Neuropeptide distribution in the cervico-thoracic paravertebral ganglia of the cat with particular reference to calcitonin gene-related peptide immunoreactivity

Summary Paraffin sections of cervical and upper thoracic paravertebral ganglia of the cat were investigated by immunohistochemistry using antisera directed against calcitonin gene-related peptide (CGRP). The relationships of CGRP-immunoreactive structures to those exhibiting immunoreactivity to antisera against other regulatory peptides and dopamine--hydroxylase (DBH), respectively, were studied in consecutive sections. Singly scattered CGRP-immunoreactive neuronal perikarya were observed in the superior and middle cervical ganglia as well as in the stellate ganglion. These neurons also displayed immunoreactivity to vasoactive intestinal polypeptide (VIP), and some additionally exhibited faint substance-P immunoreactivity. DBH- and neuropeptide Y-immunoreactive ganglion cells were not identical with CGRP-immunoreactive neuronal cell bodies.According to the immunoreactive properties of varicosities, which abut on CGRP/VIP-immunoreactive perikarya, three types of CGRP/VIP-immunoreactive ganglion cells could be distinguished: (1) CGRP/VIP-immunoreactive neurons being surrounded by somatostatin-immunoreactive nerve fibers, (2) neurons being approached by both DBH- and met-enkephalin-immunoreactive varicosities, and (3) neurons receiving both DBH- and neurotensin-immunoreactive fibers. The stellate and upper thoracic ganglia harbored clusters of intensely VIP-immunoreactive somata, which lacked CGRP-immunoreactivity. Fine somatostatin-immunoreactive and coarse CGRP-immunoreactive fibers were distributed within these clusters, whereas patches of neurotensin-immunoreactive fibers were complementarily arranged. At all segmental levels investigated, a few postganglionic neurons were approached by both CGRP-immunoreactive and substance P-immunoreactive varicosities, but lacked a VIP-immunoreactive innervation. Therefore, CGRP/substance P-immunoreactive fiber baskets appeared rather to be of extraganglionic origin than to emerge from intraganglionic CGRP/VIP/SP neurons. CGRP-immunoreactive cell bodies or fibers were absent in clusters of small paraganglionic cells, but some of the solitary paraganglionic cells displayed CGRP-immunoreactivity. Our findings establish the presence of CGRP-immunoreactivity in a population of sympathetic neurons in the cat. A highly differentiated, segment-dependent organizational pattern of neuropeptides in cervico-thoracic paravertebral ganglia was demonstrated.Supported by Deutsche Forschungsgemeinschaft grant He 919/6-2     

Histochemical evidence of chemical sympathectomy by guanethidine in newborn rats

Synopsis Guanethidine is known to cause a loss of catecholamines from sympathetically innervated tissues and sympathetic ganglia in adult animals but its effect on newborn animals has not been examined.Newborn rats were injected daily with guanethidine (20 mg/kg body weight) for 8 days. They were killed when 1 month-old along with untreated litter mate controls. Catecholamines were demonstrated in the iris, in the pineal body and in sympathetic ganglia, using the formaldehyde-induced fluorescence method.In the guanethidine-treated rats there was a complete loss of fluorescent nerve fibres from the pineal body and an almost complete loss of similar fibres from the iris. The sympathetic ganglia were reduced to less than 10% of the control ganglia, and the number of nerve cell bodies per unit area was decreased in the ganglion remnants.It is concluded that guanethidine causes, in newborn rats, an irreversible destruction of most sympathetic neurons, i.e. a chemical sympathectomy closely resembling that obtainable in newborn animals by injections of 6-hydroxydopamine or antiserum to nerve growth factor.     

Neuropeptide Y: presence in sympathetic and parasympathetic innervation of the nasal mucosa

Summary The occurrence of neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in the sympathetic and parasympathetic innervation of the nasal mucosa was studied in various species including man. A dense network of NPY-immunoreactive (IR) fibres was present around arteries and arterioles in the nasal mucosa of all species studied. NPY was also located in nerves around seromucous glands in pig and guinea-pig, but not in rat, cat and man. The NPY-IR glandular innervation corresponded to about 20% of the NPY content of the nasal mucosa as revealed by remaining NPY content determined by radioimmunoassay after sympathectomy. These periglandular NPY-positive fibres had a distribution similar to the VIP-IR and PHI-IR nerves but not to the noradrenergic markers tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH). The NPY nerves around glands and some perivascular fibres were not influenced by sympathectomy and probably originated in the sphenopalatine ganglion where NPY-IR and VIP-IR ganglion cells were present. The venous sinusoids were innervated by NPY-positive fibres in all species except the cat. Dense NPY and DBH-positive innervation was seen around thick-walled vessels in the pig nasal mucosa; the latter may represent arterio-venous shunts. Double-labelling experiments using TH and DBH, and surgical sympathectomy revealed that the majority of NPY-IR fibres around blood vessels were probably noradrenergic. The NPY-positive perivascular nerves that remained after sympathectomy in the pig nasal mucosa also contained VIP/PHI-IR. The major nasal blood vessels, i.e. sphenopalatine artery and vein, were also densely innervated by NPY-IR fibres of sympathetic origin. Perivascular VIP-IR fibres were present around small arteries, arterioles, venous sinusoids and arterio-venous shunt vessels of the nasal mucosa whereas major nasal vessels received only single VIP-positive nerves. The trigeminal ganglion of the species studied contained only single TH-IR or VIP-IR but no NPY-positive ganglion cells. It is concluded that NPY in the nasal mucosa is mainly present in perivascular nerves of sympathetic origin. In some species, such as pig, glandular and perivascular parasympathetic nerves, probably of VIP/PHI nature, also contain NPY.     

The response of the atrium to direct mechanical wounding in the adult heart of the newt,Notophthalmus viridescens

Summary Wound repair and proliferation were examined in the injured newt atrium with light- and electron-microscopic techniques including autoradiography. Hearts were injured by removing a piece approximately 0.5 mm² of the atrial wall. The five-day wound was an endothelial and mesothelial-lined blood clot bordered by a 150-m necrotic zone. Repair progressed from the periphery inward with areas of macrophage activity replaced by fibroblasts and connective tissue. The wound at 25 days consisted of a scar with few myocytes. There was no difference in the proliferative behavior between the right and left atria. Proliferative cells were localized to a 500-m reactive zone surrounding the wound. The maximum mesothelial cell thymidine-labeling index of 20.5% and mitotic index of 1.4% were seen 5 days after injury. The peak connective tissue cell thymidine-labeling index of 10.2% and mitotic index of 0.4% were seen 10 days after wounding. The peak thymidine-labeling index of 9.8% for myocardial cells was recorded 10 days after injury with a mitotic index of 0.2%. Proliferation returned to control levels by 25 days post-injury. Electron microscopy demonstrated that myocytes engaged in DNA synthesis were indistinguishable from control myocytes. Z-band material was not observed in mitotic myocytes, but myofilaments and junctions were present.     

Distribution of NADPH-diaphorase activity in rat paravertebral,prevertebral and pelvic sympathetic ganglia

Summary Paravertebral (superior cervical and stellate), prevertebral (coeliac-superior mesenteric, inferior mesenteric) and pelvic (hypogastric) sympathetic ganglia of the rat were investigated by enzyme histochemistry to ascertain the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity. In the paravertebral ganglia the majority of the sympathetic neuronal perikarya contained lightly and homogeneously distributed formazan reaction product but there was a range of staining intensities amongst the neuron population. In contrast, in the prevertebral ganglia, intense NADPH-diaphorase staining was present in certain neurons. Firstly, a population of neurons of the coeliac-superior mesenteric ganglion complex were surrounded by densely NADPH-diaphorase-positive baskets of fibres and other stained fibres were seen in interstitial nerve bundles and in nerve trunks connected to the ganglion complex. Secondly, in both the inferior mesenteric ganglion and hypogastric ganglion there were many very intensely NADPH-diaphorase positive neurons. Stained dendritic and axonal processes emerged from these cell bodies. In both ganglia this population of neurons was smaller in size than the lightly stained ganglionic neurons and commonly had only one long (presumably axonal) process. The similarity of these highly NADPH-diaphorase-positive neurons with previously described postganglionic parasympathetic neurons in the hypogastric ganglion is discussed.     

Autoradiographic assessment of 3H-proline uptake by osteoblasts following guanethidine-induced sympathectomy in the rat

Summary Sympathectomy was carried out in rats by injections of guanethidine-sulfate from birth to 14 days of age. At 45 days of age, the activity of osteoblastic cells was monitored by ³H-proline autoradiography. Effectiveness of sympathectomy was verified by light-microscopic examination of superior cervical and celiac ganglia. Grain counts over periosteal osteoblasts of the femoral diaphysis and osteoblasts mesial to the first molar in the mandible demonstrated a significantly reduced uptake of ³H-proline in the sympathectomized rats. The data provide direct evidence of sympathetic influence on osteoblastic activity and suggest that sympathectomy may result in the loss of a trophic influence which is important in the regulation of osteogenesis.Supported by N.I.D.R. grant DEO 4557 (R.M.K.), N.I.H. grant 5-SO1RR-5373 to K.U.M.C., and N.I.H. grant RR-05332 to N.Y.U. (U.S.). We thank Charles A. Brownley of CIBA-Geigy, Summit, N.J. for the guanethidine sulfate     

Compensatory responses and development of the nodose ganglion following ablation of placodal precursors in the embryonic chick (Gallus domesticus)

The nodose ganglion is the distal cranial ganglion of the vagus nerve which provides sensory innervation to the heart and other viscera. In this study, removal of the neuronal precursors which normally populate the right nodose ganglion was accomplished by ablating the right nodese placode in stage 9 chick embryos. Subsequent histological evaluation showed that in 54% of lesioned embryos surviving to day 6, the right ganglion was absent. Most embryos surviving to day 12, however, had identifiable right ganglia. In day 12 embryos, the right ganglion which developed was abnormal, with ganglion volume and ganglion cell diameter reduced by 50% and 20%, respectively, compared to control ganglia. To investigate the source of the neuron population in the regenerated ganglion, we combined nodose placode ablation with bilateral replacement of chick with quail cardiac neural crest (from mid-otic placode to somite 3). These cells normally provide only non-neuronal cells to the nodose ganglion, but produce neurons in other regions. At day 9, quail-derived neurons were identified in the right nodose ganglia of these chimeras, indicating that cardiac neural crest cells can generate neurons in the ganglion when placode-derived neurons are absent or reduced in number. On the other hand, we found that sympathetic neural crest (from somites 10 to 20) does not support ganglion development, suggesting that only neural crest cells normally present in the ganglion participate in reconstituting its neuronal population. Our previous work has shown that right nodose placode ablation produces abnormal cardiac function, which mimics a life-threatening human heart condition known as long QT syndrome. The present results suggest that the presence of neural crest-derived neurons in the developing right nodose ganglion may contribute to the functional abnormality in long QT syndrome.This work was supported by grant PO1 HL 36059     

Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

Summary Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10g/ml) resulted in an increased SIF cell fluorescence (P<0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2×10^–6 M; 2×10^–5 M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2×10^–5 M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.This work was supported by a grant from the Medical Research Council. We thank Mrs. G. O'Shea, Mr. T.T. Lee and Mr. P.F. Hire for their valuable technical assistance     

Consecutive demonstration of catecholamines and dopamine-β-hydroxylase within the same specimen

Summary A combined method was developed for characterization and differentiation of catecholamines in neuron populations containing more than one catecholamine, e.g. dopamine and norepinephrine. Its application to small intensely fluorescent (SIF-) cell clusters in sympathetic ganglia allows the successive demonstration of glyoxylic acid-induced catecholamine fluorescence and dopamine--hydroxylase by indirect immunofluorescence within the same tissue section. Applying this technique to an example, two types of SIF-cells were demonstrated in the guinea pig superior cervical ganglion.Supported by the Deutsche Forschungsgemeinschaft grant He 919/4     

Morphometric study of the superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

To compare the functional state of the superior cervical (SCG) and stellate sympathetic ganglia (SG) of spontaneously hypertensive rats (SHR) with those of age-matched normotensive Wistar Kyoto rats (WKY), ganglion cell volume and area occupied by ganglion cells relative to each whole ganglionic area were morphometrically examined using the Texture Analyse System (TAS) in rats at 0, 10 and 30 days of age. The weight of each ganglion relative to animal weight was also measured. The ganglion cell volume and the relative area of ganglionic cells in both ganglia of SHR were significantly larger (P<0.05) than those of age-matched WKY at ages 0 and 10 days after birth. The relative ganglionic weights of SHR were significantly larger (P<0.01) compared with those of WKY at all ages examined, except for SG at 0 days after birth. These results show that the relative volume of sympathetic ganglion cells is greater in both SCG and SG of SHR than that of WKY, suggesting that hyperfunction of sympathetic ganglia occurs at the prehypertensive stage as a primary factor in the development of hypertension in SHR.     

Microfluorimetric quantitation of catecholamine turnover in the sympathetic neurons of rat

Summary The quantitative aspects of the formaldehydeinduced fluorescence and the turnover of catecholamines in the sympathetic neuronal perikaryon of different sympathetic ganglia were studied after a blockade of the amine synthesis with -methyltyrosine. The concentration of catecholamines was determined by microfluorimetric quantitation method. The half-life of catecholamines in sympathetic neuronal perikarya was short and depended on the ganglion studied. The turnover rate of catecholamines in sympathetic neurons was highest in superior cervical and lowest in coeliac ganglion. Brightly fluorescent fibers were still seen five hours after the amine synthesis blockade, whereas almost all cell bodies had lost their fluorescence. Also small intensely fluorescent cells were still brightly fluorescent after the follow-up period. Microfluorimetrically determined turnover of catecholamines gave more detailed information about the turnover of catecholamines in sympathetic nervous system when compared to the biochemical methods used earlier.     

Alteration of neonatal rat parotid gland acinar cell proliferation by guandethidine-induced sympathectomy.

Newborn rats were injected with guanethidine-sulfate (20 micrograms/g body weight) every 48 hr from 12 hr after birth until day 14 (eight injections per animal). The guanethidine treatment resulted in an 86% absolute reduction in cell number in the superior cervical ganglia of 15 day old rats. The cells which remained after guanethidine treatment showed destruction of mitochondria and an extensive decrease in endoplasmic reticulum. Chemical sympathectomy with guanethidine induced a 3.1 hr lengthening of the acinar cell generation cycle time (17.4 hr to 20.5 hr), resulting from a longer G1 period (6.9 hr in the control group as compared to 10.5 hr in the guanethidine-treated group), as well as a cecrease in the mean percentage of [3H]thymidine-labeled acinar cells (22.3 +/- 0.5% to 19.3 +/- 0.5%) and mean acinar cell mitotic index (2.6 +/- 0.2% to 2.1 +/- 0.1%). A circadian rhythm was found to exist in parotid gland acinar cell mitotic activity of 15 day old rats and the amplitude of the rhythm was reduced from 26.5% to 14.9% in guanethidine-treated rats. This study indicates that the diminution of sympathetic influence on the developing parotid gland results in a slight, but significant alteration in acnar cell proliferation.     

The development of the neurons of the glossopharyngeal (IX) and vagal (X) sensory ganglia in chick embryos

The timetable of cell generation, neuronal death and neuron numbers in the fused proximal glossopharyngeal (IX) and vagal (X) ganglion and distal IX and X ganglia were studied in normal and nerve growth factor (NGF) treated chick embryos. 3H-thymidine was injected between the 3rd and 7th days of incubation and embryos sacrificed on the 11th day. Neurons in the distal IX and X ganglia were generated between the 2nd and 5th days of incubation, the peak mitotic activity occurring on the 4th and 3rd days, respectively. Neurons of the proximal IX and X ganglion were generated between the 4th and 7th days, with maximum neuron generation on the 5th day of incubation. Counts of neurons in the 3 ganglia between the 5th and 18th days of incubation showed a maximum of 22,000 on the 8th day in the proximal IX and X ganglion and this decreased to 12,000 by the 13th day. In the distal IX ganglion, the neuron number decreased by 44% from 4,500 on the 6th day to 2,500 by the 11th day. A similar decrease of 43% was found in the distal X ganglion, the neuron number falling from 11,500 on the 7th day to 6,500 by the 11th day of incubation. Neuronal cell death in these ganglia extended from the 5th to the 12th day of incubation, maximum cell death occurring at or after the cessation of mitotic activity. NGF administration from the 5th to the 11th day of incubation did not have a measurable effect on the neurons of proximal IX and X and distal IX ganglia, but increased neuronal survival by 30% in the distal X ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometric study of the superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

To compare the functional state of the superior cervical (SCG) and stellate sympathetic ganglia (SG) of spontaneously hypertensive rats (SHR) with those of age-matched normotensive Wistar Kyoto rats (WKY), ganglion cell volume and area occupied by ganglion cells relative to each whole ganglionic area were morphometrically examined using the Texture Analyse System (TAS) in rats at 0, 10 and 30 days of age. The weight of each ganglion relative to animal weight was also measured. The ganglion cell volume and the relative area of ganglionic cells in both ganglia of SHR were significantly larger (P less than 0.05) than those of age-matched WKY at ages 0 and 10 days after birth. The relative ganglionic weights of SHR were significantly larger (P less than 0.01) compared with those of WKY at all ages examined, except for SG at 0 days after birth. These results show that the relative volume of sympathetic ganglion cells is greater in both SCG and SG of SHR than that of WKY, suggesting that hyperfunction of sympathetic ganglia occurs at the prehypertensive stage as a primary factor in the development of hypertension in SHR.     

ALTERATION OF NEONATAL RAT PAROTID GLAND ACINAR CELL PROLIFERATION BY GUANETHIDINE-INDUCED SYMPATHECTOMY

Newborn rats were injected with guanethidine-sulfate (20 μg/g body weight) every 48 hr from 12 hr after birth until day 14 (eight injections per animal). The guanethidine treatment resulted in an 86% absolute reduction in cell number in the superior cervical ganglia of 15 day old rats. The cells which remained after guanethidine treatment showed destruction of mitochondria and an extensive decrease in endoplasmic reticulum. Chemical sympathectomy with guanethidine induced a 3.1 hr lengthening of the acinar cell generation cycle time (17.4 hr to 20.5 hr), resulting from a longer G₁ period (6.9 hr in the control group as compared to 10.5 hr in the guanethidine-treated group), as well as a decrease in the mean percentage of [³H]thymidine-labeled acinar cells (22.3 ± 0.5% to 19.3 ± 0.5%) and mean acinar cell mitotic index (2.6 ± 0.2% to 2.1 ± 0.1%). A circadian rhythm was found to exist in parotid gland acinar cell mitotic activity of 15 day old rats and the amplitude of the rhythm was reduced from 26.5% to 14.9% in guanethidine-treated rats. This study indicates that the diminution of sympathetic influence on the developing parotid gland results in a slight, but significant alteration in acinar cell proliferation.     

Innervation of dog ciliary ganglion

There are species differences with regard to the composition of the ciliary ganglion. For instance, in rabbits and cats it consists solely of oculomotor nerves and has no sympathetic or sensory innervation. The purpose of this study is to clarify the participation of these nerves in the ciliary ganglion of the dog by histochemical methods. Cholinesterase (ChE) activity was studied by Karnovsky's method and catecholamine fluorescence by the glyoxylic acid method. Furthermore, the origins of the respective nerves were investigated by a serial preparation method, involving unilateral cervical sympathectomy and tracer dye injection in the ganglion. The results obtained were: (1) Ciliary ganglion cells showed intense ChE activity. Oculomotor nerve fibers leading to the ganglion showed moderate ChE activity, while the reaction in the short ciliary nerves was strong. (2) Aminergic nerves were present in the intercellular space of the ciliary ganglion, and bilateral or central innervation was suggested by the results of cervical sympathectomy. (3) Connection between the ciliary and trigeminal ganglia was proved by the dye tracer study. The results show that the ciliary ganglion in dogs is composed of oculomotor, trigeminal and sympathetic nerves.     

Innervation of dog ciliary ganglion

Summary There are species differences with regard to the composition of the ciliary ganglion. For instance, in rabbits and cats it consists solely of oculomotor nerves and has no sympathetic or sensory innervation. The purpose of this study is to clarify the participation of these nerves in the ciliary ganglion of the dog by histochemical methods. Cholinesterase (ChE) activity was studied by Karnovsky's method and catecholamine fluorescence by the glyoxylic acid method. Furthermore, the origins of the respective nerves were investigated by a serial preparation method, involving unilateral cervical sympathectomy and tracer dye injection in the ganglion. The results obtained were: (1) Ciliary ganglion cells showed intense ChE activity. Oculomotor nerve fibers leading to the ganglion showed moderate ChE activity, while the reaction in the short ciliary nerves was strong. (2) Aminergic nerves were present in the intercellular space of the ciliary ganglion, and bilateral or central innervation was suggested by the results of cervical sympathectomy. (3) Connection between the ciliary and trigeminal ganglia was proved by the dye tracer study. The results show that the ciliary ganglion in dogs is composed of oculomotor, trigeminal and sympathetic nerves.     

The effect of an LH pulse on 3H-thymidine incorporation into cultured ovaries of metestrous rats

Summary The effect of an LH pulse on the rate at which ³H-thymidine is incorporated into cultured ovaries of metestrous rats was studied. In comparison to ovaries cultured with tonic LH, an LH pulse (1) rescued follicles from atresia, (2) induced thecal cell proliferation, and (3) increased the rate at which granulosa cells enter mitosis. It is concluded that LH pulses increase follicular growth by first triggering thecal cell proliferation and then inducing mitotic divisions within the granulosa cells of both atretic and non-atretic follicles.     

选择性切除交感神经对大鼠心内神经节中去甲肾上腺素、乙酰胆碱和NPY的影响

为了探讨心内神经节中去甲肾上腺素（ＮＡ）、乙酰胆碱（ＡＣｈ）和ＮＰＹ的相互作用,本实验应用６－ＯＨ－ＤＡ选择性切除大鼠心脏交感神经纤维,然后应用荧光和酶组织化学法、免疫组织化学结合图像分析法观察了大鼠心内神经节ＮＡ、ＡＣｈＥ活性和ＮＰＹ的变化。结果显示：实验组大鼠心内神经节中儿茶酚胺荧光反应阳性和ＮＰＹ免疫反应（ＮＰＹ－ＩＲ）阳性的神经纤维明显减少,ＡＣｈＥ阳性神经纤维明显增多,ＡＣｈＥ反应性神经元积分光密度增加,而儿茶酚胺荧光反应和ＮＰＹ－ＩＲ阳性神经元变化不明显。结果提示：１．交感神经化学切除后大鼠心内神经节中ＮＡ、ＡＣｈＥ活性和ＮＰＹ出现不同的变化,体现了心脏交感神经和副交感神经的相互抑制作用;２．心内神经节可能含有两种性质的ＮＰＹ,即交感性和非交感性ＮＰＹ。     

Neuronal competition determines the spatial pattern of neuropeptide expression by identified neurons of the leech.

Staining adult and embryonic leech ventral nerve cords with antibodies raised against the molluscan neuropeptides small cardioactive peptide B (SCP) and FMRFamide results in segment-specific and bilaterally asymmetric patterns of cell staining. One immunoreactive neuron, the RAS interneuron, is present in only four rostral segmental ganglia, while another, the CAS interneuron, is restricted to the four most caudal abdominal ganglia and tail. In addition to their segment-specific distributions, only one RAS or CAS cell is found in each segmental ganglion, and they alternate sides between adjacent ganglia (either L-R-L-R or R-L-R-L) with a fidelity of about 95%. This paper utilizes cell deletion techniques to investigate the determination of the asymmetric and alternating pattern of RAS and CAS neurons. We show that developmentally equivalent RAS and CAS homologs are present on both sides of the appropriate ganglia, and that within each ganglion one of the initially paired homologs loses the ability to assume the immunoreactive RAS or CAS fate 2-3 days after axonogenesis has begun. These experiments suggest that there is a competitive interaction between bilateral homologs which ensures that only one mature RAS/CAS neuron is formed per ganglion, and that contralateral RAS/CAS neurons are not required in the same or adjacent ganglia for the determination of the RAS or CAS developmental pathways. Nerve cord transections between ganglia in the CAS domain can alter the spatial pattern of CAS neuron determination, confirming that both bilateral homologs retain the ability to express neuropeptide until late embryonic stages, and suggesting that the alternating pattern of RAS/CAS cells requires communication between adjacent ganglia through the longitudinal connectives.