Peripheral ganglia supplying the genital smooth musculature in the female pig: an experimental study

The aim of the present study was to locate the sensory and autonomic ganglia innervating the female genital musculature in pigs. The retrograde neuronal tracers horseradish peroxidase (HRP) or fast blue (FB) were injected into the left retractor clitoridis muscle (RCM), which was treated as a typical model of the genital smooth musculature. Labelled cells were found in ipsilateral dorsal root ganglia Sl–S4, in bilateral sympathetic paravertebral ganglia from L5–L6 or L6–L7 to S3 and in the left and right caudal mesenteric ganglion. In two of the five animals treated, presumably preganglionic parasympathetic cells were labelled in the ipsilateral intermediate grey substance of the segments Sl–S2.     

Striated Perineal Muscles: Location of Somatic and Autonomic Neurons Projecting to the Male Pig Ischiocavernous Muscle. Neurochemical Features of the Sympathetic Subset

The location, number and size of the central and peripheral neurons innervating the ischiocavernous muscle (ICM) were studied in male pigs by means of Fast Blue (FB) retrograde neuronal tracing. Moreover the immunohistochemical properties of the sympathetic ganglia were investigated combining the double immunolabeling method. After injection of FB into the left ICM, a mean number of 245.3 ± 134.9 labeled neurons were found in the ipsilateral ventral horn of the S1–S3 segments of the spinal cord (SC), 129.7 ± 45.5 in the L6‐S3 ipsilateral and S2–S3 contralateral spinal ganglia (SGs), 2279.3 ± 622.1 in the ipsilateral L2‐S2 and contralateral L5‐S2 sympathetic trunk ganglia (STGs), 541.7 ± 158 in the bilateral caudal mesenteric ganglia (CMGs), and 78.3 ± 35.8 in the microganglia of the pelvic plexus (PGs). The mean area of the ICM projecting neurons was 1217 ± 69.7 μm² in the SC, 2737.5 ± 176.5 μm² in the SGs, 982.8 ± 36.8 μm² in the STGs, 865.9 ± 39.14 μm² in the CMGs and 426.2 ± 24.72 μm² in the PGs. The FB positive neurons of autonomic ganglia contained Dopamine β hydroxylase, vesicular acetylcholine transporter, neuronal nitric oxyde sinthase, calcitonine gene related peptide, leu‐enkephaline, neuropeptide Y, substance P, vasoactive intestinal polypeptide, and somatostatine often colocalized with tyrosine hydroxylase. The particular localization of the motor somatic nucleus, the abundant autonomic innervation and the qualitatively different content of ICM projecting sympathetic neurons suggest a complex regulation of this striated muscle involved in involuntary functions, such as the erection, ejaculation, micturition and defecation. Anat Rec, 301:837–848, 2018. © 2017 Wiley Periodicals, Inc.     

Striated Perineal Muscles: Location of Autonomic,Sensory, and Somatic Neurons Projecting to the Male Pig Bulbospongiosus Muscle

The location, number, and size of the neurons innervating the bulbospongiosus muscle (BSM) were studied in male pigs, by means of Fast Blue (FB) retrograde transport. After injection of FB into the left BSM, labeled neurons were found bilaterally in the L2‐S4 sympathetic trunk ganglia (STGs), in the caudal mesenteric ganglia (CMGs), in the microganglia of the pelvic plexus (PGs), in a dorsolateral area with respect to the central canal of S1‐S3 segments of the spinal cord (SC) and in the S1‐S4 ipsilateral and S2‐S3 contralateral spinal ganglia (SGs). The mean number of labeled FB cells was 3,122 ± 1,968 in STGs, 979 ± 667 in CMGs, 108 ± 104 in PGs, 89 ± 39 in SC and 77 ± 23 in SGs. The area of the multipolar neurons was 852 ± 22 μm² in the STGs, 878 ± 23 μm² in the CMGs and 922 ± 31 μm² in the PGs. The multipolar SC neurons had an area of 1,057 ± 38 μm², while pseudounipolar SG cells had dimensions of 2,281 ± 129 μm². Our research enables us to highlight two peculiarities regarding the innervation of the boar BSM: the very high number of labeled autonomic neurons and the particular localization of the motor somatic nucleus. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Sensory, motor somatic, and autonomic neurons projecting to the porcine cremaster muscle

The location of sensory, somatic, and autonomic neurons projecting to the pig cremaster muscle (CM) was studied by means of the retrograde neuronal tracer Fast Blue (FB) technique. FB was randomly injected in the left CM of four impuberal pigs and serial sections of sensory and autonomic ganglia and spinal cord were examined under a fluorescence microscope. Additionally, some indications about the number and size of labeled neurons were given. Sensory pseudounipolar somata were located ipsilaterally in the L2-L6 and S1-S2 dorsal root ganglia, their total number ranging between 125 and 194, their mean diameter between 24 and 89 microm. Somatic multipolar motoneurons were located ipsilaterally in the L2-L4 neuromeres of the spinal cord, their total number ranging between 53 and 169, their mean diameter between 29 and 53 microm. Autonomic multipolar paravertebral ganglia neurons were located ipsilaterally from L1 to S4 and contralaterally from L2 to S2. Their total number ranged from 2,015 to 3,067 and their mean diameter between 25 and 55 microm. The multipolar caudal mesenteric ganglia neurons were located bilaterally, their total number ranging between 14 and 1,408 and their diameter from 22 to 39 microm. In two subjects only, multipolar neurons were also found ipsilaterally in the microganglia of pelvic plexus (2 and 13 neurons). Their mean diameter ranged between 28 and 54 microm. Our study documented that the CM-projecting neurons were located at different neural levels, with a predominance in the autonomic ganglia.     

大鼠胃的神经支配——荧光双标记法研究

采用荧光双标记法,研究大鼠胃体与幽门的神经支配。延髓迷走神经背核(DmnX)、疑核(nA)和孤束核(nTs)均有标记神经元,部分标本脊髓胸段(T_(3～9))灰质后角V层、脊神经节(T_(0～8))和胸交感链中亦有标记神经元。结合观察结果,对胃的神经支配和外周神经标记的荧光染料选择等问题,进行了讨论。根据DmnX标记细胞的情况分析,表明胃体与幽门分别有迷走神经分支支配,部分神经纤维既有末梢分布于胃体,亦有末梢分布于幽门。支配胃体的神经纤维密度远较幽门部为大。根据胸交感链中存在标记神经元的情况,提出支配胃的节前交感纤维有部分是在交感链中换神经元。     

Sensory and Autonomic Neurons Project Both to the Smooth Retractor Penis and to the Striated Bulbospongiosus Muscles. Neurochemical Features of the Sympathetic Subset

Aim of the present study was to verify, by means of double retrograde neuronal tracers technique, the hypothesis that a subpopulation of sensory and autonomic neurons send collateral axons to both smooth and striated genital muscles. We also wanted to define the neurochemical content of the eventually retrogradelly double labeled (RDL) neurons in the sympathetic trunk ganglia (STG). We used six intact pigs and we injected the tracer Diamidino Yellow (DY) in the smooth left retractor penis muscle (RPM) and the tracer Fast Blue (FB) in the striated left bulbospongiosus muscle (BSM). Rare (2 ± 0.6) RDL neurons were found in the ipsilateral S2 spinal ganglion (SG), 220 ± 42 in the ipsilateral STGs, from L3 to S3, 19 ± 15 in the contralateral S1–S2 ones and 22 ± 5 in the bilateral caudal mesenteric ganglia (CMG). The RDL neurons of the STG were IR for TH (85 ± 13%), DβH (69 ± 17%), NPY (69 ± 23%), nNOS (60 ± 11%), LENK (54 ± 19%), VIP (53±26%), SOM (40 ± 8%), CGRP (34 ± 12%), SP (31 ± 16%), and VAChT (28 ± 3%). Our research highlights the presence of sensory and sympathetic neurons with qualitatively different neurochemical content sending axons both to the smooth RPM and to the striated BSM of the pig. These RDL neurons are likely to project to the smooth vasal musculature to create the ideal physiological conditions in which these muscles can optimize the erectile function. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

一级感觉神经元周围突分支支配多个内脏器官的研究——荧光素逆行双标及三标法

将大鼠颈、胸、腹、盆腔多个脏器配对:食管-甲状腺、食管-气管、心-胃、肺-胃、左肾-脾、膀胱-直肠、膀胱-子宫、子宫-直肠。在配对脏器中先后注入碘化丙啶(PI)或快蓝(FB)及双苯甲亚胺(Bb)或核黄(NY),于迷走神经上、下节及相应背根神经节内出现PI与Bb(或FB与NY)的双标细胞。另设膀胱-子宫-直肠组,分别注入PI、FB及NY,在L_(1～2)背根节出现极少量PI、FB与NY三标细胞。结果提示,大鼠体内普遍存在内脏-内脏感觉在同一感觉细胞的汇聚。     

Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry

The innervation of rat and guinea pig urinary tract was examined using immunohistochemistry, radioimmunoassay and True Blue retrograde tracing techniques and was further assessed following both surgical and chemical denervation experiments. Substantial amounts of calcitonin gene-related peptide-like immunoreactivity (range 20-150 pmol/g) were detected in tissue extracts and localised to nerve fibres distributed throughout the urinary tract of both species, these being concentrated in the ureter and base of the bladder. In the guinea pig, the number and distribution pattern of calcitonin gene-related peptide-like immunoreactive nerves appeared to be identical to that of substance P-containing nerves, whereas in the rat the former predominated. Seven days after injection of the fluorescent dye True Blue into tissues of the urinary tract, retrogradely labelled cells were found in the dorsal root ganglia. These cells had a segmental distribution pattern which was specific for each of the injection sites. Thus, after injection of True Blue into the left kidney hilum a single group of labelled cells were found in the ipsilateral T10-L2 dorsal root ganglia. In contrast, injection into the left ureter produced labelled cells in two separate groups of ipsilateral ganglia (T11-L3 and L6-S1). Injection into the wall of the bladder and upper urethra resulted in bilateral labelling, with most labelled cells occurring in L6 and S1 ganglia. Approximately 90% of labelled cells in T10-L3 dorsal root ganglia displayed calcitonin gene-related peptide-like immunoreactivity, but only 60% of retrogradely labelled bladder neurons in L6-S1 ganglia were immunoreactive for this peptide. Adult guinea pigs and neonatal rats injected systemically with capsaicin subsequently exhibited a marked reduction both in the amount of calcitonin gene-related peptide immunostaining and the concentration of immunoreactive material in the urinary tract, dorsal root ganglia and spinal cord. In rats treated neonatally with capsaicin, there was a significant reduction in the number of retrogradely labelled cells and a hypertrophy of the bladder. Sectioning of the pelvic and hypogastric nerves in the rat also resulted in a depletion of calcitonin gene-related peptide-like immunoreactive nerves in the bladder, whereas chemical sympathectomy appeared to have no effect. The results indicate that calcitonin gene-related peptide immunoreactivity occurs in a major proportion of afferent neurons supplying the urinary tract of the rat and guinea pig.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of protein tracers in the nervous system of the crayfish (Astacus astacus L.) following systemic and local application

Summary A study was made on the penetration and cellular uptake of two protein tracers, albumin labelled with Evans blue (EBA) and horseradish peroxidase (HP), in the nervous system of the crayfish following systemic and local administration. Followingsystemic injection, EBA did not diffuse freely from the cerebral vessels into the brain parenchyma. When the tracers werelocally applied on the surface of the ventral nerve cord their penetration into the nervous parenchyma was to some extent restricted by the nerve sheath. However, unlike the perineurium of vertebrate peripheral nerves, which acts as an efficient diffusion barrier, the crayfish nerve sheath allowed the diffusion of small amounts of tracers into the ganglia. The tracers could more readily penetrate into peripheral nerves in the vicinity of ganglia. Inside the ganglion the tracers spread in extracellular spaces, between glial cell membranes and reached the neuronal surfaces. The proteins were taken up by pinocytosis in glial cells, and also in axons.     

Immunohistochemical Properties of the Peripheral Neurons Projecting to the Pig Bulbospongiosus Muscle

Retrograde neuronal tracing and single labelling immunofluorescence methods were used to define the neurochemical content of the peripheral autonomic and sensitive neurons projecting to the male pig striated bulbospongiosus muscle (BSM). The retrograde fluorescent neuronal tracer Fast Blue (FB) was injected into the left bulbospongiosus muscle of four intact impuberal pigs. After a 10‐day survival time, the ipsilateral sacral sympathetic trunk ganglia (STGs), the caudal mesenteric ganglion (CMG), and the sacral spinal ganglia (SGs) were collected from each animal. In FB+ neurons of these ganglia, the presence of cathecolamine‐ (tyrosine hydroxylase‐TH), acetylcholine‐ (vesicular acetylcholine transporter‐VChAT), or nitric oxide‐synthesizing (neuronal Nitric Oxide Synthase‐nNOS) enzymes and of some biologically active peptides (calcitonine gene‐related peptide‐CGRP, Leu‐Enkephaline‐LENK, Neuropeptide Y‐NPY, Substance P‐SP and Vasoactive Intestinal Peptide‐VIP) were studied. The ipsilateral STGs FB+ neurons showed immunoreactivity principally for TH and NPY and in decreasing order for VIP, VChAT, SP, CGRP, nNOS, and LENK. The left CMG FB+ neurons were immunoreactive to TH and NPY, and in smaller proportions for VIP, LENK, VChAT, CGRP, nNOS, and SP. The ipsilateral SGs FB+ neurons resulted immunoractive for CGRP, LENK, NPY, nNOS, SP, and VChAT. The heterogeneous neurochemical content of the peripheral neurons projecting to the pig BSM allows us to hypothesize the involvement of autonomic ganglia in the activity of both blood vessels and striated fibers of the muscle and the involvement of sensory ganglia in the afferent transmission from the muscle to spinal cord and in antidromic mechanisms that causes the relaxation of the BSM blood vessels. Anat Rec, 299:1192–1202, 2016. © 2016 Wiley Periodicals, Inc.     

Substance P and calcitonin gene-related peptide immunoreactive sensory DRG neurons innervating the lumbar intervertebral discs in rats.

The rat L5/6 disc is innervated from T13 to L6 dorsal root ganglia (DRGs) multisegmentally. Sensory fibers from T13, L1 and L2 DRGs have been reported to innervate through the paravertebral sympathetic trunks, whereas those from L3 to L6 DRGs innervate directly through sinuvertebral nerves on the posterior longitudinal ligament (PLL). The presence of substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive (ir) nerve fibers has been demonstrated in the lumbar intervertebral discs, but their percentages in DRG neurons have not been studied. Fluoro-gold (F-G) labeled neurons innervating the L5/6 disc were distributed throughout DRGs from T13 to L6 levels. Of F-G labeled neurons innervating the L5/6 disc, the percentage of SP-ir T13 to L6 DRG neurons was 30%, and that of CGRP-ir neurons was 47%. The mean cross-sectional area of the cell of SP-ir neurons was 696+/-66 microm2 (mean +/- S. E.), and that of CGRP-ir neurons was 695+/-72 microm2 (mean +/- S. E.). SP- and CGRP-ir were mainly observed in small neurons. The percentages of SP- or CGRP-ir neurons in L1 and L2 DRGs innervating the L5/6 disc were not different from those in L3, L4 or L5 DRGs. In the physiological condition in rats, DRG neurons at all levels may have the same significant role in pain sensation of the disc.     

Inflammation in sympathetic ganglia proximal to sciatic nerve transection in rats

Comparison was made between recruitment of T-lymphocytes and macrophages into lumbar sympathetic ganglia (SGs) and dorsal root ganglia (DRGs) following sciatic nerve transection in rats. In both control and lesioned SGs, resident (ED2+) macrophages expressed less major histocompatibility complex class II (MHC II), but MHC II+ macrophage density was higher, than in equivalent DRGs. The influx of T-cells was larger and the influx and activation of macrophages were more sustained in SGs than in DRGs. Only two of the five subtypes of macrophage that invade lesioned DRGs were recruited to SGs. While some MHC II+ cells phagocytosed dead sympathetic neurones, most phagocytes in SGs lacked a macrophage marker. The different patterns of response between ganglia may provide clues about macrophage involvement in neuronal death and hyperexcitability after peripheral nerve lesions.     

The origin of extrinsic nitrergic axons supplying the human eye

Nitrergic nerve fibres of intrinsic and extrinsic origin constitute an important component of the autonomic innervation in the human eye. The intrinsic source of nitrergic nerves are the ganglion cells in choroid and ciliary muscle. In order to obtain more information on the origin of extrinsic nitrergic nerves in the human eye, we obtained superior cervical, ciliary, pterygopalatine and trigeminal ganglia from six human donors, and stained them for neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D). In the superior cervical ganglia, nNOS/NADPH-D-positive varicose axons were observed whereas perikarya were consistently negative. Fewer than 1% of perikarya in the ciliary ganglia were labelled for nNOS/NADPH-D. The diameter of nNOS/NADPH-D-positive ciliary perikarya was between 8 and 10 microm, which was markedly smaller than the diameter of the vast majority of negative perikarya in the ciliary ganglion. More than 70% of perikarya in the pterygopalatine ganglia were intensely labelled for both nNOS and NADPH-D. In trigeminal ganglia, 18% of perikarya were nNOS/NADPH-D-positive. The average diameter of trigeminal nNOS/NADPH-D perikarya was between 25 and 45 microm. Pterygopalatine and trigeminal ganglia are the most likely sources for extrinsic nerve fibres to the human eye.     

The differentiation of the skin and its appendages. II. Altered development of papillary ridges following neuralectomy.

In order to test the hypothesis that the nervous system is an important determinant of skin differentiation, deletions of the left lumbosacral dorsal root ganglia (DRGs), the sources of cutaneous afferents to the left hindpaw, were performed on opossum pups at day 1 when hindpaws have just begun to be innervated. At birth, each lumbosacral DRG measures about 200 microns rostrocaudally and a deletion measuring 1 mm would span 4-5 DRGs. Following survival periods of 5-24 days, serial sections through the trunk documented partial left lumbosacral DRG deletion and a variable degree of spinal cord destruction. The blood supply to the trunk and hindpaws was preserved. Bilateral enlargement of residual DRGs was observed and regenerating skin at the site of the deletion was hyperplastic and hyperinnervated. The skin of the plantar pads of the hindpaws was studied following the neuralectomies. Statistically significant differences were observed between the left (experimental) and right (control) hindpaws. The density of innervation of the left hindpaw was reduced compared to the right hindpaw, development of papillary ridges was retarded by 3-4 days, and non-innervated Merkel cells were hypogranulated. This period of delay in ridge development is probably a reflection of the expansion of residual DRGs into the peripheral domains of deleted DRGs. The present study confirms a role for afferent nerves in the timing of cutaneous differentiation and a mutual trophic dependence between cutaneous nerves and Merkel cells in the epidermis.     

The formation of the superior and jugular ganglia: Insights into the generation of sensory neurons by the neural crest

The superior and jugular ganglia (S/JG) are the proximal ganglia of the IXth and Xth cranial nerves and the sensory neurons of these ganglia are neural crest derived. However, it has been unclear the extent to which their differentiation resembles that of the Dorsal Root Ganglia (DRGs). In the DRGs, neural crest cells undergo neuronal differentiation just after the onset of migration and there is evidence suggesting that these cells are pre‐specified towards a sensory fate. We have analysed sensory neuronal differentiation in the S/JG. We show, in keeping with previous studies, that neuronal differentiation initiates long after the cessation of neural crest migration. We also find no evidence for the existence of migratory neural crest cells pre‐specified towards a sensory phenotype prior to ganglion formation. Rather our results suggest that sensory neuronal differentiation in the S/JG is the result of localised spatiotemporal cues. Developmental Dynamics 239:439–445, 2010. © 2009 Wiley‐Liss, Inc.     

Morphometric evaluation of the sacral dorsal root ganglia: A cadaveric study

An anatomic study of the sacral dorsal root ganglia (DRG) was performed to determine the location and dimensions of the S1-4 DRGs and to correlate this information to sacral nerve root ganglion lesions. S1 DRGs were located in the intraforaminal region in 55–60% and in the intracanalar region in 40–45%. S2 DRGs were in the intraforaminal region in 15–50% and in the intracanalar region in 50–85%. All the S3 and S4 DRGs were located in the intracanalar region. None of the sacral DRGs was located in the extraforaminal region. The intraforaminal position of the S1 and S2 ganglia renders them vulnerable to compression caused by sacral fractures involving the sacral foramina because of the little space available for these ganglia in the foraminal region. The S1 DRG, with its its relatively larger dimensions and its intracanalar position relative to the other sacral DRGs, may be susceptible to compression by L5-S1 disk herniation. Its intraforaminal position may predispose it to injury during S1 or S2 pedicle screw placement.     

The cat cervical dorsal root ganglia: general cell-size characteristics and comparative study of neck muscle, neck cutaneous and phrenic afferents.

Sizes of neuronal somata in the cat cervical dorsal root ganglia were determined at different levels (C1-C8). The average value and class distribution of mean cell diameter were analyzed. The ganglia from C1 to C5 could be clearly distinguished from those at levels of brachial plexus afferents (C6-C8) with respect to cell size range, distribution and average. The size distribution, most often limited to 70 microns from C1 to C5, skewed to more than 90 microns from C6 to C8. Cells in the 35-50 microns range of diameter constituted the main portion of the cell population (49-52%) at the C1-C5 levels, whereas from C6 to C8 51-77% of the ganglion cell bodies were more than 50 microns in diameter. The cell size distribution of afferents projecting from C1 to C5 and supplying different muscle or cutaneous targets was studied following retrograde labeling with horseradish peroxidase conjugated to wheatgerm agglutinin. Sizes of cell bodies of biventer cervicis (postural muscle), phrenic (purely respiratory muscle) and cutaneous afferents were all similar. The labelled cell bodies were in the majority (51-64%) less than 35 microns in diameter and ranged towards smaller diameters than counterstained cells in the corresponding ganglia. In spite of similarities in cell size distribution it was estimated from the fiber caliber spectra of the labelled afferents that both unmyelinated and myelinated cutaneous afferents originate from larger cell bodies than muscle afferents in the same diameter range.     

The sensory innervation of the ovary: A horseradish peroxidase study in the rat

Injections of horseradish peroxidase (HRP) into the right or left ovary of the rat produced labeling of perikarya in both nodose ganglia and ipsilateral dorsal root ganglia (DRGs) from T₁₀ to L₂. The greatest concentration of labeled cells was in T₁₃ and L₁, DRGs. It is suggested that visceral afferent fibers from the ovary may mediate visceral reflexes that modulate ovarian function.     

Origin of the primary efferent neurons projecting to the prostate of the dog.

The retrograde tracing technique of neuronal tracer Fast Blue was used to determine sources of origin of efferent nerve fibers supplying the prostate of the dog. After injection of Fast Blue into the canine prostate retrogradely labelled neurons were found in bilateral L3-S3 sympathetic chain ganglia, bilateral caudal mesenteric ganglion and in bilateral pelvic plexus ganglia. No Fast Blue-positive neurons were present in bilateral L1-L2 sympathetic chain ganglia and in coeliac-mesenteric ganglion complex. The vast majority of Fast Blue-positive efferent prostate-projecting neurons (56.2% +/- 1.7) were located in bilateral caudal mesenteric ganglion, whereas 28.7% +/- 1.5 of them were located in bilateral pelvic plexus ganglia and 14.9% +/- 0.5 in bilateral L3-S3 sympathetic chain ganglia. Immunohistochemical staining for tyrosine hydroxylase and dopamine beta-hydroxylase was applied to determine the neurochemical character of Fast Blue-positive efferent neurons. Immunohistochemistry revealed that in all tyrosine hydroxylase immunoreactive Fast Blue-positive neurons immunoreactivity for dopamine beta-hydroxylase was also found (noradrenergic neurons) while all tyrosine hydroxylase-negative Fast Blue-positive neurons did not express dopamine beta-hydroxylase (non-noradrenergic neurons). In bilateral sympathetic chain ganglia, 96.4% +/- 2.1 of the prostate-projecting neurons were adrenergic and in bilateral caudal mesenteric ganglion this frequency amounted to 95.6% +/- 1.6. In bilateral pelvic plexus ganglia, 26.7% +/- 1.5 of the prostate-supplying efferent neurons did not express either tyrosine hydroxylase or dopamine beta-hydroxylase immunoreactivity which makes discussion of their cholinergic character possible.