Preganglionic and sensory origins of calcitonin gene-related peptide-like and substance P-like immunoreactivities in bullfrog sympathetic ganglia

These experiments further define the organization of peptidergic pathways in the paravertebral sympathetic system of the bullfrog. Populations of axons and synaptic boutons in sympathetic ganglia 9 and 10 were found to express calcitonin gene-related peptide-like immunoreactivity (CGRP-IR) and substance P-like immunoreactivity (SP-IR). CGRP-IR is present in fibers that run through the ganglia and in boutons that make contact with almost half of the principal neurons. SP-IR is also present in fibers within the ganglia and in a rare class of synaptic boutons that are found on less than 1% of the principal neurons. Both forms of immunoreactivity are coexpressed in some nerve fibers and in the rare synaptic boutons that contain SP-IR. Neuropeptide Y-like immunoreactivity (NPY-IR), a marker for C-type postganglionic neurons, was used to identify the postsynaptic targets of boutons containing CGRP-IR and SP-IR. Ninety-five percent of the ganglion cells contacted by CGRP-IR boutons were negative for NPY-IR and are therefore likely to be B-type postganglionic neurons. Similarly, 100% of the ganglion cells contacted by boutons containing SP-IR were negative for NPY-IR. Lesions of the sympathetic chain demonstrated that synaptic boutons containing CGRP-IR arise from neurons whose axons enter the chain rostral to ganglion 7. Cutting the chain between ganglia 8 and 9 eliminates all preganglionic B and C inputs to ganglia 9 and 10. The destruction of the preganglionic C pathway by this lesion was verified by staining ganglia 9 and 10 for luteinizing hormone releasing hormone (LHRH). This lesion also eliminated boutons containing CGRP-IR and drastically reduced the number of ganglionic fibers that stained for CGRP-IR and SP-IR. By contrast, cutting the sympathetic chain between ganglia 6 and 7 spared LHRH-IR in the preganglionic C pathway but still eliminated the boutons that normally express CGRP-IR and reduced the amount of staining for SP-IR. CGRP-IR in the sympathetic ganglia arises from preganglionic and sensory neurons whereas ganglionic SP-IR is purely sensory in origin. In the spinal cord, the preganglionic B and C neurons that innervate ganglia 9 and 10 are located in different segments. In segments that contain preganglionic B cells, but not those that contain C cells, there were 243 +/- 37 (mean +/- SD) neurons in the intermediolateral cell column that express CGRP-IR. However, no cell bodies containing SP-IR were found in this region of the spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spinal origins of preganglionic B and C neurons that innervate paravertebral sympathetic ganglia nine and ten of the bullfrog

These experiments were designed to characterize the distribution, morphology, and number of spinal preganglionic neurons that selectively innervate the B- and C-type sympathetic neurons in paravertebral ganglia 9 and 10 of the bullfrog. For this purpose, horseradish peroxidase (HRP) was applied to the anterior end of the sectioned sympathetic chain between ganglia 8 and 9. Subsequent retrograde axonal transport of the HRP labeled ipsilateral spinal neurons whose cell bodies form a column having rostral and caudal boundaries that are, respectively, just caudal to the level of spinal nerve 4 and midway between the entry zones of spinal nerves 7 and 8. In all segments, the labeled preganglionic somata were found in the lateral half of the spinal gray and slightly dorsal to the central canal; a position analogous to that of the intermediolateral cell column in mammals. Most labeled preganglionic neurons were multipolar in shape, and the cell bodies lying between spinal nerves 4 and 5 were, on average, larger than those found between spinal nerves 7 and 8. In transverse sections that were cut near spinal nerve 5, the axons of preganglionic neurons could be seen to exit the cord through ventral roots. Counts of labeled preganglionic neurons indicate that an average +/- S.D. of 338 +/- 89 cells innervate ganglia 9 and 10. Selective labeling of preganglionic B neurons, by cutting spinal nerves 7 and 8 central to their rami communicantes at the time of HRP application, revealed an average +/- S.D. of 137 +/- 31 cells that lie exclusively between spinal nerves 4 and 6. By contrast, selective labeling of preganglionic C neurons, by cutting the sympathetic chain rostral to ganglion 7 at the time of HRP application, revealed an average +/- S.D. of 187 +/- 77 cells in an adjacent portion of the preganglionic column that is bounded by spinal nerve 6 and by a point midway between spinal nerves 7 and 8. These results thus demonstrate a clear segmental segregation between the preganglionic B and C neurons that innervate ganglia 9 and 10.     

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.     

Differential responses of VIPergic and nitrergic neurons in paediatric patients with Crohn's disease

Inflammatory bowel disease is a recurrent intestinal inflammatory disorder that in adults has been associated with changes in enteric nervous system neuropeptide expression. The aim of the present study was to determine whether similar changes were observed in paediatric Crohn's disease. The distribution of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) was determined in colonic tissues from children with ileo-colonic (n=4) and colonic (n=3) Crohn's disease. The submucosal plexus of inflamed regions showed significant increase in density of VIP immunoreactive neurons (margin, 48% vs. inflamed tissue, 82% of HuC/D positive neurons). The density of submucosal plexus nNOS immunoreactive neurons was too low to be reliably quantified. Using the pan-neuronal marker HuC/D, no significant difference in numbers of HuC/D positive submucosal neurons was evident except where neurons were normalized to length of tissue (margins, 3.6+/-0.7 vs. inflamed tissue, 4.0+/-0.6 neurons/ganglia, p=0.33; margins, 2.7+/-0.4 vs. inflamed tissue, 5.7+/-1.2, neurons/mm, p=0.03). In the myenteric plexus, there was a significant increase in the percent of NOS neurons (38% vs. 82% of HuC/D positive neurons) while there was no significant difference in percent of VIP neurons (4% vs. 8%). No difference in number of HuC/D positive myenteric neurons among margin and inflamed tissues was observed (margin, 12.2+/-3.0 vs. inflamed tissue, 12.5+/-5.1 neurons/ganglia, p=0.50; margins 9.1+/-2.1 vs. inflamed tissue, 13.7+/-2.3 neurons/mm, p=0.11). These data demonstrate that inflammation is associated with a differential expression of VIP and nNOS neuronal subpopulations within the two major enteric plexi, likely due to phenotypic switch. Such changes might contribute to the pathogenesis of IBD and ongoing symptoms even in quiescent disease.     

Peptide 19 in the rat vagal and glossopharyngeal sensory ganglia

Peptide 19 (PEP 19) is a 7.6-kDa polypeptide which binds to calmodulin and inhibits calcium-calmodulin signaling. In this study, PEP 19-immunoreactivity (PEP 19-IR) was examined in the rat vagal and glossopharyngeal sensory ganglia. Twenty-nine percent, 59%, and 41% of sensory neurons contained PEP 19-IR in the jugular, petrosal, and nodose ganglia, respectively. These neurons were of various sizes (jugular, mean +/- SD = 635.8 +/- 392.6 microm2, range = 105.9-1695.9 microm2; petrosal, mean +/- SD = 370.9 +/- 228.5 microm2, range = 57.7-1662.7 microm2; nodose, mean +/- SD = 380.5 +/- 157 microm2, range = 87.5-950.4 microm2) and scattered throughout these ganglia. Double immunofluorescence method revealed that PEP 19-IR neurons which had parvalbumin-IR were rare in the ganglia (jugular, 4%; petrosal, 10%; nodose, 8%). PEP 19-IR neurons which contained calbindin D-28k were abundant in the petrosal (20%) and nodose (22%) ganglia but not in the jugular ganglion (8%). Retrograde tracing method indicated that many PEP 19-IR neurons projected to the circumvallate papilla and soft palate. In the soft palate, taste buds were innervated by PEP 19-IR nerve fibers. The present study suggests that PEP 19-IR neurons include chemoreceptors in the vagal and glossopharyngeal sensory ganglia.     

Proenkephalin opioid peptide product in the sensory ganglia of the rat: a developmental immunohistochemical study

The distribution and development of Met-enkephalin-Arg6-Gly7-Leu8 (Enk-8)-containing neurons in the sensory ganglia of the rat were investigated by means of immunocytochemistry using specific antiserum to this octapeptide. Enk-8-like immunoreactivity first appeared in neurons of the trigeminal ganglia of the 18-day embryo, then in the dorsal root ganglia of the 21-day embryo, thus exhibiting a rostrocaudal gradient in terms of appearance and abundance. The number of immunoreactive neurons in these sensory ganglia peaked on the 5th-7th postnatal days, with several small ones observed in each section (1.0-1.4% of total cell number). About 30-40% of these Enk-8-like immunoreactive neurons were also immunoreactive to substance P. Subsequently, Enk-8-like immunoreactivity in the sensory ganglia was decreased and was rarely detected in adult animals. However, colchicine treatment revealed the presence of several Enk-8-containing neurons per section prepared from mature rat. All these neurons were small (12.5-25 microns; mean +/- S.E.M., 19.86 +/- 3.26 microns). Some of these were also immunoreactive to substance P. These results strongly suggest that the preproenkephalin A system exists in subpopulations of both developing and matured sensory cells in the rat. Functional significance of this is discussed.     

Substance P content in cultured neonatal rat vagal sensory neurons: the effect of nerve growth factor

To begin to study the factors regulating the synthesis and release of substance P (SP) in the sensory vagus nerve, cultures of neonatal rat nodose ganglia were developed. In microexplant cultures, obtained from small fragments of nodose ganglia, SP was present in low amounts: after 3 weeks, 141 +/- 36 pg per well, 10 ganglia equivalents per well. To enhance neuron survival, nodose ganglia were enzymatically dissociated using neutral protease. Estimated survival at 5 days was 20-30%, with 800-1200 surviving neurons per plated ganglion, and decreased slowly thereafter. Specific SP immunostaining was present in 10-20% of neurons, mostly of small diameter (18-22 micron). SP content was low for 5 days then rose progressively after 14 days to 80-150 pg per plated ganglion. The addition of nerve growth factor (NGF, 100 ng/ml) to the culture medium did not alter neuron survival. However, SP content was doubled in the presence of NGF, or fell rapidly to one-half control levels following its withdrawal: e.g. following 12 days in culture with NGF 1185 +/- 176 pg/well vs NGF withdrawn day 8-12, 592 +/- 118 pg/well, mean +/- S.D., P less than 0.01. Somatostatin, present in one-sixth the amount of SP, was unaltered by NGF. In subsequent studies, plating of neurons onto previously dissociated rat atriacytes increased survival by 50% but did not alter SP content per surviving neurons. These studies demonstrate that SP is present in dissociated cultures of rat vagal sensory neurons; the quantities and estimated net synthesis rate correspond to previous observations in vivo. The studies also demonstrate that SP content but not neuron survival are regulated by NGF in nodose ganglion neurons. This model may prove valuable for the study of SP and other sensory neuropeptides in this important class of visceral afferent neurons.     

Extensive co-occurrence of substance P and dynorphin in striatal projection neurons: an evolutionarily conserved feature of basal ganglia organization.

A number of different neuroactive substances have been found in striatal projection neurons and in fibers and terminals in their target areas, including substance P (SP), enkephalin (ENK), and dynorphin (DYN). In a preliminary report on birds and reptiles, we have suggested that SP and DYN are to a large extent found in the same striatal projection neurons and that ENK is found in a separate population of striatal projection neurons. In the present study, we have examined this issue in more detail in pigeons and turtles. Further, we have also explored this issue in rats to determine whether this is a phylogenetically conserved feature of basal ganglia organization. Simultaneous immunofluorescence double-labeling procedures were employed to explore the colocalization of SP and DYN, SP and ENK, and ENK and DYN in striatal neurons and in striatal, nigral, and pallidal fibers in pigeons, turtles, and rats. To guard against possible cross-reactivity of DYN and ENK antisera with each others' antigens, separate double-label studies were carried out with several different antisera that were specific for DYN peptides (e.g., dynorphin A 1-17, dynorphin B, leumorphin) or ENK peptides (leucine-enkephalin, metenkephalin-arg6-gly7-leu8, methionine-enkephalin-arg6-phe7). The results showed that SP and DYN co-occur extensively in specific populations of striatal projection neurons, whereas ENK typically is present in different populations of striatal projection neurons. In pigeons, 95-99% of all striatal neurons containing DYN were found to contain SP and vice versa. In contrast, only 1-3% of the SP+ striatal neurons and no DYN neurons contained ENK. Similarly, in turtles, greater than 75% of the SP+ neurons were DYN+ and vice versa, whereas ENK was observed in fewer than 5% of the SP+ neurons and 2% of the DYN+ neurons. Finally, in rats, more than 70% of the SP+ neurons contained DYN and vice versa, but ENK was found in only 5% of the SP+ neurons and in none of the DYN+ perikarya. Fiber double-labeling in the striatum and its target areas (the pallidum and substantia nigra) was also consonant with these observations in pigeons, turtles, and rats. These results, in conjunction with studies in cats by M.-J. Besson, A.M. Graybiel, and B. Quinn (1986; Soc Neurosci. Abs. 12:876) strongly indicate that the co-occurrence of SP and DYN in large numbers of striatonigral and striatopallidal projection neurons in a phylogenetically widespread, and therefore evolutionarily conserved, feature of basal ganglia organization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brn-3a deficiency increases tyrosine hydroxylase-immunoreactive neurons in the dorsal root ganglion

Immunohistochemistry for tyrosine hydroxylase (TH) was performed on the dorsal root ganglia (DRG) in wild-type, heterozygous and Brn-3a knockout mice at embryonic day 18.5. TH-immunoreactive (-IR) neurons were detected in the DRG of wild-type and heterozygous mice, but their proportion was greatly increased by the loss of Brn-3a function (wild-type and heterozygot, 8.4%; knockout, 20.9%). IR neurons were of various sizes in wild-type (mean+/-S.D.=118.1+/-55.4 microm2, range=26.6-306.3 microm2) and heterozygous mice. In the knockout mice, however, TH-IR neurons were mostly small (mean+/-S.D.=68.2+/-34.3 microm2, range=11.8-166.8 microm2). The present study suggests that Brn-3a may normally suppress TH expression in many small DRG neurons but activate TH expression in large DRG neurons.     

Delta-opioid receptor-immunoreactive neurons in the rat cranial sensory ganglia

Immunohistochemistry for delta-opioid receptor (DOR) was performed on the rat cranial sensory ganglia. The immunoreactivity was detected in 16%, 19% and 11% of neurons in the trigeminal, jugular and petrosal ganglia, respectively. The nodose ganglion was devoid of such neurons. DOR-immunoreactive (IR) neurons were mostly small to medium-sized (trigeminal, range = 62-851 microm(2), mean +/- SD = 359 +/- 175 microm(2); jugular, range = 120-854 microm(2), mean +/- SD = 409 +/- 196 microm(2); petrosal, range = 167-1146 microm(2), mean +/- SD = 423 +/- 233 microm(2)). Double immunofluorescence method revealed that all DOR-IR neurons were also immunoreactive for calcitonin gene-related peptide. The cutaneous and mucosal epithelia in the oro-facial region, tooth pulp, taste bud and carotid body were innervated by DOR-IR nerve fibers. In the brainstem, IR nerve terminals were located in the superficial medullary dorsal horn and dorsomedial part of the subnucleus oralis as well as the solitary tract nucleus. The present study suggests that DOR-IR neurons may be associated with nociceptive and/or chemoreceptive function in the cranial sensory ganglia.     

Effect of guanethidine on dopamine in small intensely fluorescent cells of the superior cervical ganglion of the rat.

To determine the portion of ganglionic dopamine stored in the small intensely fluorescent (SIF) cells of the superior cervical ganglion, rats were treated chronically with the neurotoxin guanethidine (50 mg/kg i.p. daily for 6, 13 or 18 days) which destroys noradrenergic neurons. The guanethidine effect was assessed in the ganglion using biochemistry of dopamine and norepinephrine and immunocytochemistry of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). After 18 days of treatment, the ganglionic norepinephrine content was reduced by 80%, but the dopamine content was reduced by only 20%. Morphologic analysis of ganglia stained to distinguish noradrenergic neurons (TH positive, DBH positive) and SIF cells (TH positive, DBH negative) indicated that guanethidine treatment reduced the number of noradrenergic neurons by 70%, dropping from 19413 +/- 1402 to 6515 +/- 1296 per ganglion, but increased the number of dopaminergic SIF cells by 80% from 578 +/- 150 to 1056 +/- 151 per ganglion. Based on these findings, it is concluded that a substantial portion of the dopamine in the rat superior cervical ganglion is located outside the noradrenergic neurons, i.e. in the SIF cells. Extrapolating the data obtained using guanethidine versus control rat leads to infer that although the proportion of SIF cells in the superior cervical ganglion is small (3 +/- 1% of the SIF and noradrenergic neurons combined), about 40% of the total ganglionic dopamine resides in SIF cells, with the remainder serving as precursor in noradrenergic neurons.     

Comparison of capsaicin-evoked calcium transients between rat nodose and jugular ganglion neurons

The objectives of this study were to describe the size distribution of capsaicin-sensitive neurons in nodose and jugular ganglia and to determine whether there is a difference in capsaicin sensitivity between these two types of ganglia. Functional identification was made by measurement of the capsaicin-evoked calcium (Ca2+) transients in cultured vagal sensory neurons of young adult Sprague-Dawley rats using the Fura-2-based ratiometric imaging technique. In the first study series, cells on the second day of culture were perfused with capsaicin solution (10(-7) M) for 15 s, and the Ca2+ transients were continuously recorded before, during, and after the capsaicin challenge. Out of 603 viable neurons, 57.5% were capsaicin-sensitive; the percentages of capsaicin-sensitive cells in the nodose and jugular ganglia were 59.8% and 55.4%, respectively. Capsaicin sensitivity predominated in the small- and medium-sized neurons; the capsaicin-sensitive cells generally had a diameter less than 35 microm in both types of ganglia. Although the results did not indicate any differences in the size distribution of capsaicin-sensitive neurons between the two ganglia, results of our second study series showed that a near-maximal concentration of capsaicin (3 x 10(-6) M) evoked a significantly greater peak Ca2+ transient in jugular neurons (382.5 +/- 85.5 nM) than in nodose neurons (134.3 +/- 17.5 nM). In summary, our results showed that an increase in cell diameter was accompanied by a decreasing trend in percentage of capsaicin-sensitive neurons in both vagal ganglia. Capsaicin at high concentration evoked a greater peak Ca2+ transient in jugular ganglion neurons, despite no difference in the responses to KCl between these two types of ganglion neurons.     

Electrophysiological properties and cholinergic responses in guinea-pig celiac ganglion neurons in primary culture.

Prevertebral neurons enzymatically dissociated from celiac ganglia of adult guinea-pigs were maintained in long-term primary culture. Cells were plated at a density of 95 +/- 15 cm-2, and intracellular electrical activity was measured between 2 and 7 weeks after dissociation. Neurite outgrowth began within 24 h of enzymatic dissociation. Cell survival dropped below 50% after more than two weeks in culture. The resting potential (-53 mV +/- 0.8), time constant (12 ms +/- 1.3), input resistance (47 M omega +/- 8.6), rheobase (0.33 nA +/- 0.02), degree of accommodation, spike amplitude (70 mV +/- 3.0), after hyperpolarization amplitude (-9.5 mV +/- 0.55), and after hyperpolarization duration (88 ms +/- 7.6) in these cells were not different from those recorded from neurons in intact celiac ganglia. A larger proportion (greater than 90%) of cells exhibited fast accommodation (phasic) in response to depolarizing current pulses. Unevoked (spontaneous) depolarizations and action potentials were observed. The cells responded to pressure ejected acetylcholine. Two types of responses consisted of an early rapid depolarization which was attenuated by hexamethonium and a later slow depolarization which was attenuated by atropine. We conclude that prevertebral neurons from guinea-pigs can be maintained in long-term primary culture, that they retain electrophysiological properties similar to intact ganglia and exhibit complex responsivity to acetylcholine.     

The expression of antigens by embryonic neurons and glia in segmental ganglia of the leech Haemopis marmorata

Monoclonal antibodies (mAbs) raised against adult leech nervous systems were screened on embryos of the leech Haemopis marmorata in order to determine when in development specific antigens are first expressed and the order in which they are expressed by different cells or tissues. Three of the mAbs produced by Zipser and McKay (Zipser, B., and R. McKay (1981) Nature 289: 549-554) were screened: Lan3-1, Lan3-5, and Lan3-6. Each mAb shows a different pattern of labeling in the adult leech nerve cord (Zipser, B. (1982) J. Neurosci. 2: 1453-1464). The embryonic stages studied were from 5 days after egg deposition to 30 days (emergence from the cocoon). The pattern of labeling was assayed in whole mounts using horseradish peroxidase-conjugated second antibodies. The principal results are as follows. (1) Antigens recognized by Lan3-5 are first expressed by the glia of the roots of the anterior segmental ganglia at 6 to 7 days, several days later by the interganglionic connective glia, and near the end of embryonic development by ganglionic neurons. An anterior to posterior temporal gradient is observed in the expression of these antigens. In addition, Lan3-5 also labels the protonephridia and nephridia from early development onward. (2) Antigens recognized by Lan3-6 are first expressed by a pair of neurons in each segmental ganglion and later in development by additional neurons. By the time of emergence, however, only about half of the neurons that label in the adult have done so, implying that some neurons express these antigens postembryonically. Labeling with Lan3-6 is first seen in neuronal somata and only later in neuronal processes. (3) Antigens recognized by Lan3-1 and expressed by segmentally specific neurons in ganglia 5 and 6 are not detectable during embryonic development, but are so at early postembryonic stages. Thus, these three mAbs provide an approach to study different aspects of the development of the leech nervous system, specifically the relation between glial and neuronal differentiation and the genesis of segmentally specific phenotypes.     

Immunohistochemical localization of bombesin/gastrin-releasing peptide and substance P in primary sensory neurons

The existence of bombesin/gastrin-releasing peptide-like immunoreactivity (BN-GRP-LI) in rat sensory ganglia and spinal cord was confirmed using immunocytochemistry, gel filtration chromatography, and high performance liquid chromatography combined with radioimmunoassay. Immunohistochemical studies showed that in the spinal sensory ganglia of the rat about 5% of the neurons exhibited BN-GRP-LI, whereas about 20% of the neurons exhibited substance P-like immunoreactivity (SP-LI). The two immunoreactivities were found in different cells, but both were located in small ganglion cells. In the posterior horn of the spinal cord, BN-GRP-LI and SP-LI were located in the superficial layers, and this distribution was different from that of Met5-enkephalin-like immunoreactivity. The results are in agreement with the concept that there is a primary sensory pathway from the sensory ganglia to the spinal cord which contains BN-GRP-LI and that these neurons are separate from those containing substance P. In extracts prepared from spinal ganglia, two molecular weight forms of BN-GRP-LI were found using gel filtration chromatography. The high molecular weight form coeluted with porcine GRP and the low molecular weight form was smaller than bombesin. The low molecular weight BN-GRP-LI extracted from spinal cord was more hydrophilic than bombesin or ranatensin.     

Cell types and axonal sizes of calcitonin gene-related peptide-containing primary sensory neurons of the rat

Neurons with immunoreactivity (IR) for calcitonin gene-related peptide (CGRP) in the rat dorsal root ganglia were examined by immunoelectron microscopy. About one-half of the neurons of the L5 dorsal root ganglia of animals treated with colchicine had CGRP-IR, and 40% of these were large neurons of type A. The proximal parts of their peripheral axons were, however, unmyelinated (91%) or thinly myelinated (9%). Thickly myelinated axons observed in the same sections were always devoid of CGRP-IR. The CGRP-IR neurons were various subtypes of type A and B neurons. No specific morphological characteristics were associated with CGRP-IR.     

Calbindin D28k-containing splanchnic and cutaneous dorsal root ganglion neurons of the rat

Calbindin D28k (CaBP)-containing splanchnic and cutaneous sensory neurons in the rat dorsal root ganglia (DRGs) were investigated immunocytochemically in combination with a fluorescent dye tracer (Fluoro gold). About 15% of the DRG neurons at Th9-10 levels showed CaBP-like immunoreactivity. Eighty-four % of the splanchnic sensory neurons were immunoreactive to CaBP, while only 3% of the cutaneous sensory neurons were. The diameters of the splanchnic and cutaneous sensory neurons containing CaBP were 23.4 +/- 6.3 microns and 38.4 +/- 8.8 microns, respectively. Splanchnic sensory neurons containing CaBP were sensitive to capsaicin while cutaneous ones were not. These findings suggest that CaBP-containing splanchnic and cutaneous sensory neurons constitute different subgroups among the DRG neurons at the lower thoracic level.     

Allatotropin-like neuropeptide in the cockroach abdominal nervous system: myotropic actions, sexually dimorphic distribution and colocalization with serotonin

Allatotropin (AT) was isolated from the moth Manduca sexta as a peptide stimulating biosynthesis of juvenile hormone in the corpora allata, but has also been shown to be cardioactive in the same species. Here, we have investigated the presence and biological activity of AT-like peptide in the cockroaches Leucophaea maderae and Periplaneta americana with focus on abdominal ganglia and their target tissues. An antiserum to M. sexta AT was used for immunocytochemical mapping of neurons in the abdominal ganglia. A small number of interneurons and efferent neurons were found AT-like immunoreactive (AT-LI) in each of the abdominal ganglia. A prominent sexual dimorphism was detected in the terminal abdominal ganglion: in L. maderae the male ganglion there are approximately 18 AT-LI neurons with cell bodies posteriorly and efferent axons in the genital nerves; in the female ganglion 4-5 AT-LI cell bodies (with efferent axons) were found in the same region. Correlated with the extra efferents in males, the male accessory glands are richly supplied by AT-LI fibers and in females a less prominent innervation was seen in oviduct muscle. A similar dimorphism was seen in abdominal ganglia of P. americana. A sexual dimorphism was also detected in the abdominal ganglia A4-A6 of L. maderae. In each of these ganglia, approximately 8-10 large AT-LI neuronal cell bodies were found along the midline; in females these neurons have significantly larger cell bodies than in males. In both sexes, and both cockroach species, two large dorsal midline neurons were detected in A-5 and 6, which seem to send axons to the hindgut: the rectal pads of the hindgut are supplied by arborizing AT-LI axons. In males and females of both species, efferent AT-LI axons from midline neurons in A3-A6 supply the lateral heart nerves and other neurohemal release sites with arborizations. The efferent midline neurons of females contain colocalized serotonin-immunoreactivity. We tested the in vitro actions of M. sexta AT on muscle contractions in the L. maderae hindgut and the abdominal heart of both species. The frequency of contractions in the hindgut increased dose dependently when applying AT at 5 x 10(-8) to 5 x 10(-6) M (maximal response at 5 x 10(-7) M). Also the frequency of contractions of the heart increased by application of AT (threshold response at 5 x 10(-9) M). This effect was more prominent in males of both species (maximal response was a 35-40% increase in males and 10-20% in females). In conclusion, an AT-like peptide is present in neurons and neurosecretory cells of cockroach abdominal ganglia and seems to play a role in control of contractions in the hindgut and heart and also to have some function in male accessory glands and oviduct.     

Coexistence of glutamate and substance P in dorsal root ganglion neurons of the rat and monkey

Antisera to a glutamate (Glu) conjugate, to glutaminase (GLN), and to substance P (SP) have been used to investigate the issue of putative glutamergic neurons in the dorsal root ganglia (DRG) and the possible coexistence in these neurons of Glu and SP. The Glu antiserum, characterized by immunoadsorption and immunoblot tests, is highly selective for Glu out of a number of other amino acids including aspartate. Quantitative data were gathered from consecutive 4-microns-thick paraffin sections from cervical ganglia of rats with or without spinal injections of colchicine and from one monkey not treated with colchicine. Neurons containing more than one antigen could be identified on adjacent sections tested with the three different antisera. Neurons labeled by the Glu-antiserum represent 15-30% of the DRG population in untreated rats. They include most of the small neurons (with mean perikaryal area around 300-400 microns) but also larger neurons (with perikaryal area greater than 600 microns). DRG neurons labeled by either the GLN or the SP antiserum are small (mean area 335-375 microns and 356-374 microns, respectively) and account for approximately 40 and 15%, respectively, of the sampled neurons. In colchicine-treated rats the number of Glu-positive neurons increases up to about 70%. The same treatment also increases the number of SP-positive neurons but not that of GLN-positive neurons. In the monkey about half of the DRG neurons are Glu positive and, as in rats, they are mostly small. GLN-positive neurons in the same species account for about half of the DRG population and are only small. In rats, about 60-80% of Glu-positive neurons are also GLN-positive and most GLN-positive neurons are Glu positive. In colchicine-treated rats, close to 90% of SP-positive neurons are also positive for Glu and about 60% of SP-positive neurons are triple labeled. In the monkey's DRGs, whose sections were not processed for SP, most Glu- or GLN-positive neurons are also positive for the other antiserum used. Several considerations argue against the possibility that, under the present experimental conditions, the Glu antiserum is a metabolic marker. It is therefore suggested that Glu-positive neurons may use Glu as neurotransmitter. Coexistence of Glu and SP in some DRG neurons suggests that both agents may be released by terminals of primary afferents from the same DRG neuron.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic influence on phenotypic differentiation in adult hippocampal neurogenesis

Regulation of adult hippocampal neurogenesis has different regulatory levels, including cell proliferation, survival and differentiation. Cell proliferation and survival are differentially influenced by inheritable traits and the genetic background determines which regulatory levels of adult hippocampal neurogenesis are preferentially involved in a neurogenic response to environmental stimuli. We here compared baseline adult neurogenesis in wild-derived strain Mus spretus and three inbred laboratory strains: A/J, C3H/HeJ and DBA/2J. Proliferation of was similar in the four strains, with the extremes being A/J, which had about 2100+/-570 (mean+/-S.D.) labeled newborn cells per dentate gyrus (after 6 days of bromodeoxyuridine injections), and DBA/2J, which had approximately 1400+/-260. C3H/HeJ had approximately 1500+/-600 and M. spretus had 1550+/-270. Survival of new cells after 4 weeks was 19% in A/J and DBA/2J, and 21% in M. spretus, but 37% in C3H/HeJ. Survival in C3H/HeJ was significantly different from DBA/2. Phenotypic analysis revealed that DBA/2J produced significantly fewer new neurons than A/J and C3H/HeJ (47% vs. 63% and 67%) but significantly more new astrocytes than A/J and C3H/HeJ (28% vs. 9% and 11%). In absolute terms there were 370+/-120 new neurons in C3H/HeJ, 250+/-60 in A/J, 130+/-50 in DBA/2J, and 190+/-130 in M. spretus. Our results indicate that regulation of adult hippocampal neurogenesis affects the level of phenotypic differentiation. At the present time it cannot be determined whether this regulation occurs by influencing cell fate decisions or by promoting selective survival.