Mechanisms underlying the gut-brain communication: How enterochromaffin (EC) cells activate vagal afferent nerve endings in the small intestine.

How the gastrointestinal tract communicates with the brain, via sensory nerves, is of significant interest for our understanding of human health and disease. Enterochromaffin (EC) cells in the gut mucosa release a variety of neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT) in the body. How 5-HT and other substances released from EC cells activate sensory nerve endings in the gut wall remains a major unresolved mystery. We used in vivo anterograde tracing from nodose ganglia to determine the spatial relationship between 5-HT synthesizing and peptide-YY (PYY)-synthesizing EC cells and their proximity to vagal afferent nerve endings that project to the mucosa of mouse small intestine. The shortest mean distances between single 5-HT- and PYY-synthesizing EC cells and the nearest vagal afferent nerve endings in the mucosa were 33.1 ± 14.4 µm (n = 56; N = 6) and 70.3 ± 32.3 µm (n = 16; N = 6). No morphological evidence was found to suggest that 5-HT- or PYY-containing EC cells form close morphological associations with vagal afferents endings, or varicose axons of passage. The large distances between EC cells and vagal afferent endings are many hundreds of times greater than those known to underlie synaptic transmission in the nervous system (typically 10-15 nm). Taken together, the findings lead to the inescapable conclusion that communication between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa of the mouse small intestinal occurs in a paracrine fashion, via diffusion. New and Noteworthy None of the findings here are consistent with a view that close physical contacts occur between 5-HT-containing EC cells and vagal afferent nerve endings in mouse small intestine. Rather, the findings suggest that gut-brain communication between EC cells and vagal afferent endings occurs via passive diffusion. The morphological data presented do not support the view that EC cells are physically close enough to vagal afferent endings to communicate via fast synaptic transmission.

Smelling TNT: Trends of the Terminal Nerve.

There is very little knowledge regarding the terminal nerve, from its implications in the involvement and pathogenesis of certain conditions, to its embryological origin. With this review, we try to summarize the most important evidence on the terminal nerve, aiming to clarify its anatomy and the various functions attributed to it, to better interpret its potential involvement in pathological processes. Recent studies have also suggested its potential role in the control of human reproductive functions and behaviors. It has been hypothesized that it plays a role in the unconscious perception of specific odors that influence autonomic and reproductive hormonal systems through the hypothalamic-pituitary-gonadal axis. We used the PubMed database and found different articles which were then selected independently by three authors. We found 166 articles, of which, after careful selection, only 21 were analyzed. The terminal nerve was always thought to be unimportant in our body. It was well studied in different types of animals, but few studies have been completed in humans. For this reason, its function remains unknown. Studies suggest a possible implication in olfaction due to the anatomical proximity with the olfactive nerve. Others suggest a more important role in reproduction and sexual behaviors. New emerging information suggests a possible role in Kallmann syndrome and COVID-19.

Using tissue clearing and light sheet fluorescence microscopy for the three-dimensional analysis of sensory and sympathetic nerve endings that innervate bone and dental tissue of mice.

Bone and dental tissues are richly innervated by sensory and sympathetic neurons. However, the characterization of the morphology, molecular phenotype, and distribution of nerves that innervate hard tissue has so far mostly been limited to thin histological sections. This approach does not adequately capture dispersed neuronal projections due to the loss of important structural information during three-dimensional (3D) reconstruction. In this study, we modified the immunolabeling-enabled imaging of solvent-cleared organs (iDISCO/iDISCO+) clearing protocol to image high-resolution neuronal structures in whole femurs and mandibles collected from perfused C57Bl/6 mice. Axons and their nerve terminal endings were immunolabeled with antibodies directed against protein gene product 9.5 (pan-neuronal marker), calcitonin gene-related peptide (peptidergic nociceptor marker), or tyrosine hydroxylase (sympathetic neuron marker). Volume imaging was performed using light sheet fluorescence microscopy. We report high-quality immunolabeling of the axons and nerve terminal endings for both sensory and sympathetic neurons that innervate the mouse femur and mandible. Importantly, we are able to follow their projections through full 3D volumes, highlight how extensive their distribution is, and show regional differences in innervation patterns for different parts of each bone (and surrounding tissues). Mapping the distribution of sensory and sympathetic axons, and their nerve terminal endings, in different bony compartments may be important in further elucidating their roles in health and disease.

Distribution of nerve endings in human thumb interphalangeal joint.

This study aims to quantitatively analyze the distribution of encapsulated nerve endings in the human thumb interphalangeal (IP) joint capsule. There are three types of nerve endings. Type-I nerve endings (Ruffini-like ending) sense pressure changes, Type II (Pacini-like ending) nerve endings contribute to the kinesthetic sense, and Type III (Golgi-like ending) nerve ending provides proprioceptive information. We dissected five right thumbs IP joints from freshly frozen cadavers (5 men). The mean age of the cadavers at the time of death was 63.4 years (55-73). Sections were stained with the hematoxylin-eosin and antiprotein gene product 9.5 (PGP9.5) to identify encapsulated nerve endings. Transverse sections were cut and divided into volar, dorsal, and then into two equal parts, proximal and distal. The density of encapsulated nerve endings compared to volar versus dorsal and proximal versus distal regions was examined. This study showed that type 1 nerve endings were more common in the distal parts of the IP joint (p < 0.05). Also, type 3 nerve endings were observed in the thumb IP joint. There was no difference between regions in type II and type III nerve endings. The current study demonstrates that the distribution of encapsulated nerve endings in the IP joint is different from the PIP and DIP joints. Moreover, further studies are required to understand the thumb's physiology.

The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

Three-dimensional architecture of the subepithelial corpuscular nerve ending in the rat epiglottis reconstructed by array tomography with scanning electron microscopy.

In the rat laryngeal mucosa, subepithelial corpuscular nerve endings, called laminar nerve endings, are distributed in the epiglottis and arytenoid region and are activated by the pressure changes of the laryngeal cavity. They are also suggested to play a role in efferent regulation because of secretory vesicles in the axoplasm. In the present study, the laminar nerve endings in the rat laryngeal mucosa were analyzed by 3D reconstruction from serial ultrathin sections in addition to immunohistochemistry for synapsin 1. In the light microscopy, synapsin 1-immunoreactive flattened or bulbous terminal parts of the laminar endings were also immunoreactive with VGLUT1, and were surrounded by S100- or S100B-immunoreactive Schwann cells and vimentin-immunoreactive fibroblasts. In the electron microscopy, 3D reconstruction views showed that laminar endings were composed of flattened terminal parts sized 2-5 µm in longitudinal length, overlapping in three to five multiple layers. The terminal parts of the endings were incompletely wrapped by flat cytoplasmic processes of the Schwann cells. In addition, the fibroblast network surrounded the complex of nerve endings and the Schwann cells. Several terminal parts entered through the basement membrane into the epithelial layer and attached to the basal epithelial cells, suggesting that interaction between epithelial cells and laminar nerve endings plays an important role in sensing the pressure changes in the laryngeal cavity. Secretory vesicles were unevenly distributed throughout the terminal part of the laminar nerve endings. The secretory vesicles were frequently observed in the peripheral limb of the terminal parts. It suggests that the laminar nerve endings in the larynx may release glutamate to maintain continuous discharge during the stretching of the laryngeal mucosa.

Cyclosporine A Decreases Dryness-Induced Hyperexcitability of Corneal Cold-Sensitive Nerve Terminals.

Cyclosporine A (CsA) is used for the treatment of dry eye (DE) with good clinical results, improving tear secretion and decreasing subjective symptoms. These effects are attributed to the improved tear film dynamics, but there are no data on the effect of CsA on the abnormal sensory nerve activity characteristic in DE. Our purpose was to evaluate the CsA effect on the enhanced activity of corneal cold thermoreceptors in a tear-deficient DE animal model using in vitro extracellular recording of cold thermoreceptors nerve terminal impulses (NTIs) before and in the presence of CsA. NTI shape was also analyzed. Blinking frequency and tearing rate were also measured in awake animals before and after topical CsA. CsA increased the tearing and blinking of treated animals. CsA significantly decreased the peak response to cold of cold thermoreceptors. Neither their spontaneous NTIs discharge rate nor their cooling threshold were modified. CsA also seemed to reverse some of the changes in NTI shape induced by tear deficiency. These data suggest that, at least in part, the beneficial clinical effects of CsA in DE can be attributed to a direct effect on sensory nerve endings, although the precise mechanisms underlying this effect need further studies to be fully clarified.

The size, number, and distribution of nerve endings around and within the human epiglottis, focusing on tracheal intubation maneuvers.

The aim of this study was to examine the distribution of nerve endings in the mucosa, submucosa, and cartilage of the epiglottis and the vallecula area and to quantify them. The findings could inform the choice of laryngoscope blades for intubation procedures. Fourteen neck slices from seven unembalmed, cryopreserved human cadavers were analyzed. The slices were stained, and cross and longitudinal sections were obtained from each. The nerve endings and cartilage were identified. The primary metrics recorded were the number, area, and circumference of nerve endings located in the mucosa and submucosa of the pharyngeal and laryngeal sides of the epiglottis, epiglottis cartilage, and epiglottic vallecula zone. The length and thickness of the epiglottis and cartilage were also measured. The elastic cartilage of the epiglottis was primarily continuous; however, it contained several fragments. It was covered with dense collagen fibers and surrounded by adipose cells from the pharyngeal and laryngeal submucosa. Nerve endings were found within the submucosa of pharyngeal and laryngeal epiglottis and epiglottic vallecula. There were significantly more nerve endings on the posterior surface of the epiglottis than on the anterior surface. The epiglottic cartilage was twice the length of the epiglottis. The study demonstrated that the distribution of nerve endings in the epiglottis differed significantly between the posterior and anterior sides; there were considerably more in the former. The findings have implications for tracheal intubation and laryngoscope blade selection and design.

Lipid-dependent regulation of neurotransmitter release from sympathetic nerve endings in mice atria.

Neurotransmitter release from sympathetic terminals is a key avenue for heart regulation. Herein, presynaptic exocytotic activity was monitored in mice atrial tissue using a false fluorescent neurotransmitter FFN511, a substrate for monoamine transporters. FFN511 labeling had similarity with tyrosine hydroxylase immunostaining. High [K+]o depolarization caused FFN511 release, which was augmented by reserpine, an inhibitor of neurotransmitter uptake. However, reserpine lost the ability to increase depolarization-induced FFN511 unloading after depletion of ready releasable pool with hyperosmotic sucrose. Cholesterol oxidase and sphingomyelinase modified atrial membranes, changing in opposite manner fluorescence of lipid ordering-sensitive probe. Plasmalemmal cholesterol oxidation increased FFN511 release upon K+-depolarization and more markedly potentiated FFN511 unloading in the presence of reserpine. Hydrolysis of plasmalemmal sphingomyelin profoundly enhanced the rate of FFN511 loss due to K+-depolarization, but completely prevented potentiating action of reserpine on FFN511 unloading. If cholesterol oxidase or sphingomyelinase got access to membranes of recycling synaptic vesicles, then the enzyme effects were suppressed. Hence, a fast neurotransmitter reuptake dependent on exocytosis of vesicles from ready releasable pool occurs during presynaptic activity. This reuptake can be enhanced or inhibited by plasmalemmal cholesterol oxidation or sphingomyelin hydrolysis, respectively. These modifications of plasmalemmal (but not vesicular) lipids increase the evoked neurotransmitter release.

Photons Induce Vesicular Exocytotic Release of Glutamate in a Power-Dependent Way.

Increasing evidence indicates that photobiomodulation, based on tissue irradiation with photons in the red to near-infrared spectrum, may be an effective therapeutic approach to central nervous system disorders. Although nervous system functionality has been shown to be affected by photons in animal models, as well as in preliminary evidence in healthy subjects or in patients with neuropsychiatric disorders, the mechanisms involved in the photobiomodulation effects have not yet been clarified. We previously observed that photobiomodulation could stimulate glutamate release. Here, we investigate mechanisms potentially involved in the glutamate-releasing effect of photons from adult mouse cerebrocortical nerve terminals. We report evidence of photon ability to induce an exocytotic vesicular release of glutamate from the terminals of glutamatergic neurons in a power-dependent way. It can be hypothesized that photobiomodulation, depending on the potency, can release glutamate in a potentially neurotoxic or physiological range.

Study of the nerve endings and mechanoreceptors of the medial meniscotibial ligament of the knee: A structural and distribution analysis.

BACKGROUND: The aim of the present study is to describe the morphology and distribution of the nerve endings of the meniscotibial ligament (MTL) of the knee, in order to understand the interaction between the proprioceptive system and knee mechanics. METHODS: Twenty medial MTLs were obtained from deceased organ donors. The ligaments were measured, weighed and cut. Sections (10 mm) were prepared on hematoxylin and eosin-stained slides for analysis of tissue integrity, and 50 mm sections were submitted to immunofluorescence with the protein gene product (PGP) 9.5 as primary antibody and Alexa Fluor 488 as secondary antibody, followed by microscopic analysis. RESULTS: The medial MTL was identified in 100% of the dissections, with average length, width, thickness and weight of 7.07 ± 1.34 mm, 32.25 ± 3.09 mm, 3.53 ± 0.27 mm and 0.67 ± 0.13 g, respectively. The hematoxylin and eosin-stained histological sections exhibited typical ligament structure, with dense well-organized collagen fibers and vascular tissue. All the specimens analyzed contained type I (Ruffini) mechanoreceptors and free (type IV) nerve endings, varying from parallel to intertwined fibers. Nerve endings not classified with different irregular shapes were also found. Most type I mechanoreceptors were found close to the MTL insertions on the tibial plateau, while the free nerve endings were found adjacent to the capsule. CONCLUSION: The medial MTL showed a peripheral nerve structure, primarily type I and IV mechanoreceptors. These findings suggest that the medial MTL is important for proprioception and medial knee stabilization.

Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

Immunolocalization of vesicular glutamate transporter 2 and exocytosis-related proteins in afferent nerve endings innervating taste buds in the rat incisive papilla.

The present study aimed to investigate the immunolocalization of vesicular glutamate transporter (VGLUT) 1 and 2, and proteins associated with exocytosis, i.e., core components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex (synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2) and synaptotagmin-1 (Syt1), in incisive papillary taste buds of rats using double-indirect immunofluorescence. No VGLUT1 immunoreactivity was observed, whereas VGLUT2-immunoreactive punctate products were closely associated with guanine nucleotide-binding protein G(t) subunit α3-immmunoreactive cells in taste buds. VGLUT2 was immunolocalized in P2X3 purinoceptor-expressing afferent nerve endings. Synaptosomal-associated protein of 25 kDa, syntaxin 1, and vesicle-associated membrane protein 2 were immunolocalized in nerve endings containing VGLUT2-immunoreactive products as well as a few cells in taste buds. VGLUT2 was co-immunolocalized in some intragemmal nerve endings immunoreactive for Syt1, a calcium sensor implicated in vesicle membrane fusion. The present results suggest that afferent nerve endings innervating incisive taste buds release glutamate by exocytosis to modulate taste cell function.

Quantification of catecholamine neurotransmitters released from cutaneous vasoconstrictor nerve endings in men with cervical spinal cord injury.

Control of cutaneous circulation is critically important to maintain thermoregulation, especially in individuals with cervical spinal cord injury (CSCI) who have no or less central thermoregulatory drive. However, the peripheral vasoconstrictor mechanism and capability have not been fully investigated after CSCI. Post- and presynaptic sensitivities of the cutaneous vasoconstrictor system were investigated in 8 CSCI and 7 sedentary able-bodied (AB) men using an intradermal microdialysis technique. Eight doses of norepinephrine (NE, 10-8 to 10-1 M) and five doses of tyramine (TY, 10-8, 10-5 to 10-2 M) were administered into the anterior right and left thigh, respectively. Endogenous catecholamines, noradrenaline, and dopamine, collected at the TY site, were determined by high-performance liquid chromatography with electrochemical detection. Regardless of vasoconstrictor agents, cutaneous vascular conductance decreased dose-dependently and responsiveness was similar between the groups (NE: Group P = 0.255, Dose P = 0.014; TY: Group P = 0.468, Dose P < 0.001), whereas the highest dose of each drug induced cutaneous vasodilation. Administration of TY promoted the release of noradrenaline and dopamine in both groups. Notably, the amount of noradrenaline released was similar between the groups (P = 0.819), although the concentration of dopamine was significantly greater in individuals with CSCI than in AB individuals (P = 0.004). These results suggest that both vasoconstrictor responsiveness and neural functions are maintained after CSCI, and dopamine in the skin is likely to induce cutaneous vasodilation.

Analysis of Mechanoreceptors and Free Nerve Endings of the Transverse Carpal Ligament.

Background: The treatment of carpal tunnel syndrome (CTS) by sectioning the transverse carpal ligament (TCL) is not exempt from complications. Some nerve branches may be damaged by the incision. The aim of this study is to identify and map the TCL nerve endings, serving as a guide for sectioning this structure in a zone with less nerve ending density. Methods: Ten TCLs were obtained from fresh frozen cadavers. The TCLs were measured, divided into 3 equal bands (radial, central, and ulnar), and submitted to cryostat sectioning. The sections were subjected to immunofluorescence with the protein gene product (PGP) 9.5 and confocal microscopy analysis. Results: All the specimens contained type I and type IV mechanoreceptors. Neural elements occupied 0.695 ± 0.056% of the ligament area. The density of the neural elements was greater in the radial, followed by the ulnar and central bands, with 0.730 ± 0.083%, 0.686 ± 0.009%, and 0.669 ± 0.031%, respectively. Conclusion: The present findings suggest that the region with the least potential for neural element injury during TCL release is the central third near the transition with the ulnar third. When performed distally to proximally with a slight inclination from the radial to the ulnar, this release compromises the lowest nerve element density. Topographically, the proximal limit of the release is the distal wrist crease, while the distal limit is the intersection of Kaplan cardinal line and the axis of the third webspace.

Frequency-Dependent Engagement of Synaptic Vesicle Pools in the Mice Motor Nerve Terminals.

Nerve terminals contain numerous synaptic vesicles (SVs) whose exo-endocytic cycling maintains neurotransmitter release. SVs may have different properties, thereby constituting separate pools. However, behavior of SV pools remains elusive in many synapses. To fill this gap, we studied the functioning of SV pools at both low- and higher-frequency stimulations utilizing microelectrode recording and dual-labeling of SVs with FM-dyes at the mice motor nerve terminals. It was found that higher-frequency stimulation caused exocytosis of different kinds of SVs. One type of SVs contributed to exocytosis exclusively at intense activities and their exocytotic rate was depended on the order in which these SVs were recovered by endocytosis. Another type of SVs can sustain the release in response to both low- and higher-frequency stimulations, but increasing activity did not lead to enhanced exocytotic rate of these SVs. In addition, depression of neurotransmitter release induced by 20 Hz stimulation occurred independent on previous episode of 10 Hz activity. We suggest that during prolonged stimulation at least two SV pools can operate. One termed "house-keeping" that would be active at different frequencies and the other termed "plug-in" that would respond to increasing activity.

Density mapping of nerve endings in the skin of the palm and flexor retinaculum of the hand. Application to open carpal tunnel release.

In order to re-evaluate the safest area to incise skin and the flexor retinaculum (FR) when performing a carpal tunnel release (CTR), we carried out a mapping study of the nerve endings in the skin and FR on cadaver specimens, which, unlike previous studies for the first time, includes histomorphometry and image digital analysis. After dividing the skin and FR into 20 and 12 sections, respectively, we carried out a histomorphological analysis of nerve endings. The analysis was performed by two neutral observers on 4-µm histological sections stained with hematoxylin-eosin (H-E), and Klüver-Barrera with picrosirius red (KB + PR) methods. A semi-automatic image digital analysis was also used to estimate the percentage of area occupied per nerve. We observed a lower quantity of nerve endings in the skin of the palm of the hand in line with the ulnar aspect of the 4th finger. The ulnar aspect of the FR was the most densely innervated. However, there are no statistically significant differences between sections in the percentage of area occupied per nerve both in the skin and in the FR. We concluded that there is not a safe area to incise when performing carpal tunnel surgery, but taking into account the quantity of nerve endings present in skin and FR, we recommend an incision on the axis of the ulnar aspect of 4th finger when incising skin and on the middle third of the FR for CTR.

Distribution of proteins for synaptic release in nerve endings associated with the trachealis muscle of rats.

The immunohistochemical localization of proteins for synaptic release was examined in smooth muscle-associated sensory nerve endings using whole-mount preparations of the rat trachea. Plant-like smooth muscle-associated nerve endings with immunoreactivity for Na+-K+-ATPase, α3-subunit were identified in the trachealis muscle. VGLUT1, synapsin1, t-SNARE proteins (SNAP25 and syntaxin1), v-SNARE proteins (VAMP1 and VAMP2), and a presynaptic active zone-related protein (piccolo) were detected in the terminal parts of these endings. These results suggest that smooth muscle-associated nerve endings secrete glutamate to modulate sensorimotor functions in the lung deflation reflex.

Eye Movements But Not Vision Drive the Development of Palisade Endings.

Purpose: To test whether visual experience and/or eye movements drive the postnatal development of palisade endings in extraocular muscles. Methods: In three newborn cats, the right eye was covered until 30 days from postnatal (P) day 7 (before opening their eyes), and in three cats both eyes were covered until 45 days, also from P7. To block eye movements, another seven cats received a retrobulbar injection of botulinum neurotoxin A (BoNT-A) into the left orbit at birth and survived for 45 days (three cats) and 95 days (four cats). The distal third of the rectus muscles containing the palisade endings was used for whole-mount preparation and triple-fluorescence labeling with anti-neurofilament along with (1) anti-synaptophysin and phalloidin or (2) anti-growth associated protein 43 (GAP43) and phalloidin. Immunolabeled specimens were analyzed in the confocal laser scanning microscope. Results: After unilateral and bilateral dark rearing, palisade endings were qualitatively and quantitatively equal to those from age-matched controls. After BoNT-A induced eye immobilization for 45 or 95 days, palisade endings were absent in the superior rectus and lateral rectus muscles and only present in the inferior rectus and medial rectus muscle. These BoNT-A-treated palisade endings were rudimentary and reduced in number, and the expression of the neuronal developmental protein GAP43 was significantly reduced. Conclusions: This study demonstrates that eye immobilization, but not visual deprivation, affects palisade ending development. Palisade endings develop in the first month of life, and the present findings indicate that, during this time window, palisade endings are prone to oculomotor perturbations.