Vitamin C deficiency in osteogenic disorder Shionogi/Shi Jcl-od/od rats: effects on sour taste preferences, lick rates, chorda tympani nerve responses, and taste transduction elements.

Animals use sour taste to avoid spoiled food and to choose foods containing vitamins and minerals. To investigate the response to sour taste substances during vitamin C (ascorbic acid; AA) deficiency, we conducted behavioral, neural, anatomical, and molecular biological experiments with osteogenic disorder Shionogi/Shi Jcl-od/od rats, which lack the ability to synthesize AA. Rats had higher 3 mM citric acid and 10 mM AA preference scores when AA-deficient than when replete. Licking rates for sour taste solutions [AA, citric acid, acetic acid, tartaric acid, and HCl] were significantly increased during AA deficiency relative to pre- and postdeficiency. Chorda tympani nerve recordings were conducted to evaluate organic acid taste responses in the AA-deficient and replete rats. Nerve responses to citric acid, acetic acid, and tartaric acid were significantly diminished in AA-deficient rats relative to replete controls. There was no significant difference in the number of fungiform papillae taste buds per unit area in the AA-deficient rats relative to the replete rats. However, mRNA expression levels of Gnat3 (NM_173139.1), Trpm5 (NM_001191896.1), Tas1r1 (NM_053305.1), Car4 (NM_019174.3), and Gad1 (NM_017007.1) in fungiform papillae taste bud cells from AA-deficient rats were significantly lower than those in replete rats. Our data suggest that AA deficiency decreases avoidance of acids and reduces chorda tympani nerve responses to acids. AA deficiency downregulates some taste-related genes in fungiform papillae taste bud cells. However, the results also reveal that the mRNA expression of some putative sour taste receptors in fungiform papillae taste bud cells is not affected by AA deficiency.

Effect of the duration of a conditioned stimulus on component recognition in binary taste mixtures in rats.

OBJECTIVES: The aim of this behavioral study was to investigate the duration of a conditioned stimulus (CS-duration) necessary for rats to recognize the components of a binary taste mixture in a conditioned taste aversion (CTA) paradigm as well as the relationship between CS-duration and their spontaneous recovery. METHODS: The experimental rats were categorized under conditioned and control groups and further divided into three groups according to the CS-duration: 10, 30, and 60 s. As the test stimuli, a mixture of 100 mM sucrose (S) + 30 µM quinine hydrochloride (Q) and its components were used. RESULTS: On day 1 of the CTA test, the number of licks (NL) for S + Q and S in all conditioned groups was significantly lower than that of the control group presented with CS for 60 s (CON-60), which was the representative control group determined by the initial CTA test. For Q, there was no significant difference between NL of the CTA group presented with CS for 10 s and that of CON-60; however, NL in the other two CTA groups, i.e., CTA-30 and CTA-60, was significantly lower than that of CON-60. When the rats were presented with a shorter CS-duration, they showed spontaneous recovery earlier depending on the CS-duration. CONCLUSIONS: These results suggest that rats can recognize a binary taste mixture and its components using a CS-duration of more than 30 s and that spontaneous recovery from CTA learning depends on the CS- duration.

Behavioral and Neural Responses to Vitamin C Solution in Vitamin C-deficient Osteogenic Disorder Shionogi/Shi Jcl-od/od Rats.

To investigate the appetite for vitamin C (VC), we conducted behavioral and neural experiments using osteogenic disorder Shionogi/Shi Jcl-od/od (od/od) rats, which lack the ability to synthesize VC, and their wild-type controls osteogenic disorder Shionogi/Shi Jcl- +/+ (+/+) rats. In the behavioral study, rats were deprived of VC for 25 days and then received two-bottle preference tests with a choice between water and 10 mM VC. The preference for 10 mM VC solution of od/od rats was significantly greater than that of +/+ rats. In the neural study, the relative magnitudes of the whole chorda tympani nerve (CTN) responses to 100-1000 mM VC, 3-10 mM HCl, 100-1000 mM NaCl, and 20 mM quinine▪HCl in the VC-deficient rats were significantly smaller than those in the nondeficient ones. Further, we conducted additional behavioral experiments to investigate the appetite for sour and salty taste solutions of VC-deficient od/od rats. Preference scores for 3 mM citric acid increased in od/od rats after VC removal, compared with before, whereas preference scores for 100 and 150 mM NaCl were decreased in VC-deficient od/od rats. The preference for 300 mM NaCl was not changed. Hence, our results suggest that the reduction of the aversive taste of VC during VC deficiency may have involved the reduction of CTN responses to acids. Overall, our results indicate that VC-deficient rats ingest sufficient VC to relieve their deficiency and that VC deficiency causes changes in peripheral sensitivity to acids, but nongustatory factors may also affect VC intake and choice.

Recognition by Rats of Binary Taste Solutions and Their Components.

This behavioral study investigated how rats conditioned to binary mixtures of preferred and aversive taste stimuli, respectively, responded to the individual components in a conditioned taste aversion (CTA) paradigm. The preference of stimuli was determined based on the initial results of 2 bottle preference test. The preferred stimuli included 5mM sodium saccharin (Sacc), 0.03M NaCl (Na), 0.1M Na, 5mM Sacc + 0.03M Na, and 5mM Sacc + 0.2mM quinine hydrochloride (Q), whereas the aversive stimuli tested were 1.0M Na, 0.2mM Q, 0.3mM Q, 5mM Sacc + 1.0M Na, and 5mM Sacc + 0.3mM Q. In CTA tests where LiCl was the unconditioned stimulus, the number of licks to the preferred binary mixtures and to all tested preferred components were significantly less than in control rats. No significant difference resulted between the number of licks to the aversive binary mixtures or to all tested aversive components. However, when rats pre-exposed to the aversive components contained of the aversive binary mixtures were conditioned to these mixtures, the number of licks to all the tested stimuli was significantly less than in controls. Rats conditioned to components of the aversive binary mixtures generalized to the binary mixtures containing those components. These results suggest that rats recognize and remember preferred and aversive taste mixtures as well as the preferred and aversive components of the binary mixtures, and that pre-exposure before CTA is an available method to study the recognition of aversive taste stimuli.

Excitatory and inhibitory synaptic function in the rostral nucleus of the solitary tract in embryonic rat.

The embryonic development of synapses in the rostral nucleus of the solitary tract (rNST) was investigated in rat to determine when synapses begin to function. Using a brain slice preparation we studied appearance of synaptic receptors on second order rNST neurons and investigated the development of postsynaptic responses elicited by afferent nerve stimulation. Prenatal excitatory and inhibitory synaptic responses were recorded as early as E14. Glutamatergic and GABAergic postsynaptic responses were detected as early as E16. Both NMDA and AMPA receptors contributed to glutamatergic postsynaptic responses. GABAergic postsynaptic responses resulted primarily from activation of GABA(A) receptors. However, functional GABA(C) receptors were also demonstrated. A glycinergic postsynaptic response was not found although functional glycine receptors were demonstrated at E16. Solitary tract (ST) stimulation-evoked EPSCs, first detected at E16, were eliminated by glutamate receptor antagonists. ST-evoked IPSPs, also detected at E16, were eliminated by GABA(A) receptor antagonist. Thus, considerable prenatal development of rNST synaptic connections occurs and this will ensure postnatal function of central taste processing circuits.

Pre- and postnatal differences in membrane, action potential, and ion channel properties of rostral nucleus of the solitary tract neurons.

There is little known about the prenatal development of the rostral nucleus of the solitary tract (rNST) neurons in rodents or the factors that influence circuit formation. With morphological and electrophysiological techniques in vitro, we investigated differences in the biophysical properties of rNST neurons in pre- and postnatal rats from embryonic day 14 (E14) through postnatal day 20. Developmental changes in passive membrane and action potential (AP) properties and the emergence and maturation of ion channels important in neuron function were characterized. Morphological maturation of rNST neurons parallels changes in passive membrane properties. Mean soma size, dendritic branch points, neurite endings, and neurite length all increase prenatally. whereas neuron resting membrane potential, input resistance, and time constant decrease. Dendritic spines, on the other hand, develop after birth. AP discharge patterns alter in pre- and postnatal stages. At E14, neurons generated a single TTX-sensitive, voltage-gated Na(+) AP when depolarized; a higher discharge rate appeared at older stages. AP amplitude, half-width, and rise and fall times all change during development. Responses to current injection revealed a number of voltage-gated conductances in embryonic rNST, including a hyperpolarization-activated inward current and a low-threshold Ca(2+) current that initiated Ca(2+) spikes. A hyperpolarization-activated, transient outward potassium current was also present in the developing neurons. Although the properties of these channels change during development, they are present before synapses form and therefore, can contribute to initial establishment of neural circuits, as well as to the changing electrophysiological properties in developing rNST neurons.

Characteristics of rostral solitary tract nucleus neurons with identified afferent connections that project to the parabrachial nucleus in rats.

Afferent information derived from oral chemoreceptors is transmitted to second-order neurons in the rostral solitary tract nucleus (rNST) and then relayed to other CNS locations responsible for complex sensory and motor behaviors. Here we investigate the characteristics of rNST neurons sending information rostrally to the parabrachial nucleus (PBN). Afferent connections to these rNST-PBN projection neurons were identified by anterograde labeling of the chorda tympani (CT), glossopharyngeal (IX), and lingual (LV) nerves. We used voltage- and current-clamp recordings in brain slices to characterize the expression of both the transient A-type potassium current, IKA and the hyperpolarization-activated inward current, Ih, important determinants of neuronal repetitive discharge characteristics. The majority of rNST-PBN neurons express IKA, and these IKA-expressing neurons predominate in CT and IX terminal fields but were expressed in approximately half of the neurons in the LV field. rNST-PBN neurons expressing Ih were evenly distributed among CT, IX and LV terminal fields. However, expression patterns of IKA and Ih differed among CT, IX, and LV fields. IKA-expressing neurons frequently coexpress Ih in CT and IX terminal fields, whereas neurons in LV terminal field often express only Ih. After GABAA receptor block all rNST-PBN neurons responded to afferent stimulation with all-or-none excitatory synaptic responses. rNST-PBN neurons had either multipolar or elongate morphologies and were distributed throughout the rNST, but multipolar neurons were more often encountered in CT and IX terminal fields. No correlation was found between the biophysical and morphological characteristics of the rNST-PBN projection neurons in each terminal field.

Synaptic responses of neurons controlling the parotid and von Ebner salivary glands in rats to stimulation of the solitary nucleus and tract.

Salivary secretion results from reflex stimulation of autonomic neurons via afferent sensory information relayed to neurons in the rostral nucleus of the solitary tract (rNST), which synapse with autonomic neurons of the salivatory nuclei. We investigated the synaptic properties of the afferent sensory connection to neurons in the inferior salivatory nucleus (ISN) controlling the parotid and von Ebner salivary glands. Mean synaptic latency recorded from parotid gland neurons was significantly shorter than von Ebner gland neurons. Superfusion of GABA and glycine resulted in a concentration-dependent membrane hyperpolarization. Use of glutamate receptor antagonists indicated that both AMPA and N-methyl-D-aspartate (NMDA) receptors are involved in the evoked excitatory postsynaptic potentials (EPSPs). Inhibitory postsynaptic potential (IPSP) amplitude increased with higher intensity ST stimulation. Addition of the glycine antagonist strychnine did not affect the amplitude of the IPSPs significantly. The GABA(A) receptor antagonist, bicuculline (BMI) or mixture of strychnine and BMI abolished the IPSPs in all neurons. IPSP latency was longer than EPSP latency, suggesting that more than one synapse is involved in the inhibitory pathway. Results show that ISN neurons receive both excitatory and inhibitory afferent input mediated by glutamate and GABA respectively. The ISN neuron response to glycine probably derives from descending connections. Difference in the synaptic characteristics of ISN neurons controlling the parotid and von Ebner glands may relate to the different function of these two glands.

Effects of 5-hydroxytryptamine and substance P on neurons of the inferior salivatory nucleus.

The parasympathetic secretomotor innervation of the salivary glands originates from a longitudinal column of neurons in the medulla called the salivatory nucleus. The neurons innervating the parotid and von Ebner salivary glands are situated in the caudal extremity of the column designated as the inferior salivatory nucleus (ISN). Immunocytochemical investigations have demonstrated the presence of a number of neuropeptides surrounding the ISN neurons. We have examined the neurophysiological effect of two of these neuropeptides on neurons of the ISN identified by retrograde transport of a fluorescent label. Both serotonin (5-HT) and substance P (SP) excited virtually all neurons in the ISN. Application of these neuropeptides resulted in membrane depolarization that was concentration dependent. Although the majority of ISN neurons that were depolarized by SP application exhibited an increase in input resistance, application of 5-HT induced widely varied change in input resistance. Membrane depolarization elicited action potential discharges that increased in frequency with increasing concentration of 5-HT and SP. Blocking action potential conduction from surrounding neurons did not eliminate the depolarizing effects of 5-HT and SP, indicating that both neuropeptides acted directly on the ISN neurons. Finally, the use of 5-HT agonists and antagonists indicates that 5-HT acts via a 5-HT(2A) receptor, and the use of SP agonists suggests that SP acts via neuokinin-1 and -2 receptors. These data show that 5-HT and SP excite most of the ISN neurons innervating the lingual von Ebner glands possibly modulating the synaptic drive to these neurons derived from afferent gustatory input.

Voltage-gated inward currents of morphologically identified cells of the frog taste disc.

We used the patch clamp technique to record from taste cells in vertical slices of the bullfrog (Rana catesbeiana) taste disc. Cell types were identified by staining with Lucifer yellow in a pipette after recording their electrophysiological properties. Cells could be divided into the following three groups: type Ib (wing) cells with sheet-like apical processes, type II (rod) cells with single thick rod-like apical processes and type III (rod) cells with thin rod-like apical processes. No dye-coupling was seen either between cells of the same type or between cells of different types. We focused on the voltage-gated inward currents of the three types of cells. Type Ib and type II cells exhibited tetrodotoxin (TTX)-sensitive voltage-gated Na+ currents. Surprisingly, type III cells showed TTX-resistant voltage-gated Na+ currents and exhibited a lack of TTX-sensitive Na+ currents. TTX-resistant voltage-gated Na+ currents in taste cells are reported for the first time here. The time constant for the inactivating portion of the voltage-gated inward Na+ currents of type III cells was much larger than that of type Ib and type II cells. Therefore, slow inactivation of inward Na+ currents characterizes type III cells. Amplitudes of the maximum peak inward currents of type III cells were smaller than those of type Ib and type II cells. However, the density (pA/pF) of the maximum peak inward currents of type III cells was much higher than that of type Ib cells and close to that of type II cells. No evidence of the presence of voltage-gated Ca2+ channels in frog taste cells has been presented up to now. In this study, voltage-gated Ba2+ currents were observed in type III cells but not in type Ib and type II cells when the bath solution was a standard Ba2+ solution containing 25 mM Ba2+. Voltage-gated Ba2+ currents were blocked by addition of 2 mM CoCl2 to the standard Ba2+ solution, suggesting that type III cells possess the voltage-gated Ca2+ channels and they do classical (calcium-influx) synaptic transmission. It appears that type III cells are taste receptor cells.