesicle-rich profiles and the connective-limiting basal laminae were observed. The results of this study demonstrate that gallbladder ganglia are similar, ultrastructurally, to enteric ganglia in the CNS-like composition of the neuropil. However, the greater degree of glial investment, lesser degree of innervation, and simpler neurons indicated differences from the enteric nervous system that may be functionally significant.     

Galanin-like immunoreactivity within Ch2 neurons in the vertical limb of the diagonal band of Broca in aging and Alzheimer's disease

Summary The neuropeptide galanin is known to inhibit the evoked release of acetylcholine in ventral hippocampus of the rat. Co-localization of this peptide with choline acetyltransferase in neurons of the cholinergic septal nuclei has been demonstrated in the rat and non-human primate. The severe deficiency of the cholinergic hippocampal projection system arising mainly from the vertical limb nucleus of the diagonal band of Broca, also referred to as Ch2 region, is a constant finding in Alzheimer's disease, a disorder which is neuropathologically characterized by the appearance of senile plaques, neurofibrillary tangles and congophilic angiopathy in neo- and archicortical structures. In the present study for the first time galanin immunoreactivity in the human Ch2 region is morphologically investigated and related to the severity of hippocampal plaques and neurofibrillary tangles in Alzheimer's disease. An inverse relationship between decreasing galanin immunoreactivity in the Ch2 region and amounts of senile plaques and neurofibrillary tangles in the hippocampus is indicated. Considering the cholinergic deficiency in Alzheimer's disease as a secondary phenomenon to primary cortical and hippocampal lesions, and realizing the inhibitory effect of galanin upon acetylcholine release in hippocampus, this preliminary study suggests that a decreased galanin immunoreactivity in Ch2 in Alzheimer's disease reflects a possible negative feedback mechanism to a degenerating cholinergic projection system.Supported fully by a research grant from the JANIVO Foundation     

Osteocalcin-immunoreactive neurons in the vagal and glossopharyngeal sensory ganglia of the rat

Immunohistochemistry for osteocalcin (OC) was performed on the rat vagal and glossopharyngeal sensory ganglia. OC-immunoreactive (IR) neurons were detected in the jugular (10%), petrosal (11%) and nodose ganglia (6%). The cell size analysis demonstrated that OC-IR neurons were predominantly small to medium-sized in the jugular ganglion (mean+/-S.D.=356.3+/-192.2 microm(2), range=86.5-831.5 microm(2)). On the other hand, such neurons were medium-sized to large in the petrosal (mean+/-S.D.=725.6+/-280.7 microm(2), range=124.7-1540.4 microm(2)) and nodose ganglia (mean+/-S.D.=857.5+/-330.2 microm(2), range=367.1-1608.0 microm(2)). In the circumvallate papilla, OC-IR nerve fibers were located in the vicinity of taste buds. Some taste bud cells were also immunoreactive for the calcium-binding protein (CaBP). In the carotid body, however, OC-IR nerve fibers could not be detected. Retrograde tracing with fluorogold revealed that OC-IR nerve fibers in the circumvallate papilla mainly originated from the petrosal ganglion. These findings may suggest that OC-IR petrosal neurons have chemoreceptive function in the tongue.     

Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord

The pattern of expression of nerve growth factor (NGF)-receptor-like immunoreactivity during postnatal development in rat central nervous system (CNS) was analyzed using immunohistochemical localization of the receptor. Interestingly, in addition to the expected staining in basal forebrain, several structures were strongly labelled only during specific postnatal developmental stages. These structures included fibers in the thalamus, the external granule cell layer of the cerebellum and motor neurons, indicating that specific neurotrophic mechanisms might play an important role for the labelled cells during a precisely defined period.     

Changes in features of degenerating primary sensory neurons with time after capsaicin treatment

Summary Capsaicin (50 mg/kg) was injected into new born mice and 5 and 12 h, and 1, 2, 3, and 5 days later, their lumbar dorsal root ganglia (DRG) with the nerve roots were fixed by immersion. The morphological changes which ensued with time after treatment were examined by light and electron microscopy. The findings were as follows: (a) rapid degeneration of certain smaller B-type neurons, indicating their prompt death, was seen 5 h after treatment. Later, accumulated neurofilaments appeared in larger B-type neurons. Fissures of the cytoplasm and cell fragmentation were also observed as particular features of degeneration. Finally, these degenerating neurons, destined to die, appeared as small round figures with a disorganized nucleus. Severely degenerated neurons were seen throughout the survival time after treatment, but seemed to be most numerous after 2–3 days. (b) Three days after treatment the Nissl substances of large A-type neurons appeared dispersed, forming ring-like bundles in the periphery of cells. Cytoplasmic rupture and large membrane-bound spaces with fine granular or fibrillar materials, indicating peripheral cytolysis, were also conspicuous. Some of these cells showed severe degeneration clearly leading to cell death. The A-type neurons began to degenerate later than the B-type neurons. (c) Satellite cells showed an increased amount of electron-opaque cytoplasm that contained large vacuoles and neuronal cell debris. Mitotic figures were increased in satellite cells 3 days after treatment. (d) Unmyelinated axons in the dorsal root of mice treated with capsaicin became enlarged with accumulation of neurofilaments, synaptic vesicles or various kinds of vesicles, multivesicular bodies amd mitochondria. Numerous dense lamellar bodies appeared in the unmyelinated axons within DRG 3 days after treatment, but were scarcely seen in the dorsal roots. Degeneration of the myelinated fibers increased with time. Interestingly, capsaicin seemed to have both a direct and indirect action on DRG neurons: its direct action induced rapid degeneration of the smaller neurons, whereas its indirect action induced relatively slow degeneration of the larger neurons, causing chromatolytic changes similar to those induced by periphal nerve axotomy. The injury to DRG neurons due to the indirect action seemed to be induced retrogradely.     

Expression of orphanin FQ/nociceptin and its receptor in rat peripheral ganglia and spinal cord

Expression of the neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, the opioid receptor-like receptor (ORL1), have been found to have a wide distribution in the central nervous system, and in brain areas involved in sensory perception in particular. The effects of OFQ/N on, e.g., sensory transmission are very complex, and a modulatory effect on pain perception has been suggested. We therefore wanted to investigate the distribution of OFQ/N and ORL1 in the spinal cord and DRG, and also in SCG and some other peripheral tissues. The methods used were in situ hybridization, immunohistochemistry and ligand binding. We found that OFQ/N and ORL1 mRNA are expressed in DRG; primarily in small and large neurons, respectively. In spinal cord, mRNA for OFQ/N and ORL1 is expressed in neurons in laminae I, II and X, and in ventral horn neurons. Further, immunoreactivity for OFQ/N is observed in fibers and neurons in the superficial laminae of the dorsal horn and around the central canal, and also in neurons in the ventral horn of the spinal cord. Receptor ligand binding to the spinal cord grey matter is demonstrated, primarily concentrated to the dorsal horn and around the central canal, and also to medium and large size DRG neurons. These findings on the morphological distribution pattern of OFQ/N and ORL1 at the cellular level may support the notion that OFQ/N is involved in modulating pain transmission. Further, expression of OFQ/N and ORL1 mRNA was also found in SCG, whereas expression was undetectable in skin.     

Quantitative changes in mitochondria of spinal ganglion neurons in aged rabbits

Within the context of our research on the age-related structural changes in spinal ganglia, we studied the mitochondria of the neuronal perikaryon in the spinal ganglia of 12-, 42-, and 79-month-old rabbits. Both the volume of the perikaryon and the total mitochondrial mass within the perikaryon increased significantly passing from young adult to old animals. Hence, there is no net loss of mitochondria in these neurons with age. Since, however, the volume of the perikaryon increased by more than 63% while the total mitochondrial mass within the perikaryon increased by only 18%, the mean percentage of perikaryal volume occupied by mitochondria decreased with age. This decrease is only in very minor part a consequence of lipofuscin accumulation, so that the ratio between the total mitochondrial mass and the functionally active volume of cytoplasm decreased with age. Possible causes of this decrease are discussed briefly. Moreover, while the mitochondrial structure did not change, mitochondrial size increased with age. Finally, in each of the three age groups both the mean percentage volume of mitochondria and the mean mitochondrial size were very similar in large light and in small dark neurons.     

Nicotine activates NPY and catecholaminergic neurons in brainstem regions involved in ACTH secretion

Nicotine rapidly and potently stimulates ACTH secretion via a centrally mediated mechanism. The purpose of the current study was to identify the phenotype of nicotine-sensitive neurons in brainstem catecholaminergic regions previously shown to be responsive to nicotine. Immunocytochemical double-labeling was used to detect c-Fos expression in neurons positive for activin, galanin, or neuropeptide Y (NPY), in comparison to those containing tyrosine hydroxylase (TH, catecholaminergic biosynthetic enzyme). These neuropeptides were chosen because (1) each is located in nicotine-sensitive brainstem regions, (2) neurons containing each of these peptides project to the hypothalamic paraventricular nucleus, and (3) each has been shown to affect ACTH secretion. Freely moving, adult, male rats received an intravenous (i.v.) infusion of saline or nicotine (0.045 mg/kg over 30 s or 0.135 mg/kg over 90 s) and were cardiac perfused 60 min thereafter. Nicotine significantly increased c-Fos expression in a dose-dependent manner in the brainstem regions examined. In nucleus tractus solitarius (NTS)-A2 and NTS-C2, both NPY⁺ and TH⁺ neurons responded to the lower dose of nicotine, whereas the activin and galanin neurons in these regions were unresponsive to either dose of nicotine. In contrast, the higher dose of nicotine was required to activate NPY⁺ neurons in the A1 region and both NPY⁺ and galanin⁺ neurons in the locus coeruleus; the C1 region was unresponsive to nicotine. Since plasma ACTH is elevated by the low dose of nicotine and only NTS neurons are activated by this dose, NPY projections from the NTS are likely to contribute to nicotine-stimulated ACTH secretion, in addition to the previously described catecholaminergic neurons.     

Somato-, branchio- and viscero-motor neurons contain glutaminase-like immunoreactivity

Immunocytochemistry combined with a fluorescent dye tracer method revealed that somatic, branchial and visceral motoneurons in the brainstem and spinal cord of the rat contain phosphate-activated glutaminase (PAG). An excitatory neurotransmitter glutamate is synthesized mainly through this enzyme. Among these motoneurons, neurons in the dorsal motor nucleus of the vagus nerve (dmnX), autonomic preganglionic neurons in the spinal cord and urethral sphincter motoneurons (DL) were most intensely immunostained. PAG is co-expressed with choline acetyltransferase, calcitonin gene-related peptide or galanin in these neurons. These findings, together with the findings that motor endplates in urethral sphincter muscle contain PAG and PAG-like immunostaining in dmnX motoneurons was decreased after axotomy, suggest that glutamate is a co-transmitter of acetylcholine in motoneurons. Brainstem motoneurons were moderately stained, while somatic motoneurons in the spinal cord other than DL, showed very weak staining for PAG. However, they showed intense PAG-like immunoreactivity at their premature stage, suggesting that glutamate has some effects on the maturation of these neurons. A variety of functional roles of glutamate in motoneurons is discussed.     

Adenosine A2 receptor-mediated excitation of a subset of AH/Type 2 neurons and elevation of cAMP levels in myenteric ganglia of guinea-pig ileum

Abstract The aim of the study was to test the hypothesis that excitatory A₂ and inhibitory A₁ receptors coexist on myenteric AHIType 2 neurons, and are positively coupled to adenylate cyclase to stimulate cAMP formation. The A₂ agonists NECA and CGS 21680 increased excitability and depolarized the membrane in 40% of 71 AH/Type 2 neurons. In the remainder, the agonists depressed excitability and hyperpolarized the neurons. In 13% of neurons, A₂ agonists caused a concentration-dependent depolarization at nanomolar concentrations, followed by hyperpolarization at higher concentrations. CGS 21680 (EC₅₀=0.15 nM) was 133-fold more potent than NECA (EC₅₀= 20 nM) in depolarizing AH/Type 2 neurons. The A₁ agonist, CCPA, caused hyperpolarization and depressed excitability in more than 90% of neurons. The potency profile of agonists for depolarization was CGS 21680 ≫ NECA ≫> CCPA. NECA augmented at nanomolar and inhibited at micromolar concentrations, excitatory depolarizing responses to forskolin in AH/Type 2 neurons; whereas, CCPA only inhibited the action of forskolin. In parallel studies on enzymatically dissociated myenteric ganglia, when the ganglia were exposed to priming concentrations of forskolin (5 μM) in the presence of Ro-20 1724, NECA enhanced the stimulatory action of forskolin on CAMP formation. This effect was abolished by the adenosine receptor antagonist DPSPX. The potency of NECA for stimulation of adenylate cyclase equalled that for depolarization of the AH/Type 2 neurons. The results suggest that high affinity excitutory A₂ receptors are coupled to adenylate cyclase in a minority subset of AH/Type 2 myenteric neurons, and that inhibitory A₁ and excitatory A₂ receptors are co-localized on some AH/Type 2 neurons.     

Infrared sensory neurons in the trigeminal ganglia of crotaline snakes: transganglionic HRP transport

Trigeminal neurons were labeled by inserting HRP into holes cut in the pit receptor membranes of a crotaline snake, Agkistrodon blomhoffi brevicaudus. Neurons were labeled in the ophthalmic ganglion and the maxillary division of the maxillo-mandibular ganglion, and the HRP was further transported across the ganglia and through the lateral descending trigeminal tract (dlv) to label axon terminals exclusively in the dlv nucleus (DLV). In 6 successful preparations, 7.1-19.3% of totals of 5568-5986 cells in the maxillary division of the ganglion were labeled, but none at all were labeled in the mandibular division. Only a few or none at all were labeled in the ophthalmic ganglion. Cells in the two ganglia ranged in size from 10 to 55 micrometers, but large cells (greater than or equal to 40 micrometers) were scarce (4.9% of the total population). All HRP-labeled neurons fell in the median range of 20-39 micrometers. We concluded that these ganglion cells were infrared neurons, and were therefore the origin of the A delta fibers in the pit membrane. There were no HRP-labeled neurons above or below this range, in spite of the fact that smaller cells (less than or equal to 19 micrometers) made up 35.8% of the total population. In normal Nissl preparations we found both light- and dark-staining cells, but the size range of neither corresponded to the size range of infrared neurons.