Chemical stimulation of the laryngopharynx increases Fos-like immunoreactivity in the rat hypothalamus and amygdala.

Using immunohistochemical detection of the Fos protein as a cellular marker of neuronal activation, we examined forebrain areas that may be activated upon chemical stimulation of the laryngeal opening. Anesthetized rats were subject to multiple infusions of a chemical solution into the laryngopharynx. These animals were compared to two control groups: a surgical control group in which the animals were subject to the surgical procedure but received no stimulus infusions and a flow control group in which physiological saline replaced the chemical stimulus. Comparing the numbers of Fos-like-immunoreactive neurons in regions of the forebrain across groups revealed that infusing the chemical stimulus solution into the laryngopharyngeal opening selectively increased the number of Fos-like-immunoreactive nuclei in the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala, two autonomic-visceral related forebrain regions. Within the paraventricular nucleus of the hypothalamus, Fos-like-immunoreactive nuclei were significantly increased in the parvocellular subdivision while in the central nucleus of the amygdala, significant increases in Fos-like-immunoreactive nuclei were limited to the lateral capsular subdivision. These data suggest that in the rat laryngopharyngeal chemosensory stimulation activates forebrain regions that receive oral sensory information and are involved in visceral and autonomic functions.

Distribution of GABA and glycine in the lamb nucleus of the solitary tract.

The inhibitory amino acids gamma-aminobutyric acid (GABA) and glycine are involved in several nucleus of the solitary tract (NST)-mediated functions. The distribution of these amino acids in the NST of the lamb, a species frequently used in investigations of NST-mediated behaviors, has not been described. Therefore, this study was designed to investigate the distribution of GABA and glycine in the lamb NST using immunohistochemistry. Both GABA and glycine immunoreactive cells and puncta were unevenly distributed in the lamb NST. The highest density of GABA immunoreactive cells was found in the intermediate zone of the NST, medial to the solitary tract (ST). High to moderate levels of puncta labeling were observed throughout the NST, particularly around the ST in intermediate and caudal zones. Moderate to low levels of glycine immunoreactivity were observed, with most glycine immunoreactive cells and puncta found in the caudal two-thirds of the NST in the medial, ventrolateral and dorsomedial NST. Only a few glycine immunoreactive cells and puncta were found in the rostral zone of the NST. The widespread distribution of GABA and glycine immunoreactivity in intermediate and caudal zones of the NST suggests that these inhibitory amino acids play an important role in modulating NST-mediated functions like swallowing, respiration and cardiovascular regulation in the lamb. The much higher density of GABA immunoreactivity compared to glycine immunoreactivity in the rostral zone of the NST suggests that GABA, but not glycine, is an important neurotransmitter in the processing of taste information by the lamb NST.

Distribution of aspartate and glutamate in the nucleus of the solitary tract of the lamb.

Excitatory amino acids have been implicated in several nucleus of the solitary tract (NST)-mediated functions. The distribution of the excitatory amino acids aspartate and glutamate has been described in both cat and rat. However, the distribution of these amino acids has not been described for the lamb, a species frequently used in the investigation of NST-mediated behaviors. Thus, this study was designed to investigate the distribution of aspartate-like (ASP) and glutamate-like (GLU) immunoreactivity in the lamb NST using pre- and post-embedding immunohistochemistry. Both ASP- and GLU-immunoreactive cells and puncta were observed throughout the rostral to caudal extent of the lamb NST. The most intense ASP- and GLU-immunoreactive cell and puncta staining was found ventromedial, ventral and ventrolateral to the solitary tract at intermediate and caudal levels of the lamb NST. The relative numbers of both cells and puncta stained were lower at rostral levels of the NST corresponding to the gustatory NST. The intense ASP and GLU immunoreactivity observed in areas of the lamb intermediate and caudal NST that are involved in respiration, deglutition and cardiovascular functions suggests excitatory amino acids plays an important role in NST neural processing that underlies these behaviors in lamb.

Fine structure of taste buds located on the lamb epiglottis.

BACKGROUND: Taste buds located on the aryepiglottal folds and laryngeal surface of the epiglottis are the principal receptors responsible for the initiation of the laryngeal chemoreflex. In contrast to the wealth of information available concerning the ultrastructure of oral taste buds, little comparable data exists for taste buds located at the entrance to the larynx. Therefore, the present study was designed to investigate the fine structure of taste buds located on the lamb epiglottis. MATERIALS: Stained thick and semi-serial thin sections from taste buds located on the lamb epiglottis were examined with light and electron microscopy. RESULTS: Based on morphological criteria, three types of cells could be identified in the taste bud: Type I, Type II, and basal cells. Both Type I and Type II cells extended into the apical taste pore, but there were differences between these two cell types with regard to nuclear profiles, electron density, and the relative density of ribosomes, apical mitochondria, and rough and smooth endoplasmic reticulum. Basal cells did not extend a process into the taste pore. Nerve processes were observed throughout the taste bud. Synapses were observed between both Type I and Type II cells and nerve fibers. These synapses exhibited membrane thickenings and accumulations of clear and dense-cored vesicles of varying proportions in the taste cell cytoplasm adjacent to membrane specializations. CONCLUSIONS: The taste buds located on the lamb epiglottis share several structural similarities to taste buds located in the oral cavity and other regions of the pharynx and larynx of many mammalian species. The presence of synapses on both Type I and Type II cells of the lamb epiglottal taste bud suggests that both cell types are involved in laryngeal chemoreception.

Response characteristics of lamb pontine neurons to stimulation of the oral cavity and epiglottis with different sensory modalities.

1. To better understand sensory information processing in pontine neurons that receive afferent fiber terminations from oral cavity and upper airway receptors, we investigated the response characteristics of single neurons to stimulation of the oral cavity and epiglottis with different stimulus modalities. These response characteristics were then compared with previously recorded response properties of neurons located in other brain stem regions that receive oral cavity and upper airway sensory inputs. 2. Receptive field sizes of pontine neurons were mapped, and responses to mechanical, thermal, and chemical stimuli were determined. A total of 47 neurons were isolated and most neurons were located near the dorsomedial border of the rostral trigeminal subnucleus oralis and caudal principal trigeminal nucleus. The likelihood that a particular stimulus modality would elicit a response was somewhat dependent on a neuron's location. Neurons that responded to chemical stimuli were always located outside the trigeminal nucleus, whereas neurons that responded exclusively to mechanical or thermal stimuli were more frequently located in the trigeminal nucleus. Receptive fields were mapped for 45 of the 47 neurons. Forty-three of the neurons had a single ipsilateral receptive field and > 80% of the receptive fields were > 100 mm2. The majority of neurons responded to only one of the three stimulus modalities. The remaining neurons were multimodal and the combination of stimulus modalities most frequently observed was mechanical and chemical. 3. Mechanical stimuli were the most effective of the three stimulus modalities, eliciting responses in > 65% of the neurons. Neurons that responded to mechanical stimuli were generally rapidly adapting and a moving stimulus was more effective than a punctate stimulus. Mechanosensitive neurons that also responded to chemical stimuli exhibited larger mean response frequencies than mechanosensitive neurons that did not respond to chemical stimuli. Chemical stimuli elicited responses in about half the neurons. A greater percentage of neurons with receptive fields on the epiglottis than neurons with oral cavity receptive fields responded to chemical stimuli. The effectiveness of a chemical stimulus was dependent on a neuron's receptive field. NH4Cl was the most effective stimulus for neurons with receptive fields located in the oral cavity, whereas KCl was more effective for neurons with receptive fields on the epiglottis. Thermal stimuli were relatively ineffective whatever the location of a neuron's receptive field. The majority of neurons showed an increase in response frequency to cooling the receptive field and in all thermosensitive neurons the response was restricted to the dynamic phase of thermal stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Separation of neuron types in the gustatory zone of the nucleus tractus solitarii on the basis of intrinsic firing properties.

1. Whole-cell current-clamp recordings were made from neurons in the rostral nucleus tractus solitarii (NTS) in an in vitro brain slice preparation in rats. On the basis of previous investigations, these neurons are believed to be involved with processing of gustatory as well as somatosensory information. 2. Rostral NTS neurons had a mean resting membrane potential of -47 mV. The mean input resistance was 336 M omega, and by fitting a double exponential function the membrane time constant had fast (2.3 ms) and slow (20.6 ms) components. 3. Neurons were separated into four different groups on the basis of their responses to a current injection pulse paradigm consisting of membrane hyperpolarization of different magnitudes and durations immediately followed by a long (1.200 ms) depolarizing pulse. The regular repetitive discharge pattern of the first group of neurons (Group I neurons) was changed into an irregular spike train by membrane hyperpolarization. Hyperpolarization of Group II neurons either delayed the occurrence of the first action potential or increased the length of the first interspike interval in the action-potential train produced by membrane depolarization. The length of the delay was related both to the magnitude and duration of the hyperpolarizing prepulse. Hyperpolarization had the least effect on the discharge pattern of Group III neurons. The discharge pattern of Group IV neurons consisted of a short burst of action potentials that was often shortened by prior hyperpolarization of the neuron. 4. Differences exist in other intrinsic properties of the four neuron groups. Group I and III neurons were capable of initiating the highest frequency of action potentials to a 100-pA 1,200-ms depolarizing pulse. In response to a short depolarizing pulse. Group II neurons had the longest latency to the first spike and responded with the fewest action potentials. Group IV neurons tended to have higher input resistance and membrane time constants than the other neuron groups. A subset of neurons in each neuron group showed membrane afterhyperpolarizations (AHP) after depolarization-induced action-potential trains (postburst AHP). Postburst AHP amplitudes ranged from 1.0 to 12.9 mV and were of greatest magnitude in Group II neurons. Postburst AHP durations ranged from 75 to 3,538 ms and were of longest duration in neurons belonging to Group III. Group II neurons, which had the largest postburst AHP magnitude, had the shortest postburst AHP duration. 5. These results demonstrate that neurons in the rostral NTS can be separated on the basis of their intrinsic membrane properties.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro intracellular recordings from gustatory neurons in the rat solitary nucleus.

The passive membrane properties of neurons in the gustatory zone of the nucleus tractus solitarius (NTS) of rats were studied using an in vitro brain slice preparation. Examination of responses evoked by a 0.5 nA, 100 ms depolarizing pulse suggests that at least two different types of neurons exist in the gustatory NTS: one responding with a low and the other with a high frequency of action potentials. These two neuron groups based on membrane properties might relate to various gustatory cell types recently categorized by morphological characteristics.

Response characteristics of lamb trigeminal neurons to stimulation of the oral cavity and epiglottis with different sensory modalities.

A region of the trigeminal complex located at the border of the subnucleus interpolaris and subnucleus caudalis receives not only trigeminal nerve inputs from the face, tongue and palate, but also afferent terminations from other nerves which innervate the oral cavity and upper airway. To increase our understanding of the types of sensory information relayed to this region of the trigeminal nucleus, we investigated the response characteristics of single neurons to stimulation of the tongue, palate and epiglottis. Receptive field size and location of 83 trigeminal neurons were mapped, and responses to mechanical, thermal and chemical stimuli were recorded. About 90% of the neurons had one receptive field and no convergence between the oral cavity and epiglottis was observed. Furthermore, only about 15% of the trigeminal neurons responded to more than one stimulus modality. A moving mechanical stimulus elicited responses in over 90% of the cells, and 84% responded to moving and punctate mechanical stimuli. These mechanosensitive neurons generally exhibited rapidly adapting responses. Thermal and chemical stimuli were relatively ineffective. Cooling a receptor surface most often produced excitation, and warming inhibition. Responses to chemical stimuli were only observed for salts at high concentrations. These results suggest that, like oral cavity information relayed by the trigeminal nerve, afferent terminations in the trigeminal nucleus from other nerves subserving the oral cavity and upper airway function to relay mechanical sensory information.(ABSTRACT TRUNCATED AT 250 WORDS)

Responses of neurons in the lamb nucleus tractus solitarius to stimulation of the caudal oral cavity and epiglottis with different stimulus modalities.

Receptors located in the posterior oral cavity and on the epiglottis play an important role in the initiation of upper airway reflexes such as swallowing, gagging, coughing and apnea. Peripheral nerves which innervate these receptor areas terminate in the nucleus tractus solitarius (NTS). We have recorded the responses of 61 neurons in the lamb NTS to stimulation of the caudal tongue, palate and epiglottis with mechanical, chemical and thermal stimuli and mapped receptive field location. Although there was some overlap in the areas of the NTS from which neurons with oral cavity and epiglottal receptive fields could be recorded, a significant difference was observed in the mean recording sites of the two groups of neurons. Neurons with oral cavity receptive fields were located more rostral, lateral and ventral in the NTS than neurons with receptive fields on the epiglottis. Little convergence of sensory input onto single cells in the NTS was observed between the oral cavity and the epiglottis. Only one NTS neuron had a receptive field in both of these receptor areas. In contrast, a large number of neurons with oral cavity receptive fields received input from two receptor areas. These neurons had a receptive field on the tongue which was located directly beneath the receptive field on the palate. Mechanical stimuli were the most effective for neurons with either oral cavity or epiglottal receptive fields and thermal stimuli were the least effective. Neurons which responded to mechanical stimuli responded better to a moving stimulus than to a punctate one, and large increases in the strength of a punctate stimulus were required to elicit significant increases in response frequency. Most NTS neurons responded to more than one of the stimulus modalities. However, a significant difference in the mean number of stimulus modalities which elicited responses was observed between neurons with oral cavity and epiglottal receptive fields. The number of multimodal neurons with epiglottal receptive fields was higher than those with oral cavity receptive fields. The multimodal nature of neurons which responded to epiglottal or oral cavity stimulation combined with their location in reflexogenic areas of the NTS suggests that these neurons could be important in the integration of afferent input from the oral cavity and upper airway. If these NTS neurons are involved in the control of oral and upper airway reflexes it would be important for them to respond to as many of the stimulus cues as possible and the majority of these neurons do just that.

Responses of lamb nucleus of the solitary tract neurons to chemical stimulation of the epiglottis.

Previous research has shown that applications of chemical stimuli to the epiglottis produced distinct patterns of activity in the lamb superior laryngeal nerve. To determine the response characteristics of second-order neurons, we recorded from single cells in the lamb nucleus of the solitary tract (NST) while stimulating the epiglottis with 0.5 M KCl, NH4Cl, NaCl, LiCl, distilled water, 0.005 M citric acid and 0.01 N HCl. Most neurons responded to more than one of the chemical solutions. The order of effective stimuli was KCl = NH4Cl greater than distilled water greater than HCl greater than citric acid greater than NaCl greater than LiCl. An analysis of the variation in response frequency over time found that different chemical stimuli produced significantly different response patterns in NST neurons. A comparison of the mean neural response profiles of NST neurons and superior laryngeal nerve fibers for each of the stimuli found that only the response profiles elicited by NH4Cl were significantly different. In addition to their responses to chemical solutions, almost one-third of the NST neurons responded to the rinse following application of at least some of the stimuli and 80% of the neurons were excited by mechanical stimulation of the epiglottis with a soft brush. Also, a small number of neurons exhibited a rhythmic response coordinated with respiration. The majority of recording sites were located in areas of the NST linked to swallowing and respiration suggesting that the response patterns of NST neurons elicited by chemical stimulation of receptors on the epiglottis may play a role in upper airway reflexes.

Convergence onto hamster medullary taste neurons.

Research has shown that gustatory afferents innervating different areas of the oral cavity converge onto single neurons in the nucleus tractus solitarii (NTS). However, most studies of gustatory physiology have only stimulated the receptors on the anterior tongue. No information exists on the responses of hamster NTS neurons to stimulation of receptors located in other areas of the oral cavity. The present investigation compared responses of hamster NTS neurons to stimulation of receptors on the anterior tongue and posterior oral cavity, and to stimulation of both receptor populations together. Of the neurons, 64% responded to both anterior tongue and posterior oral cavity stimulation. The remaining neurons responded exclusively to stimulation of one area. Cells responsive to both fields of stimulation were found throughout the rostral NTS. Cells responding to stimulation of only one field were anatomically separate. Most neurons (69%) were more responsive to anterior tongue than posterior oral cavity stimulation. The neural responses to stimulation of both fields simultaneously were complex. Frequently, a cell's response was intermediate between those produced by stimulation of either receptor population alone. In other cases the response was the same as the larger of the two individual responses. The breadth of responsiveness to the 4 basic taste stimuli (sucrose, NaCl, HCl and quinine-HCl) was similar for both receptor populations, but the breadth of tuning of an individual cell for one field of stimulation was not correlated with its breadth of responsiveness for the other. In contrast, the breadth of tuning following stimulation of the entire oral cavity was correlated with that following stimulation of the anterior tongue.

Central connections of the lingual-tonsillar branch of the glossopharyngeal nerve and the superior laryngeal nerve in lamb.

Afferent and efferent central connections of the lingual-tonsillar branch of the glossopharyngeal nerve (LT-IX) and the superior laryngeal nerve (SLN) in the lamb were traced with horseradish peroxidase (HRP) histochemistry. After entering the brainstem, most LT-IX and SLN afferent fibers turned caudally in the solitary tract (ST). Some afferent fibers of LT-IX terminated in the medial nucleus of the solitary tract slightly caudal to their level of entry. The remaining fibers projected to the dorsolateral, ventrolateral, and interstitial areas of the nucleus of the solitary tract (NST) at the level of the area postrema. Superior laryngeal nerve afferent fibers terminated extensively in the medial and ventral NST at levels near the rostral pole of the area postrema. Further caudal, near the level of obex, SLN afferent terminations were concentrated in the region ventrolateral to the ST and in the interstitial NST. The caudal extent of LT-IX and the rostral extent of SLN terminals projected to similar levels of the NST, but only a relatively small proportion of the total projections overlapped. Lingual-tonsillar and SLN fibers also coursed rostrally to terminate in the caudal pons within and medial to the dorsomedial principal sensory trigeminal nucleus. Other labeled afferent fibers traveled caudally in the dorsal spinal trigeminal tract to terminate in the dorsal two-thirds of the spinal trigeminal nucleus at the level of obex. Large numbers of labeled cells with fibers in the LT-IX or SLN were located in the ipsilateral rostral nucleus ambiguus and surrounding reticular formation. Fewer labeled cells were observed in the inferior salivatory nucleus following HRP application to either the LT-IX or SLN. The LT-IX and SLN projections to areas of the NST associated with upper airway functions, like swallowing and respiration, suggest an important role for these two nerves in the initiation and control of airway reflexes.