Transcriptome profiling of UPF3B/NMD-deficient lymphoblastoid cells from patients with various forms of intellectual disability.

The nonsense-mediated mRNA decay (NMD) pathway was originally discovered by virtue of its ability to rapidly degrade aberrant mRNAs with premature termination codons. More recently, it was shown that NMD also directly regulates subsets of normal transcripts, suggesting that NMD has roles in normal biological processes. Indeed, several NMD factors have been shown to regulate neurological events (for example, neurogenesis and synaptic plasticity) in numerous vertebrate species. In man, mutations in the NMD factor gene UPF3B, which disrupts a branch of the NMD pathway, cause various forms of intellectual disability (ID). Using Epstein Barr virus-immortalized B cells, also known as lymphoblastoid cell lines (LCLs), from ID patients that have loss-of-function mutations in UPF3B, we investigated the genome-wide consequences of compromised NMD and the role of NMD in neuronal development and function. We found that ~5% of the human transcriptome is impacted in UPF3B patients. The UPF3B paralog, UPF3A, is stabilized in all UPF3B patients, and partially compensates for the loss of UPF3B function. Interestingly, UPF3A protein, but not mRNA, was stabilised in a quantitative manner that inversely correlated with the severity of patients' phenotype. This suggested that the ability to stabilize the UPF3A protein is a crucial modifier of the neurological symptoms due to loss of UPF3B. We also identified ARHGAP24, which encodes a GTPase-activating protein, as a canonical target of NMD, and we provide evidence that deregulation of this gene inhibits axon and dendrite outgrowth and branching. Our results demonstrate that the UPF3B-dependent NMD pathway is a major regulator of the transcriptome and that its targets have important roles in neuronal cells.

Urocortin 3 transgenic mice exhibit a metabolically favourable phenotype resisting obesity and hyperglycaemia on a high-fat diet.

AIMS/HYPOTHESIS: Urocortins are the endogenous ligands for the corticotropin-releasing factor receptor type 2 (CRFR2), which is implicated in regulating energy balance and/or glucose metabolism. We determined the effects of chronic CRFR2 activation on metabolism in vivo, by generating and phenotyping transgenic mice overproducing the specific CRFR2 ligand urocortin 3. METHODS: Body composition, glucose metabolism, insulin sensitivity, energy efficiency and expression of key metabolic genes were assessed in adult male urocortin 3 transgenic mice (Ucn3(+)) under control conditions and following an obesogenic high-fat diet (HFD) challenge. RESULTS: Ucn3(+) mice had increased skeletal muscle mass with myocyte hypertrophy. Accelerated peripheral glucose disposal, increased respiratory exchange ratio and hypoglycaemia on fasting demonstrated increased carbohydrate metabolism. Insulin tolerance and indices of insulin-stimulated signalling were unchanged, indicating these effects were not mediated by increased insulin sensitivity. Expression of the transgene in Crfr2 (also known as Crhr2)-null mice negated key aspects of the Ucn3(+) phenotype. Ucn3(+) mice were protected from the HFD-induced hyperglycaemia and increased adiposity seen in control mice despite consuming more energy. Expression of uncoupling proteins 2 and 3 was higher in Ucn3(+) muscle, suggesting increased catabolic processes. IGF-1 abundance was upregulated in Ucn3(+) muscle, providing a potential paracrine mechanism in which urocortin 3 acts upon CRFR2 to link the altered metabolism and muscular hypertrophy observed. CONCLUSIONS/INTERPRETATION: Urocortin 3 acting on CRFR2 in skeletal muscle of Ucn3(+) mice results in a novel metabolically favourable phenotype, with lean body composition and protection against diet-induced obesity and hyperglycaemia. Urocortins and CRFR2 may be of interest as potential therapeutic targets for obesity.

Histological and histochemical characterisation of the equine soft palate muscles.

REASONS FOR PERFORMING STUDY: Dysfunction of the muscles is implicated in the pathogenesis of intermittent dorsal displacement of the soft palate (DDSP) in exercising horses. The histological features of normal equine soft palate muscles have not been previously described. OBJECTIVE: To describe the histological and morphometric features of normal equine soft palate muscles. METHODS: The palatinus, palatopharyngeus, levator veli palatini and tensor veli palatini muscles of 6 Thoroughbred type horses were examined histologically and histochemically to assess their general morphology, fibre-type distribution and mean fibre diameter. RESULTS: The muscles of all 6 specimens showed very similar characteristics, including a low proportion of type 1 muscle fibres in the 4 different muscles examined, with the 3 muscles innervated by the pharyngeal branch of the vagus (i.e. palatopharyngeus, palatinus and levator veli palatini) having significantly fewer (P<0.005) type 1 muscle fibres compared to the tensor veli palatini. The mean fibre diameters were significantly (P<0.05) smaller in type 1 than type 2 fibres in all muscles except the palatinus. Considerable variability in mean fibre size diameter was evident in all muscles examined, with type 1 fibres in the levator veli palatini and rostral fasciculus of the palatopharyngeus and both fibre types in the caudal fasciculus of the palatopharyngeus having the most marked variation, with coefficients of variation >250 and the latter also having high levels of endomysial connective tissue. The palatinus had least variation in fibre size. CONCLUSION AND CLINICAL RELEVANCE: Morphological characterisation of the normal soft palate muscles may provide reference values for future comparative studies with samples obtained from horses with palatal dysfunction. The palatinus appears to be the best muscle to histologically examine for evidence of muscle abnormality.

A glycogen synthase 1 mutation associated with equine polysaccharide storage myopathy and exertional rhabdomyolysis occurs in a variety of UK breeds.

REASONS FOR PERFORMING STUDY: A glycogen synthase (GYS1) mutation has been described in horses with histopathological evidence of polysaccharide storage myopathy (PSSM) in the USA. It is unknown whether the same mutation is present in horses from the UK. OBJECTIVES: To determine whether the GYS1 mutation occurs in UK horses with histopathological evidence of PSSM and exertional rhabdomyolysis. HYPOTHESIS: The R309H GYS1 mutation is present in a variety of UK horse breeds and that the mutation is commonly associated with exertional rhabdomyolysis. METHODS: DNA was extracted from 47 muscle or blood samples from UK horses with histories of exertional rhabdomyolysis in which muscle biopsy diagnosis had been pursued. The proportions of GYS1 mutation positive cases were compared among histopathologically defined groups. In addition, breeds that carried the GYS1 mutation were identified from a total of 37 grade 2 (amylase-resistant) PSSM cases. RESULTS: Of 47 horses with exertional rhabdomyolysis in which a muscle biopsy diagnosis was pursued, 10 (21%) carried the GYS1 mutation. The mutation was only found in horses with grade 2 PSSM (i.e. not in horses with normal, idiopathic myopathy or grade 1 PSSM biopsy samples). In total, the GYS1 mutation was found in 24/37 (65%) of grade 2 PSSM cases. A variety of breeds, including Quarter Horse, Appaloosa, Warmblood, Connemara-cross, Cob, Polo Pony and Thoroughbred cross carried the mutation. CONCLUSIONS: The GYS1 mutation is an important cause of exertional rhabdomyolysis of UK horse breeds but does not account for all forms of PSSM. POTENTIAL RELEVANCE: Genotyping is recommended in cases of exertional rhabdomyolysis, prior to or in combination with, muscle biopsy. However a significant proportion of horses with histopathological evidence of PSSM and/or exertional rhabdomyolysis have different diseases.

Histological and ultrastructural evidence that recurrent laryngeal neuropathy is a bilateral mononeuropathy limited to recurrent laryngeal nerves.

REASONS FOR PERFORMING STUDY: Recurrent laryngeal neuropathy (RLN) is a common and debilitating peripheral nerve disease of horses, but it remains unclear if this disease is a mono- or polyneuropathy. An understanding of the distribution of the neuropathological lesions in RLN affected horses is fundamental to studying the aetiology of this very significant disease of tall horses. OBJECTIVE: To determine whether RLN should be classified as a mono- or polyneuropathy. METHODS: Multiple long peripheral nerves and their innervated muscles were examined systematically in 3 clinically affected RLN horses RESULTS: Severe lesions were evident in the left as well as right recurrent laryngeal nerves in all horses, both distally and, in one case, also proximally. No primary axonal lesions were evident in other nerves nor were changes found in their innervated muscles. CONCLUSIONS: RLN is not a polyneuropathy but should be classified as a bilateral mononeuropathy. POTENTIAL RELEVANCE: Genetic and local factors specifically affecting the recurrent laryngeal nerves in RLN-affected horses should now be investigated further.

Polyneuropathy of Finnish horses characterised by inflammatory demyelination and intracisternal Schwann cell inclusions.

REASON FOR PERFORMING STUDY: A neurological disorder characterised by pelvic limb metatarsophalangeal joint extensor paresis has been observed in numerous horses in Scandinavia for the last decade. Very little has been formally reported and there have been no detailed assessments of the neurological signs or neuropathological lesions. OBJECTIVES: To describe the epidemiological and pathological features of an outbreak of 'Scandinavian knuckling syndrome' in a riding stable in southern Finland. METHODS: Clinical neurological examination of 4 cases and neuropathological assessment of tissues of one case were performed. RESULTS: Eleven out of 17 horses fed on ryegrass from a common source showed progressive clinical signs of metatarsophalangeal extensor paresis necessitating euthanasia of 7 horses. Nervous system lesions in one horse consisted of a novel demyelinating, mildly inflammatory peripheral neuropathy, with BiP/GRP positive rough endoplasmatic reticulum Schwann cell inclusions. CONCLUSIONS: The clinical signs and lesions documented differ from any previously described equine polyneuropathy and suggest a primary Schwann cell lesion. POTENTIAL RELEVANCE: The classification of this disease as a novel demyelinating polyneuropathy may assist focused epidemiological investigations.

Differential effects on gene transcription and hematopoietic differentiation correlate with GATA2 mutant disease phenotypes.

Heterozygous GATA2 mutations underlie an array of complex hematopoietic and lymphatic diseases. Analysis of the literature reporting three recurrent GATA2 germline (g) mutations (gT354M, gR396Q and gR398W) revealed different phenotype tendencies. Although all three mutants differentially predispose to myeloid malignancies, there was no difference in leukemia-free survival for GATA2 patients. Despite intense interest, the molecular pathogenesis of GATA2 mutation is poorly understood. We functionally characterized a GATA2 mutant allelic series representing major disease phenotypes caused by germline and somatic (s) mutations in zinc finger 2 (ZF2). All GATA2 mutants, except for sL359V, displayed reduced DNA-binding affinity and transactivation compared with wild type (WT), which could be attributed to mutations of arginines critical for DNA binding or amino acids required for ZF2 domain structural integrity. Two GATA2 mutants (gT354M and gC373R) bound the key hematopoietic differentiation factor PU.1 more strongly than WT potentially perturbing differentiation via sequestration of PU.1. Unlike WT, all mutants failed to suppress colony formation and some mutants skewed cell fate to granulocytes, consistent with the monocytopenia phenotype seen in GATA2-related immunodeficiency disorders. These findings implicate perturbations of GATA2 function shaping the course of development of myeloid malignancy subtypes and strengthen complete or nearly complete haploinsufficiency for predisposition to lymphedema.

A neurologic syndrome in Golden Retrievers presenting as a sensory ataxic neuropathy.

BACKGROUND: A sensory ataxic neuropathy has been observed in Swedish Golden Retrievers recently. ANIMALS: Twenty-one affected Golden Retrievers. METHODS: Clinical and neurologic status, electrophysiologic, and pathologic status as well as pedigree analyses were evaluated. RESULTS: Clinical signs had an insidious onset between 2 and 8 months of age and a slowly progressive course. Affected dogs were ataxic and dysmetric. They had abnormal postural reactions and decreased spinal reflexes but no apparent muscle atrophy. Clinical pathology, radiography, and electrophysiology of motor systems were all within reference values. Sensory nerve conduction results of affected dogs were significantly different from those of a group of control dogs. Necropsy revealed a chronic progressive central and peripheral sensorimotor axonopathy; the proprioceptive pathways were most severely affected. CONCLUSIONS AND CLINICAL IMPORTANCE: This disease in these Golden Retrievers is distinct from other canine breed-related neurodegenerative diseases or hereditary neurodegenerative diseases described in humans. Pedigree analyses indicated a hereditary background, but the mode of inheritance could not be established.

Atypical masticatory muscle myositis in three cavalier King Charles spaniel littermates.

This case report describes a novel manifestation of the immune-mediated disease, masticatory muscle myositis. Clinical signs, including difficulty in opening the mouth (trismus), were seen in three of four 12-week-old cavalier King Charles spaniel littermates. Diagnosis was established by 2M immunohistochemistry, supported by characteristic histopathological changes in affected temporal muscle. Treatment using corticosteroids at immune-modifying doses resulted in resolution of clinical signs in all the affected animals. Masticatory muscle myositis should be considered as a differential diagnosis in groups of young dogs with clinical signs of myositis localised to the head.

A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm.

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

Horses on pasture may be affected by equine motor neuron disease.

REASONS FOR PERFORMING STUDY: Equine motor neuron disease (EMND) was diagnosed in 3 horses maintained on lush, grass-based pasture. This contrasted with North American studies which identified limited or no access to green herbage as an important risk factor for EMND. HYPOTHESIS: Grazing horses that have an apparently adequate intake of pasture herbage to meet normal equine vitamin E requirements can develop EMND. METHODS: Owners of 32 European horses diagnosed with EMND completed a questionnaire regarding intrinsic, managemental, nutritional and environmental factors that could potentially be risk factors for EMND, and also regarding clinical signs, treatments and case outcome. Plasma/serum vitamin E data for these horses were supplied by the veterinarians. No control population was studied. RESULTS: Thirteen of 32 horses (termed the 'grazing' group) had part- or full-time access to grass-based pasture at the onset of EMND (median duration at pasture 12 h/day, range 3-24 h). Five of these horses were at pasture for at least 235 h/day at the onset of EMND, 2 of which were at pasture for at least 23.5 h/day throughout the year. Despite grazing, all these horses had a low vitamin E status. The remaining 19 horses resembled those cases reported from North America, in that they had no or limited access to pasture. CONCLUSIONS AND POTENTIAL RELEVANCE: A diagnosis of EMND should not be discounted on the basis that a horse has access, even full-time, to lush grass-based pasture. Inadequate vitamin E intake was probably not the sole cause of either the EMND or the low vitamin E status in the grazing horses; the latter was probably the result of abnormal bioavailability or excessive utilisation of vitamin E.

Bodyweight change aids prediction of survival in chronic equine grass sickness.

REASONS FOR PERFORMING STUDY: Objective criteria for predicting survival of chronic grass sickness cases are currently lacking. OBJECTIVES: To determine whether the rate and/or magnitude of bodyweight change during hospitalisation of chronic grass sickness cases can provide an objective predictor of survival to discharge from hospital. Clinicians' recorded indication(s) for euthanasia were also reviewed. STUDY DESIGN: Single centre retrospective observational study. METHODS: Case records of all horses admitted for management of chronic grass sickness to The Dick Vet Equine Hospital between 1998 and 2013 were analysed. Case background, survival to hospital discharge, indication(s) for euthanasia, disease duration at admission and bodyweight changes during the hospitalisation period were analysed, and data for survivors and nonsurvivors compared. Percentage weight change was calculated for 7 day intervals up to 28 days (0-7, 7-14, 14-21, 21-28 days) and for entire periods from the first weight recorded (0-7, 0-14, 0-21, 0-28 days). These results were used to estimate survival probability conditional on weight change. RESULTS: The study sample comprised 213 horses, with 114 survivors (53.5%) and 99 (46.5%) nonsurvivors. Compared with nonsurvivors, survivors had significantly lower median maximum bodyweight loss as a percentage of first weight (survivors 5.9%, interquartile range 1.8-13.5; nonsurvivors 12.7%, 6.4-17.3). Throughout all time periods analysed, survivors had significantly lower median bodyweight loss than nonsurvivors, but no specific time period was more predictive of survival. Highest percentages of total bodyweight loss for individual horses were comparable for survivors (36%) and nonsurvivors (37%). Survival prediction curves reporting percentage survival rates for all time periods analysed provided data to aid prediction of chronic grass sickness survival. CONCLUSIONS: Overall, nonsurvivors had greater bodyweight loss than survivors. Rapidity and magnitude of bodyweight loss were equally predictive of outcome. Percentage survival prediction curves provide objective data to aid discussion of prognosis, but greater predictive specificity with associated sensitivity is required for clinical decision making in individual cases.

Expression profiling reveals functionally important genes and coordinately regulated signaling pathway genes during in vitro angiogenesis.

Angiogenesis is a complex multicellular process requiring the orchestration of many events including migration, alignment, proliferation, lumen formation, remodeling, and maturation. Such complexity indicates that not only individual genes but also entire signaling pathways will be crucial in angiogenesis. To define an angiogenic blueprint of regulated genes, we utilized our well-characterized three-dimensional collagen gel model of in vitro angiogenesis, in which the majority of cells synchronously progress through defined morphological stages culminating in the formation of capillary tubes. We developed a comprehensive three-tiered approach using microarray analysis, which allowed us to identify genes known to be involved in angiogenesis and genes hitherto unlinked to angiogenesis as well as novel genes and has proven especially useful for genes where the magnitude of change is small. Of interest is the ability to recognize complete signaling pathways that are regulated and genes clustering into ontological groups implicating the functional importance of particular processes. We have shown that consecutive members of the mitogen-activated protein kinase and leukemia inhibitory factor signaling pathways are altered at the mRNA level during in vitro angiogenesis. Thus, at least for the mitogen-activated protein kinase pathway, mRNA changes as well as the phosphorylation changes of these gene products may be important in the control of blood vessel morphogenesis. Furthermore, in this study, we demonstrated the power of virtual Northern blot analysis, as an alternative to quantitative RT-PCR, for measuring the magnitudes of differential gene expression.

Observations on the interstitial cells of Cajal and neurons in a recovered case of equine dysautonomia (grass sickness).

This paper describes the histopathological and immunohistochemical changes in the central, autonomic and enteric nervous systems in a well-documented case of equine dysautonomia (ED), after the animal had recovered without significant residual clinical signs. Evidence of neuronal degeneration, such as neuronal chromatolysis, glial scars or a decrease in density of neurons, was not observed in the midbrain, pons, medulla oblongata or spinal cord, including the nuclei of cranial nerves III, V, VII, X and XII. In addition, no evidence of muscle denervation or re-innervation, such as group atrophy of clusters of cells or grouping of cells of similar size in the triceps muscle, was observed. These findings support the hypothesis that the chromatolytic motor neurons may not progress to cell death in ED. The autonomic ganglia showed evidence of previous neuronal loss. In the myenteric and submucosal plexuses of the ileum there was significant loss of enteric neurons. However, in the ileum, a continuous network of interstitial cells of Cajal (ICC) was observed by means of immunohistochemical techniques. It is possible that recovery without significant residual clinical signs was associated with lack of damage to, or more likely recovery of, the ICC, which have a pacemaker function in the intestinal tract.

Thoracolaryngeal reflex latencies in Thoroughbred horses with recurrent laryngeal neuropathy.

Electrolaryngeography was used to study the latencies of the thoracolaryngeal adductor reflex in Thoroughbred horses with and without recurrent laryngeal neuropathy (RLN). Latencies were compared in horses with grades 1 and 2 RLN, diagnosed by endoscopy in resting horses. The reliability of the measurements, effect of sedation and correlations of latencies with age of the horse were also studied. There was no effect of sedation on reflex latency periods. The latency of the reflex period measured to a convolved peak of the electromyographic response was significantly different in horses with grades 1 and 2 disease; medians and quartile ranges were 0.067 (0.065-0.073) and 0.072 (0.068-0.074) s, respectively (P<0.05). Significant associations were found between reflex latencies and both horse age and the grade of RLN. Reflex latency measurements are reliable and sensitive, and may assist with the clinical appraisal of Thoroughbred horses with RLN.

The distribution of nerve fibres immunoreactive for vasoactive intestinal peptide, calcitonin gene-related peptide, substance P and dopamine beta-hydroxylase in the normal equine larynx.

The autonomic innervation of the mammalian respiratory system is complex, and involves a wide variety of peptide and non-peptide neurotransmitters which will have an important role in normal laryngeal function and the response to disease. This innervation has been partially described in the horse airway and lung, but there is no information on the equine larynx. This paper describes the expression and distribution of nerve fibres immunoreactive for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP) and the adrenergic enzymatic marker dopamine beta-hydroxylase (DBetaH) in the mucosa of the equine larynx. The overall relative density of nerve fibres immunoreactive for the different antigens was VIP>>CGRP>SP>>DBetaH. There were differences in the distribution of nerve fibre types, although each antigen was found in nerve fibres adjacent to blood vessels and mucous glands. VIP -like immunoreactivity (VIP -Li) was particularly extensive in association with mucous glands. SP - and CGRP -like immunoreactivity (SP -Li, CGRP -Li) were also seen close to the epithelium, with occasional nerve fibres coursing beneath and between the epithelial cells. Fragments of SP -Li and CGRP -Li fibres were also present in large nerve fibre bundles and ganglionic cell clusters, but not in the neurons themselves. The density of nerve fibres immunoreactive for DBetaH was very low and restricted to blood vessels and mucous glands. There was marked variation in the density of nerve fibres at the different sites, with the greatest density, particularly for VIP, over the arytenoid cartilage. Immunoreactive nerve fibres were less plentiful over the epiglottis, and the density of all types of nerve fibres was low over the cricoid cartilage. Overall VIP -Li nerve fibres were the most plentiful.

Studies on the experimental induction of ptosis in horses.

The precise appearance of ptosis due to lesions at different sites was investigated in experimental ponies. The angles of the eyelashes to the head was used as an objective measurement of ptosis after local anaesthesia of the sympathetic trunk or the palpebral nerve and the administration of an ocular alpha agonist or antagonist. It was shown that ptosis is not an inevitable consequence of palpebral nerve pathology, that ocular alpha antagonists can induce ptosis, and that alpha agonist eyedrops have an inconsistent effect on the equine pupil, but are consistent at reversing ptosis induced by sympathetic denervation in unsedated horses.

Measurement of the lateral thoracic reflex latency in ponies.

Lateral thoracic nerve reflex latencies values were measured in ponies using a simple, non-invasive technique. The reflex was elicited using an external triggering hammer attached to an electrodiagnostic unit. The resulting evoked, compound muscle action potentials were recorded with electrodes, which were placed over the 6th ribs or 11th rib level with the axilla. Two superimposed repeats of 4 signal-averaged sweeps of 50 or 100 milliseconds were recorded and the estimated reflex pathway was measured for each subject in order to calculate the reflex latencies and latency velocities. Mean left and right 6th rib peak latencies were not significantly different from each other (P = .609), but left 6th rib latencies were shorter than those recorded from the 11th rib (P < .0001), substantiating the existence of an indirect (central) pathway to the reflex. The calculated left and right 6th rib latency velocities were not significantly different from each other (P = .58) but left 6th rib latency velocities were different from left 11th rib (P = .009). The calculated latency velocities were within the broad range for corticospinal tract motor conduction velocities and comparable to magnetic motor evoked latency velocities. The use of lateral thoracic reflex latency measurements to objectively identify the site of spinal cord lesions is discussed.

Expression of vasoactive intestinal peptide, calcitonin gene-related peptide, substance P, and intermediate neurofilaments in nasal mucosal nerve fibers of horses without nasal disease.

OBJECTIVE: To determine the distribution of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and intermediate neurofilaments in nasal mucosa of horses. ANIMALS: 6 horses without evidence of nasal disease. PROCEDURE: Full-thickness nasal tissue specimens were obtained from the rostral portion of the nasal septum at necropsy, and fluorescence immunohistochemistry was performed to assess mucosal distribution of nerve fibers. RESULTS: Nerve fibers with CGRP-like immunoreactivity (CGRP-Li) formed a dense subepithelial network, and a large number of fibers were found coursing between epithelial cells. Fibers with CGRP-Li were also associated with blood vessels and mucous glands. Fibers with SP-like immunoreactivity (SP-Li) had a similar distribution and density. In contrast, there were few fibers with VIP-like immunoreactivity. Fibers containing intermediate neurofilaments were prominent and appeared as large nerve fiber bundles mainly adjacent to the nasal septum but also close to mucous glands and within the lamina propria. Intermediate neurofilaments were also identified in single nerve fibers at all sites, but the density of fibers with intermediate neurofilaments did not match that of fibers with CGRP- or SP-Li. CONCLUSIONS: The density and distribution of nerve fibers containing SP- or CGRP-Li in nasal mucosa of horses was similar to that reported for other species. However, expression of VIP in nerve fibers was low. Antibodies against intermediate neurofilaments identified many nerve fibers in nasal mucosa of horses but did not appear to identify small diameter fibers expressing SP or VIP.