Some Aspects of Human and Canine Macroscopic Pancreas Innervation

Both vagus nerves and the celiae ganglion complex are the main source of the pancreatic gland innervation. In both man and dog there is a distinct difference in the number of fibers ending in the different segments of the gland. In the former, the macroscopic innervation is primarily concentrated in the pancreas head and isthmus. In the latter most of the nerves enter through the upper part of the right limb (uncinate process). Both vagus nerves send direct fibers to the gland but in most of the nerves, that course in general along the different branches of the celiac and mesenteric arteries, the parasympathetic and adrenergic fibers are intermingled. The anterior hepatic plexus, continued down by the gastroduodenal, completes a nervous circle, at the lower edge of the pancreas, with the branches arising from the splenic plexus. In human pancreas most of the nerves, with the exception of the branches given off by the gastroduodenal network, enter the gland by its periphery, either through its superior or inferior border. The gastroduodenal plexus is the pathway for the duodenopancreatic and the duodenogastric are reflexes and for the "pancreatic type" of pain triggered by some posterior penetrating duodenal ulcers.     

Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors

To understand how the nervous system processes information, a map of the connections among neurons would be of great benefit. Here we describe the use of vesicular stomatitis virus (VSV) for tracing neuronal connections in vivo. We made VSV vectors that used glycoprotein (G) genes from several other viruses. The G protein from lymphocytic choriomeningitis virus endowed VSV with the ability to spread transsynaptically, specifically in an anterograde direction, whereas the rabies virus glycoprotein gave a specifically retrograde transsynaptic pattern. The use of an avian G protein fusion allowed specific targeting of cells expressing an avian receptor, which allowed a demonstration of monosynaptic anterograde tracing from defined cells. Synaptic connectivity of pairs of virally labeled cells was demonstrated by using slice cultures and electrophysiology. In vivo infections of several areas in the mouse brain led to the predicted patterns of spread for anterograde or retrograde tracers.     

Sensory substance P innervation of the stomach and pancreas. Demonstration of capsaicin-sensitive sensory neurons in the rat by combined immunohistochemistry and retrograde tracing

The sensory innervation of the stomach and pancreas has been identified by retrograde tracing using the fluorescent dye True Blue (Illing, Gross-Umstadt, Federal Republic of Germany), coupled with the immunohistochemical localization of substance P. Labeled cells were visualized in spinal ganglia, nodose ganglia, celiac ganglion, and dorsal motor nucleus of the vagus after injection of True Blue into both stomach and pancreas. Substance P immunoreactivity was found in 35%-50% of gastric spinal afferent neurons and in approximately 15% of pancreatic spinal afferents. In rats treated at birth with the sensory neurotoxin capsaicin there was a reduction of about 70% in True Blue-labeled cells in the spinal and nodose ganglia, and virtually complete loss of substance P in these ganglia. There was also a marked depletion of substance P-immunoreactive fibers in the pancreas, and in the submucosa of the stomach. The results suggest that the substance P-containing spinal afferents that project to the gastric submucosa are an important component of the gastric sensory innervation.     

Localization of cholinergic innervation in guinea pig heart by immunohistochemistry for high-affinity choline transporters

OBJECTIVE: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. METHODS: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. RESULTS: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. CONCLUSIONS: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.     

Pancreatic galanin: molecular forms and anatomical locations.

Galanin-like immunoreactivity (GalLI) was found to be present in extracts taken from human, canine, and porcine pancreata. The dominant gel filtration peak in each species co-elutes with standard synthetic porcine galanin (Gal), but an apparently smaller molecular weight Gal form was present in human pancreas and intestine and in dog intestine. Reverse-phase HPLC demonstrated identity of porcine pancreatic Gal immunoreactivity with synthetic intestinal Gal. Heterogeneity was seen on reverse-phase HPLC: Human pancreas and intestine had three peaks of immunoreactivity; the retention times were identical between the pancreas and intestinal extracts; and human Gal elutes at an earlier retention time than porcine Gal. Similarly, dog pancreatic GalLI eluted earlier than porcine Gal on reverse-phase HPLC. Immunohistochemical studies revealed the presence of specific staining for GalLI in varicose nerve fibers in the pancreas of the three species. In the dog pancreas an association between Gal-containing nerve fibers and islet cells was readily demonstrable. This was not the case with pig or human pancreas. We conclude that pancreatic Gal is present in the pancreas of the three species and that molecular heterogeneity is similar between intestinal and pancreatic forms. In the dog, a distinct anatomical relationship is demonstrable between Gal-containing nerves and islet cells.     

Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus

Detailed information regarding the contribution of individual γ-aminobutyric acid (GABA)-containing inhibitory neurons to the overall synaptic activity of single postsynaptic cells is essential to our understanding of fundamental elements of synaptic integration and operation of neuronal circuits. For example, GABA-containing cells in the thalamic reticular nucleus (nRt) provide major inhibitory innervation of thalamic relay nuclei that is critical to thalamocortical rhythm generation. To investigate the contribution of individual nRt neurons to the strength of this internuclear inhibition, we obtained whole-cell recordings of unitary inhibitory postsynaptic currents (IPSCs) evoked in ventrobasal thalamocortical (VB) neurons by stimulation of single nRt cells in rat thalamic slices, in conjunction with intracellular biocytin labeling. Two types of monosynaptic IPSCs could be distinguished. “Weak” inhibitory connections were characterized by a significant number of postsynaptic failures in response to presynaptic nRt action potentials and relatively small IPSCs. In contrast, “strong” inhibition was characterized by the absence of postsynaptic failures and significantly larger unitary IPSCs. By using miniature IPSC amplitudes to infer quantal size, we estimated that unitary IPSCs associated with weak inhibition resulted from activation of 1–3 release sites, whereas stronger inhibition would require simultaneous activation of 5–70 release sites. The inhibitory strengths were positively correlated with the density of axonal swellings of the presynaptic nRt neurons, an indicator that characterizes different nRt axonal arborization patterns. These results demonstrate that there is a heterogeneity of inhibitory interactions between nRt and VB neurons, and that variations in gross morphological features of axonal arbors in the central nervous system can be associated with significant differences in postsynaptic response characteristics.     

Cellular localization of endocrine cells in the adult pancreas of the house musk shrew, Suncus murinus: a comparative immunocytochemical study

The right and left lobes of the pancreas in the house musk shrew, Suncus murinus, were found to be completely separated. A morphologic study of the pancreas in S. murinus in terms of the blood supplies and innervation of the right and left lobes was performed in our previous study. It revealed clearly different blood supply and innervation patterns in the right and left lobes, suggesting that the right lobe of the pancreas corresponded to the ventral pancreas, and the left lobe related to the dorsal pancreas. To test this perspective from the histology, in this study, we investigated the immunolocalization of the cells of Langerhans islets in the pancreas of the animal. The distribution of insulin-, glucagon-, somatostatin-, and pancreatic polypeptide (PP)-secreting cells of the right and left lobes of the pancreas was examined in 10 animals. The glucagon-immunoreactive cells were distributed in both the right and left lobes. The PP-immunoreactive cells were extremely abundant in the right lobe and distributed throughout almost all the islets of Langerhans in the right lobe. By contrast, in the left lobe, immunoreactive PP cells were absent in the islets of Langerhans, and only very few immunoreactive PP cells were scattered in the exocrine parenchyma in part of the specimens. Therefore, these findings support our previous studies, and showed that the right and left lobes of the S. murinus pancreas could be related to an embryological origin from the ventral and dorsal pancreatic primordium, respectively, and that the S. murinus pancreas is suitable as a new experimental model to study the development of the human pancreas.     

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.     

Hypothalamic interconnections of somatostatin and growth hormone releasing factor neurons

Combined immunohistochemical labelling for neurons containing growth hormone (GH) releasing factor (GRF) or somatostatin and single labelling immunohistochemistry combined with Fluorogold retrograde transport labelling were used to examine whether somatostatin or GRF neurons might be reciprocally innervated. Occasional somatostatin-immunoreactive neurons in the periventricular preoptic area were found to be closely approached by GRF-immunoreactive fibres, providing possible evidence of scant innervation of somatostatin neurons by GRF cells. In contrast, many GRF-immunoreactive neurons in the arcuate nucleus appeared to have somatostatin-immunoreactive fibres closely applied to their perikarya suggesting that GRF neurons might be innervated by somatostatin cells. Combined retrograde tracing and fluorescence immunohistochemistry revealed few somatostatin-immunoreactive neurons doubly labelled following injections of Fluorogold in the basal hypothalamus. Occasional GRF-immunoreactive neurons in the basal hypothalamus were doubly labelled following PO/AHA injections of Fluorogold. Numerous somatostatin-immunoractive perikarya were observed in the periventricular arcuate region in colchicine-pretreated animals. We conclude that GH-regulating neurons do not have strong reciprocal innervation. The innervation of GRF neurons by somatostatin fibres may be derived from local somatostatin neurons.     

Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), but not galanin, are autonomic cotransmitters in the porcine pancreas.

The neuropeptide galanin has been identified as a potential sympathetic cotransmitter in the canine pancreas. Immunoreactive galanin, also present in nerve fibers of the pig pancreas, was therefore measured in the effluent from isolated perfused pig pancreas with preserved sympathetic (splanchnic) or parasympathetic (vagal) innervation with radioimmunoassays directed against both the N-terminus and the C-terminus of galanin. Electrical vagus stimulation increased the pancreatic exocrine secretion, the secretion of insulin and glucagon, and the release of VIP, but did not influence galanin release. Splanchnic nerve stimulation increased perfusion pressure and glucagon secretion, inhibited insulin secretion, and increased the release of NPY, but galanin release was not affected. We conclude that the pancreatic galanin nerve fibers belong neither to the sympathetic nor to the parasympathetic divisions of the efferent nerve supply to the pig pancreas.     

Presence of sorbin in human digestive tract and endocrine digestive tumours

BACKGROUND: Sorbin, a 153 amino acid peptide isolated from porcine intestine, was localised by immunohistochemistry in endocrine cells of the intestinal mucosa and pancreas and in the enteric nervous system in the pig. AIMS: To identify sorbin cells in normal human digestive tissues and to explore the expression of sorbin in 37 digestive endocrine tumours: 14 intestinal carcinoid tumours and 23 endocrine pancreatic tumours including six insulinomas. METHODS: Two polyclonal antibodies against the C-terminal and the N-terminal sequences of porcine sorbin raised in rabbit were used to evaluate sorbin expression by immunohistochemistry. RESULTS: In the human digestive tract, sorbin, characterised by both C-terminal and N-terminal immunoreactivity, was found in enterochromaffin cells of the gastric and intestinal epithelium from the pyloric junction to the descending colon. C-Terminal sorbin immunoreactivity alone was found in plexii from the enteric nervous system and in some insulin-containing cells of normal pancreas. C-Terminal and N-terminal antibodies disclosed sorbin in five of 14 intestinal carcinoid tumours; C-terminal antibody alone disclosed a C-terminal sorbin peptide in two of six insulinomas and three of 17 endocrine pancreatic tumours. The presence of sorbin was not associated with a specific clinical syndrome. CONCLUSIONS: Sorbin is present in the digestive tract in several forms. It is expressed in some intestinal and pancreatic endocrine tumours.     

Patterns of innervation of vasoactive intestinal polypeptide, neuropeptide Y, and gastrin-releasing peptide immunoreactive nerves in the feline pancreas.

In this study, we performed a detailed analysis of the immunoreactive (IR) patterns and tissue distribution of vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and gastrin-releasing peptide (GRP) in the feline pancreas by means of immunohistochemical and radioimmunological techniques. Immunoreactivity for each peptide is localized to varicose nerve fibers distributed throughout the exocrine and endocrine pancreas, with some differences in the density and pattern of fiber distribution. In the acinar and stromal compartments, VIP-IR processes have a higher density than NPY- and GRP-containing fibers, the latter being the least abundant. The vasculature receives a particularly prominent NPY innervation, while GRP- and VIP-IR fibers are found occasionally in association with blood vessels. Around ducts, NPY- and VIP-IR nerves are more numerous than those positive for GRP-IR, which are quite sparse. One of the most interesting findings of the present work is the visualization of all peptide-IRs both in neuronal cell bodies and fibers within the intrapancreatic ganglia. VIP-IR is observed in virtually all ganglion cells, while GRP- and NPY-IRs are seen in a few neuronal cells. VIP and NPY tissue levels are much higher than GRP concentrations in all regions of the pancreas. VIP content in the head and body is greater than in the tail. The morphological relationship of VIP-, NPY-, and GRP-IR fibers with different pancreatic structures is consistent with specific peptidergic neural inputs in the regulation of pancreatic functions.     

Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models

Aims/hypothesis

Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans.

Methods

We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2.

Results

The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species.

Conclusions/interpretation

Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a ‘null’ model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas.     

A case of intraductal papillary-mucinous carcinoma of the pancreas with the onset of acute pancreatitis

Intraductal papillary-mucinous carcinoma of the pancreas has been reported with increasing frequency. We report a case with intraductal papillary-mucinous carcinoma of the pancreas and discuss surgical treatment and current imaging modalities. A case with intraductal papillary-mucinous carcinoma was analyzed by radiological findings and clinical course. A 47-year-old man developed abdominal pain and nausea. Computed tomography showed a diffusely dilated main pancreatic duct. Duodenoscopy showed a patulous orifice of the pancreas with massive mucus secretion, but the pancreatic juice was not positive for malignant cells. Endoscopic retrograde cholangiopancreatography revealed a markedly dilated pancreatic duct extending from the body to the tail of the pancreas. Distal pancreatectomy was performed with splenectomy and lymph nodes dissection. Histopathological diagnosis was intraductal papillary-mucinous carcinoma. Endoscopic retrograde cholangiopancreatography is useful for diagnosing intraductal papillary mucin-producing tumors. To avoid unnecessary total pancreatectomy and preserve pancreatic function, intraoperative frozen section examination is widely available for the surgical treatment of intraductal papillary mucin-producing tumors.     

Intrinsic nerves of the pancreas after celiac and superior mesenteric ganglionectomy in rats: a morphologic study of acetylcholinesterase activity and catecholamine histofluorescence

The effect of surgical celiac and superior mesenteric ganglionectomy (GGX) on intrinsic pancreatic innervation was studied in male Wistar rats. Neurons were stained in the pancreas of control and ganglionectomized rats with histochemical methods evidencing the acetylcholinesterase (AChE) activity and the induced fluorescence of catecholamines. Two segments of the head of the pancreas ("duodenal" and "splenic" segments) were studied. The surface density of neuronal cell bodies was larger in the splenic segment than in the duodenal segment of the head of the pancreas (p less than 0.01), both in control and in ganglionectomized rats. The surface density of the neuronal cell bodies and the intensity of AChE staining were not significantly different in control and in ganglionectomized rats. Most AChE-positive fibers survived after ganglionectomy; only some of them disappeared. These disappearing fibers corresponded to the thinnest fiber bundles found in the pancreatic tissue of control rats. Only 10% of catecholamine fibers remained after ganglionectomy. Some of the remaining fibers may run through direct duodenopancreatic pathways.     

A direct neuronal connection between the subparafascicular and ventrolateral arcuate nuclei in non-lactating female rats. Could this pathway play a role in the suckling-induced prolactin release?

The neuronal pathways, through which prolactin secretion is regulated during lactation, have still not been fully explored. Studies indicate that the suckling stimulus travels through the spinal cord, the brain stem, and then reaches the hypothalamus. The focus of this present experiment is to further explore the neuronal connections between the brain stem and the arcuate nucleus that may be involved in suckling-induced prolactin release. Ante- and retrograde tracing techniques were used. To chemically characterize the explored neurons neuropeptide immunohistochemistry was applied. Previous studies have indicated that the peripeduncular nucleus is a relay of the suckling stimulus in the midbrain, conveying the information to the hypothalamus. In our experiments, we have found an additional cell group in the subparafascicular parvocellular nucleus located just behind the posterior thalamus that projects to the arcuate neurons. The injection of the retrograde tracer into the ventrolateral part of the arcuate nucleus labeled cells in the lateral subdivision of the subparafascicular parvocellular nucleus. Anterograde tracing from the subparafascicular parvocellular nucleus resulted in fiber labeling in the arcuate nucleus in close apposition with dynorphin immunopositive neurons. Double labeling revealed that a subpopulations of the subparafascicular parvocellular neurons projecting to the arcuate nucleus contained tuberoinfundibular peptide of 39 residues or calcitonin gene-related peptide. The presented findings suggest that the ascending fibers from the subparafascicular parvocellular nucleus might be in the pathway involved in the suckling-induced prolactin release.     

Heterogeneous cell renewal of pancreas in mice: [(3)H]-thymidine autoradiographic investigation

INTRODUCTION: Although cell kinetics of the gastrointestinal mucosa has been extensively examined, that of the pancreas has not been fully analyzed. AIM: To determine the renewal rate of pancreatic cells directly. METHODOLOGY: Postnatal proliferative activity and cellular renewal of the parenchymal cells in ICR mouse pancreas were studied by immunohistochemistry and [(3)H]-thymidine autoradiography. RESULTS: In the single labeling experiments, the proliferative activity of the parenchymal cells in pancreas showed peaks at a few days after birth, decreased thereafter, and reached a low level at 2 months after birth. Continuous labeling experiments revealed that, in the adult pancreas, the half lives of acinar cells, islet cells, and duct epithelial cells were approximately 70 days, 47 days, and 40 days, respectively. Moreover, in the exocrine pancreas, acinar cells of the peri-insular region proliferated more actively than those of the tele-insular region. The renewal rate of glucagon cells was more rapid than that of insulin cells or somatostatin cells. Large ducts showed a high rate of cell renewal in comparison with small ducts. CONCLUSION: The results of this study indicate that cell renewal rates of the pancreas are not homogeneous, but heterogeneous.     

Pseudorabies virus expressing enhanced green fluorescent protein: A tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified central nervous system circuits

Physiological properties of central nervous system neurons infected with a pseudorabies virus were examined in vitro by using whole-cell patch-clamp techniques. A strain of pseudorabies virus (PRV 152) isogenic with the Bartha strain of PRV was constructed to express an enhanced green fluorescent protein (EGFP) from the human cytomegalovirus immediate early promoter. Unilateral PRV 152 injections into the vitreous body of the hamster eye transsynaptically infected a restricted set of retinorecipient neurons including neurons in the hypothalamic suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) of the thalamus. Retinorecipient SCN neurons were identified in tissue slices prepared for in vitro electrophysiological analysis by their expression of EGFP. At longer postinjection times, retinal ganglion cells in the contralateral eye also expressed EGFP, becoming infected after transsynaptic uptake and retrograde transport from infected retinorecipient neurons. Retinal ganglion cells that expressed EGFP were easily identified in retinal whole mounts viewed under epifluorescence. Whole-cell patch-clamp recordings revealed that the physiological properties of PRV 152-infected SCN neurons were within the range of properties observed in noninfected SCN neurons. Physiological properties of retinal ganglion cells also appeared normal. The results suggest that PRV 152 is a powerful tool for the transsynaptic labeling of neurons in defined central nervous system circuits that allows neurons to be identified in vitro by their expression of EGFP, analyzed electrophysiologically, and described in morphological detail.     

Retrograde neuron tracing with microspheres reveals projection of CGRP-immunolabeled vestibular afferent neurons to the vestibular efferent nucleus in the brainstem of rats

OBJECTIVE: A new retrograde neuron-tracing technique with microspheres was used to explore the possible innervation of calcitonin gene-related peptide (CGRP)-immunolabeled vestibular afferent neurons in the vestibular efferent immunolabeled nucleus in the brainstem. METHODS: 0.1 microl of 5% microfluorospheres was injected into the area of the vestibular efferent nucleus, which is located lateral to the genu of the facial nerve. CGRP immunohistochemistry was processed in serial sections of the brainstem at the facial nerve genu level. Double-labeled neurons with both CGRP immunoreactivity and microfluorospheres were examined with fluorescence and confocal laser microscopy. RESULTS: Three types of labeled neurons were observed: (1) neurons only retrogradely microfluorosphere-labeled that were mainly located in the medial vestibular nucleus, lateral vestibular nucleus, superior vestibular nucleus and parvicellular reticular nucleus on the ipsilateral side of the injection; (2) neurons that were both immunolabeled with CGRP and also retrogradedly labeled with microfluorospheres, indicating that they are CGRP cells projecting to the area of vestibular efferent nucleus, these cells were mainly distributed in the superior vestibular nucleus and dorsal vestibular nucleus, and (3) cells only immunolabeled for CGRP that were scattered extensively in the brainstem. CONCLUSION: The presented methodical contribution demonstrates the suitability of fluorescein-labeled microspheres for retrograde neuronal tracing. The vestibular nuclei contain numerous afferent neurons that send projections to the vestibular efferent nucleus, some of which are CGRP cells. This afferent innervation provides morphological evidence that the vestibular efferent neurons receive input from the vestibular afferent neurons including CGRP cells. These vestibular primary CGRP afferent neurons may have an influence on vestibular efferent neurons. CGRP acts as an important co-transmitter or modulator in the afferent-mediated activity of vestibular efferent neurons, which in turn affect afferents in the vestibular end organs.