Standardization of a Videofluoroscopic Swallow Study Protocol to Investigate Dysphagia in Dogs.

BACKGROUND: Videofluoroscopic swallow study (VFSS) is the gold standard for diagnosis of dysphagia in veterinary medicine but lacks standardized protocols that emulate physiologic feeding practices. Age impacts swallow function in humans but has not been evaluated by VFSS in dogs. HYPOTHESIS/OBJECTIVES: To develop a protocol with custom kennels designed to allow free-feeding of 3 optimized formulations of contrast media and diets that address limitations of current VFSS protocols. We hypothesized that dogs evaluated by a free-feeding VFSS protocol would show differences in objective swallow metrics based on age. ANIMALS: Healthy juvenile, adult, and geriatric dogs (n = 24). METHODS: Prospective, experimental study. Custom kennels were developed to maintain natural feeding behaviors during VFSS. Three food consistencies (thin liquid, pureed food, and dry kibble) were formulated with either iohexol or barium to maximize palatability and voluntary prehension. Dogs were evaluated by 16 swallow metrics and compared across age groups. RESULTS: Development of a standardized VFSS protocol resulted in successful collection of swallow data in healthy dogs. No significant differences in swallow metrics were observed among age groups. Substantial variability was observed in healthy dogs when evaluated under these physiologic conditions. Features typically attributed to pathologic states, such as gastric reflux, were seen in healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Development of a VFSS protocol that reflects natural feeding practices may allow emulation of physiology resulting in clinical signs of dysphagia. Age did not result in significant changes in swallow metrics, but additional studies are needed, particularly in light of substantial normal variation.

Specific mesenchymal/epithelial induction of olfactory receptor, vomeronasal, and gonadotropin-releasing hormone (GnRH) neurons.

We asked whether specific mesenchymal/epithelial (M/E) induction generates olfactory receptor neurons (ORNs), vomeronasal neurons (VRNs), and gonadotropin-releasing hormone (GnRH) neurons, the major neuron classes associated with the olfactory epithelium (OE). To assess specificity of M/E-mediated neurogenesis, we compared the influence of frontonasal mesenchyme on frontonasal epithelium, which becomes the OE, with that of the forelimb bud. Despite differences in position, morphogenetic and cytogenic capacity, both mesenchymal tissues support neurogenesis, expression of several signaling molecules and neurogenic transcription factors in the frontonasal epithelium. Only frontonasal mesenchyme, however, supports OE-specific patterning and activity of a subset of signals and factors associated with OE differentiation. Moreover, only appropriate pairing of frontonasal epithelial and mesenchymal partners yields ORNs, VRNs, and GnRH neurons. Accordingly, the position and molecular identity of specialized frontonasal epithelia and mesenchyme early in gestation and subsequent inductive interactions specify the genesis and differentiation of peripheral chemosensory and neuroendocrine neurons.

Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics.

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

A speculative essay on retinoic acid regulation of neural stem cells in the developing and aging olfactory system.

Circulating signals like the acidic derivative of vitamin A: retinoic acid (RA) may regulate resident stem cells in the adult nervous system, particularly in the olfactory pathway. RA is an essential factor for inducing neural stem or precursor cells that give rise to olfactory receptor neurons (ORNs) and olfactory bulb (OB) interneurons (OBINs) during embryonic development. Similar precursors in the adult brain constantly generate new ORNs and OBINs, and embryonic signaling pathways, like that via RA, may be retained or reactivated for this purpose. We have shown that RA regulates neural precursors in the embryonic and adult olfactory pathway. Moreover, RA administration after olfactory system damage stimulates an immune response and yields a more rapid recovery of olfactory-guided behavior. We suggest that olfactory integrity may be maintained by RA-mediated regulation of neurogenesis as well as local immune responses, and that aging compromises these mechanisms. The chemical senses, particularly olfaction, decline in aged individuals, and RA (via vitamin A) levels may also decline, perhaps due to changes in appetite and food intake. This synergy may result in a high prevalence of olfactory pathology in aged individuals.

Once and again: retinoic acid signaling in the developing and regenerating olfactory pathway.

Retinoic acid (RA), a member of the steroid/thyroid superfamily of signaling molecules, is an essential regulator of morphogenesis, differentiation, and regeneration in the mammalian olfactory pathway. RA-mediated teratogenesis dramatically alters olfactory pathway development, presumably by disrupting retinoid-mediated inductive signaling that influences initial olfactory epithelium (OE) and bulb (OB) morphogenesis. Subsequently, RA modulates the genesis, growth, or stability of subsets of OE cells and OB interneurons. RA receptors, cofactors, and synthetic enzymes are expressed in the OE, OB, and anterior subventricular zone (SVZ), the site of neural precursors that generate new OB interneurons throughout adulthood. Their expression apparently accommodates RA signaling in OE cells, OB interneurons, and slowly dividing SVZ neural precursors. Deficiency of vitamin A, the dietary metabolic RA precursor, leads to cytological changes in the OE, as well as olfactory sensory deficits. Vitamin A therapy in animals with olfactory system damage can accelerate functional recovery. RA-related pathology as well as its potential therapeutic activity may reflect endogenous retinoid regulation of neuronal differentiation, stability, or regeneration in the olfactory pathway from embryogenesis through adulthood. These influences may be in register with retinoid effects on immune responses, metabolism, and modulation of food intake.

Immunolocalization of retinoic acid receptors in the mammalian olfactory system and the effects of olfactory denervation on receptor distribution.

All-trans retinoic acid (ATRA), a metabolite of vitamin A, binds to retinoic acid receptors (RARs) to mediate gene transcription in target cells. We previously found that an ATRA supplement enhanced olfactory recovery rate in adult mice after olfactory bulb deafferentation. In this study, we examined the cellular localization of RARalpha, RARbeta, and RARgamma and the effects of surgery and ATRA treatment using immunocytochemistry. Mice received a left olfactory nerve transection with the right side serving as internal control. One day after surgery, the mice were given either ATRA mixed with sesame oil or just sesame oil. In the unoperated olfactory bulb, only RARalpha immunoreactivity (ir) was observed. In the unoperated right olfactory epithelium, RARalpha-ir was found in flask-shaped cells located in the supporting cell layer, in cell clusters above the basal cell layer, in cells in the lamina propria, in some respiratory cells and in the olfactory bulb. The flask-shaped cells did not immunostain for either neurons or sustentacular cells. RARbeta-ir was localized only in the respiratory cells while no RARgamma-ir was observed in the olfactory epithelium. The density of RARalpha-ir cells was higher in the operated left olfactory epithelium and highest after ATRA treatment. This study demonstrates the presence of RARs in the olfactory system, provides additional support that the ATRA-signaling pathway may be involved in the recovery of the olfactory epithelium after injury, and suggests a role for an unstudied cell type in that process.

Preliminary results of olfactory testing in rats without deprivation.

Although rodents are nocturnal, their behavior is usually tested during the day. The authors present the results of a preliminary study, which suggest that altering the animals' day:night cycle might be the key to eliminating the need for food or water deprivation prior to testing.

Characteristics of odorant elicited calcium changes in cultured human olfactory neurons.

An important step in establishing and utilizing a cell culture system for the in vitro study of olfaction is assessing whether the cultured cells possess physiological properties similar to those of mature olfactory neurons. Various investigators have successfully established proliferating cell lines from olfactory tissue, but few have demonstrated the characteristics of odor sensitivity of these cells. We successfully established cultured cell lines from adult human olfactory tissue obtained using an olfactory biopsy procedure and measured their ability to respond to odor stimulation using calcium imaging techniques. A subset of the human olfactory cells in culture displayed a distinct morphology and specifically expressed immunocytochemical markers characteristic of mature human olfactory neurons such as OMP, G(olf), NCAM and NST. Under defined growth conditions, these cultured cells responded to odorant mixes that have been previously shown to elicit intracellular calcium changes in acutely-isolated human olfactory neurons. These odorant-elicited calcium responses displayed characteristics similar to those found in mature human olfactory neurons. First, cultured cells responded with either increases or decreases in intracellular calcium. Second, increases in calcium were abolished by removal of extracellular calcium. Third, inhibitors of the olfactory signal transduction cascades reversibly blocked these odorant-elicited intracellular calcium changes. Our results demonstrate that cultures of adult human olfactory cells established from olfactory biopsies retain some of the in vivo odorant response characteristics of acutely isolated cells from the adult olfactory epithelium. This work has important ramifications for investigation of olfactory function and dysfunction using biopsy procedures and in vitro assays of odor sensitivity.

Retinoic acid enhances the rate of olfactory recovery after olfactory nerve transection.

In the olfactory system, retinoic acid (RA) plays an important role in development and may affect growth in the adult animal. To explore the potential effects of RA on recovery after injuries, adult mice were trained in a buried food paradigm and were given a single oral supplement of RA after olfactory nerve transection. Results demonstrate that RA accelerates the recovery of olfactory functions after injury.

Expression of mRNAs encoding for two different olfactory receptors in a subset of olfactory receptor neurons.

Evidence has accumulated to support a model for odorant detection in which individual olfactory receptor neurons (ORNs) express one of a large family of G protein-coupled receptor proteins that are activated by a small number of closely related volatile chemicals. However, the issue of whether an individual ORN expresses one or multiple types of receptor proteins has yet to be definitively addressed. Physiological data indicate that some individual ORNs can be activated by odorants differing substantially in structure and/or perceived quality, suggesting multiple receptors or one nonspecific receptor per cell. In contrast, molecular biological studies favor a scheme with a single, fairly selective receptor per cell. The present studies directly assessed whether individual rat ORNs can express multiple receptors using single-cell PCR techniques with degenerate primers designed to amplify a wide variety of receptor sequences. We found that whereas only a single OR sequence was obtained from most ORNs examined, one ORN produced two distinct receptor sequences that represented different receptor gene families. Double-label in situ hybridization studies indicated that a subset of ORNs co-express two distinct receptor mRNAs. A laminar segregation analysis of the cell nuclei of ORNs labeled with the two OR mRNA probes showed that for one probe, the histogram of the distribution of the cell nuclei along the depth of the epithelium was bimodal, with one peak overlapping the (unimodal) histogram for the other probe. These results are consistent with co-expression of two OR mRNAs in a population of single ORNs.

Modulation of odor-induced increases in [Ca(2+)](i) by inhibitors of protein kinases A and C in rat and human olfactory receptor neurons.

Protein kinases A and C have been postulated to exert multiple effects on different elements of signal transduction pathways in olfactory receptor neurons. However, little is known about the modulation of olfactory responses by protein kinases in intact olfactory receptor neurons. To further elucidate the details of the modulation of odorant responsiveness by these protein kinases, we investigated the action of two protein kinase inhibitors: H89, an inhibitor of protein kinase A, and N-myristoylated EGF receptor, an inhibitor of protein kinase C, on odorant responsiveness in intact olfactory neurons. We isolated individual olfactory neurons from the adult human and rat olfactory epithelium and measured responses of the isolated cells to odorants or biochemical activators that have been shown to initiate cyclic AMP or inositol 1,4,5-trisphospate production in biochemical preparations. We employed calcium imaging techniques to measure odor-elicited changes in intracellular calcium that occur over several seconds. In human olfactory receptor neurons, the protein kinase A and C inhibitors affected the responses to different sets of odorants. In rats, however, the protein kinase C inhibitor affected responses to all odorants, while the protein kinase A inhibitor had no effect. In both species, the effect of inhibition of protein kinases was to enhance the elevation and block termination of intracellular calcium levels elicited by odorants. Our results show that protein kinases A and C may modulate odorant responses of olfactory neurons by regulating calcium fluxes that occur several seconds after odorant stimulation. The effects of protein kinase C inhibition are different in rat and human olfactory neurons, indicating that species differences are an important consideration when applying data from animal studies to apply to humans.

Cell and molecular biology of olfaction.

Genetics, experience, environment, and health can all affect the anatomic and physiologic components of the olfactory system and thereby influence olfactory performance. Large individual differences exist among subjects with respect to olfactory sensitivity and identification ability, which may result in both qualitative and quantitative differences in perceptual ability.

Olfactory neuron-specific expression of NeuroD in mouse and human nasal mucosa.

Human olfactory neuroepithelium (OE) is situated within the olfactory cleft of the nasal cavity and has the characteristic property of continually regenerating neurons during the lifetime of the individual. This regenerative ability of OE provides a unique model for neuronal differentiation, but little is known about the structure and biology of human olfactory mucosa. Thus, to better understand neurogenesis in human OE, we studied the expression of olfactory marker protein (OMP), TrkB and NeuroD in human nasal biopsies and autopsy specimens and compared these data with those obtained from normal and regenerating mouse OE. We show that NeuroD and TrkB are coordinately expressed in human OE. Thus, by using these markers we have been able to extend the known boundaries of the human OE to include the inferior middle turbinate. In normal mouse OE, TrkB and OMP expression overlap in cells closest to the superficial layer, but TrkB is expressed more strongly in the lower region of this layer. In contrast, NeuroD expression is more basally restricted in a region just above the globose basal cells. These characteristic expression patterns of OMP, TrkB and NeuroD were also observed in the regenerating mouse OE induced by axotomy. These results support a role of NeuroD and brain-derived neurotrophic actor (BDNF), the preferred ligand for TrkB, in the maintenance of the olfactory neuroepithelium in humans and mice.

Distribution and phenotype of neurons containing the ATP-sensitive K+ channel in rat brain.

Select groups of neurons within the brain alter their firing rate when ambient glucose levels change. These glucose-responsive neurons are integrated into systems which control energy balance in the body. They contain an ATP-sensitive K+ channel (KATP) which mediates this response. KATP channels are composed of an inwardly rectifying pore-forming unit (Kir6.1 or Kir6.2) and a sulfonylurea binding site. Here, we examined the anatomical distribution and phenotype of cells containing Kir6.2 mRNA within the rat brain by combinations of in situ hybridization and immunocytochemistry. Cells containing Kir6. 2 mRNA were widely distributed throughout the brain without apparent concentration in areas known to contain specific glucose-responsive neurons. Kir6.2 mRNA was present in neurons expressing neuron-specific enolase, tyrosine hydroxylase, neuropeptide Y (NPY) and the glutamic acid decarboxylase isoform, GAD65. No astrocytes expressing glial fibrillary acidic protein or oligodendrocytes expressing carbonic anhydrase II were found to co-express Kir6.2 mRNA. Virtually all of the NPY neurons in the hypothalamic arcuate n. and catecholamine neurons in the substantia nigra, pars compacta and locus coeruleus contained Kir6.2 mRNA. Epinephrine neurons in the C2 area also expressed high levels of Kir6.2, while noradrenergic neurons in A5 and A2 areas expressed lower levels. The widespread distribution of Kir6.2 mRNA suggests that the KATP channel may serve a neuroprotective role in neurons which are not directly involved in integrating signals related to the body's energy homeostasis.

The use of olfactory receptor neurons (ORNs) from biopsies to study changes in aging and neurodegenerative diseases.

A gradual loss of olfactory capability with age and in a number of neurodegenerative diseases is common, and mechanisms underlying these losses are not understood. We determined the feasibility of using ORNs obtained from olfactory epithelial biopsies to identify possible changes in ORN function that may contribute to olfactory impairment in these individuals. ORNs from nine healthy subjects (66-84 yr), three patients with Alzheimer's disease and one with multi-infarct dementia were studied with calcium imaging techniques and two odorant mixtures. Seventy-five viable ORNs were studied; 53% of these were odorant responsive, and twenty percent of these responded to both odorant mixtures. In contrast, 25% of 173 ORNs from younger subjects were odorant responsive, and none of these responded to both odorant mixtures. The proportion of cells responding to each of the odorant mixtures also differed between older and younger subjects. These studies demonstrate the feasibility of this approach to examine age or disease-associated changes in neuronal function. Further, age-related changes in ORN selectivity may contribute to changes in olfactory performance.

Selectivity and response characteristics of human olfactory neurons.

Transduction mechanisms were investigated in human olfactory neurons by determining characteristics of odorant-induced changes in intracellular calcium concentration ([Ca2+]i). Olfactory neurons were freshly isolated from nasal biopsies, allowed to attach to coverslips, and loaded with the calcium-sensitive indicator fura-2. Changes in [Ca2+]i were studied in response to exposure to individual odors, or odorant mixtures composed to distinguish between transduction pathways mediated by adenosine 3'5'-monophosphate (cAMP; mix A) or inositol 1,4,5-trisphosphate (InsP3; mix B). Overall, 52% of biopsies produced one or more odorant-responsive olfactory neurons, whereas 24% of all olfactory neurons tested responded to odorant exposure with a change in [Ca2+]i. As in olfactory neurons from other species, the data suggest that odorant exposure elicited calcium influx via second-messenger pathways involving cAMP or InsP3. Unlike olfactory neurons from other species that have been tested, some human olfactory neurons responded to odorants with decreases in [Ca2+]i. Also in contrast with olfactory neurons from other species, human olfactory neurons were better able to discriminate between odorant mixtures in that no neuron responded to more than one type of odor or mixture. These results suggest the presence of a previously unreported type of olfactory transduction mechanism, and raise the possibility that coding of odor qualities in humans may be accomplished to some degree differently than in other vertebrates, with the olfactory neuron itself making a greater contribution to the discrimination process.

Fatty acid oxidation modulates the eating response to the fructose analogue 2,5-anhydro-D-mannitol.

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM) stimulates feeding behavior in rats apparently through its effects on hepatic energy metabolism, where it reduces glucose utilization, traps phosphate, and decreases ATP. The extent to which the magnitude and duration of the eating response are dependent on the ability of the liver to switch to fat oxidation for energy production was investigated by manipulating substrate availability through dietary and pharmacological means. Rats adapted to a high-fat, low-carbohydrate diet preferentially use fat fuels for hepatic energy production and were insensitive to the effects of 2,5-AM on food intake. The lack of an eating response occurred despite similar changes in plasma fuels and liver glycogen compared with rats fed a low-fat, high-carbohydrate diet. In contrast, inhibiting fatty acid oxidation with methyl palmoxirate, which blocks transport of long-chain fatty acids to the mitochondria, potentiated the ability of 2,5-AM to stimulate feeding without altering its effects on plasma and liver fuels. These data demonstrate that the eating response to 2,5-AM is modulated by the availability of fat fuels and implicate a mechanism for initiation of feeding that is not dependent on inhibition of carbohydrate metabolism per se but rather integrates information about the use of both types of fuels.

Functionally mature olfactory neurons from two anosmic patients with Kallmann syndrome.

Patients with Kallmann syndrome (KS) exhibit hypogonadotropic hypogonadism and anosmia [Kallmann et al., Am. J. Mental Def., 48 (1944) 203-236] secondary to failure of gonadotropin-releasing hormone (GnRH)-producing neurons to migrate from the olfactory placode to the brain, and to agenesis of the olfactory bulbs. It has been hypothesized that olfactory neurons (ON) from individuals with KS are immature partly on the basis of studies in animals showing that lack of synaptic connection of ON with the olfactory bulb results in expression of immature ON [Schwob et al., J. Neurosci., 12 (1979) 880-883]. To test this assumption, we obtained olfactory tissue samples from two males diagnosed with KS on the basis of medical history and MRI studies. Both patients were anosmic. The functioning of cells isolated from biopsies taken from the upper middle turbinate and septum was studied by measuring changes in intracellular Ca2+ concentration ([Cai]) using dual excitation fluorescence microscopy. Biopsies from both patients yielded cells that morphologically appeared to be ON. Seven of 16 cells that morphologically resembled ON responded with a change in [Cai] upon stimulation with an odorant mixture. These studies show that at least some ON in KS individuals are functionally mature and suggest that complete development of the olfactory bulbs is not required for differentiation of mature human ON.

L-ethionine, an amino acid analogue, stimulates eating in rats.

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM) triggers feeding in rats through its actions in liver, which include a decrease in ATP due to trapping of phosphate. To determine whether decreasing liver ATP by a different means would also trigger feeding, we gave rats L-ethionine (ETH), an amino acid analogue that reduces ATP in liver by trapping adenosine as S-adenosyl-L-ethionine. ETH-treatment increased food intake from 4 to 8 h after administration, without affecting 24-h intake. Two hours after treatment, liver ATP was 25% lower in rats given ETH than in vehicle-treated controls. Circulating fuels and liver lactate and pyruvate were not affected by ETH treatment, whereas liver glycogen was 15% lower in ETH-treated rats. These results are the first to show that an amino acid analogue elicits feeding in rats fed protein-sufficient diets. Because a decrease in liver ATP is the only common effect of ETH and 2,5-AM observed thus far, a signal related to liver ATP status may be involved in the mechanism for initiation of feeding in rats.

Phosphate loading prevents the decrease in ATP and increase in food intake produced by 2,5-anhydro-D-mannitol.

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM) triggers feeding in rats apparently by its action in the liver. In vivo phosphorylation of this analogue decreases hepatic inorganic phosphate and ATP by trapping of phosphate in the mono- and diphosphorylated forms of 2,5-AM. To determine whether hepatic phosphate depletion and decreased ATP are involved in the eating response to 2,5-AM, rats were treated with excess sodium phosphate before injection of 2,5-AM. Phosphate loading prevented both the increase in food intake and the decrease in liver ATP, without affecting the changes seen in plasma fuels produced by 2,5-AM treatment. Phosphate loading did not influence water intake or eating elicited by insulin or 2-deoxy-D-glucose, indicating that the effect on 2,5-AM-induced eating was behaviorally specific and not due to malaise. These data suggest that 2,5-AM elicits eating by trapping phosphate and reducing ATP in liver.