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1Department of Biologic and Materials Sciences, School of Dentistry and 2Department of Molecular and Integrative Physiology, Medical School, University of Michigan, Ann Arbor, Michigan
Submitted 19 March 2004; accepted in final form 9 September 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Satomi et al. 1979). Parasympathetic fibers in the glossopharyngeal nerve also travel to the intralingual ganglia (Remak’s ganglia) in the posterior tongue from which postganglionic fibers innervate the von Ebner glands that drain into the clefts of the circumvallate and foliate papillae (Bradley et al. 1985).
The ISN lies principally along the medial edge of the nucleus of the solitary tract (NST) before extending into the ventral reticular formation at its rostral extent (Contreras et al. 1980). This configuration suggests potential interactions between the ISN and NST. However, although the general location of the ISN has been described including brief morphological descriptions of the neurons (Contreras et al. 1980; Satomi et al. 1979), there are no detailed neurophysiological descriptions of ISN neurons and until recently no morphological descriptions (Kim et al. 2004). Moreover, the electrophysiological and morphological characteristics of the ISN neurons have not been related to the glands that they innervate. The purpose of this study was to record from identified neurons in the ISN that innervate the parotid and von Ebner salivary glands, and describe the morphology of the neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Twelve-day-old male or female Sprague-Dawley rats were anesthetized with a 6% solution of halothane mixed with air (400–600 ml/min). During surgery, the depth of anesthesia was monitored by the foot withdrawal reflex. All surgical procedures were carried out under National Institutes of Health and University of Michigan Animal Care and Use Committee approved protocols.
The fluorescent retrograde tracer, Alexofluor 568 dextran (Molecular Probes) was used to label ISN neurons innervating either the von Ebner or parotid salivary glands. To label ISN neurons innervating the von Ebner glands, the lingual-tonsillar branch of the glossopharyngeal nerve was exposed by a ventral approach and cut. Crystals of the tracer were applied to the cut central end of the nerve and isolated from surrounding tissue by application of a fast setting silicone sealer (Kwik-sil, World Precision Instruments). To label ISN neurons innervating the parotid gland, the otic ganglion was exposed on the medial aspect of the mandibular division of the trigeminal nerve and crystals of the tracer placed on the ganglion and isolated with silicone. The tissues were then reapposed and the skin sutured. The rats recovered in an isolated cage on a heating pad and when ambulatory were returned to their mother’s home cage.
Brain slice preparation
After a suitable time for retrograde transport (2–4 days), the rats were reanesthetized with halothane and decapitated, and the brain was quickly removed and cooled for 6 min in oxygenated physiological saline in which NaCl was replaced with isosmotic sucrose at 4°C (Aghajanian and Rasmussen 1989). After cooling, the brain was transected at the level of the pons and just below the obex, and the cerebellum was removed. The brain stem was then secured to a Vibratome stage with cyanoacrylate glue and 300-µm-thick horizontal slices cut and transferred to standard physiological saline containing (in mM) 124 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 26 NaHCO3, 1.25 KH2PO4, and 25 glucose, gassed with 95% O2-5% CO2 to give a pH of 7.3. Slices were incubated for 
1 h in the oxygenated physiological saline at room temperature before being transferred to a recording chamber with a wide-mouthed pipette. Physiological saline at room temperature was flowed at a rate of 1.5 ml/min across the chamber and aspirated at the opposite side. The submerged slice was stabilized by a series of spaced nylon fibers on a wire frame.
Neurophysiology
Patch electrodes were pulled from 1.5 mm OD borosilicate filament glass (WPI, TW150F-4) in two stages on an electrode puller (Narashige PP 83). The pipettes were filled with a solution containing (in mM) 130 K gluconate, 10 HEPES, 10 EGTA, 1 CaCl2, 1 MgCl2, and 2 ATP, adjusted to a pH 7.2 with KOH. Lucifer yellow (0.1%, Sigma) was also included in the filling solution for intracellular labeling of the neuron. Filled pipettes had a tip resistance 3–6 M
Retrogradely labeled ISN neurons were identified visually under fluorescence illumination using a Nikon E600-FS fixed stage microscope. A x40 water-immersion objective lens was used to identify and approach a neuron. Once a labeled neuron was identified, it was visualized using infrared-differential interface contrast (IR-DIC) optics via a CCD camera (DAGE IR-1000) (Dodt 1993).
Whole cell recording were performed using an Axoclamp 2B amplifier (Axon Instruments) in bridge mode. When a giga- seal was formed and a patch ruptured, current stimulation protocols were performed and voltage data were acquired using pCLAMP software (Axon Instruments). After recording, the identity of the neuron was confirmed by fluorescence illumination.
Electrophysiological data were analyzed using the Clampfit program (Axon Instruments). Biophysical properties (input resistance, membrane time constants, action potential amplitude, and half-width) were determined by delivering a series of 100-ms hyperpolarizing and depolarizing current pulses (-100 to 75 pA). The junctional potential due to potassium gluconate (10 mV) was subtracted from the membrane potential values (Standen and Stanfield 1992).
We employed an intracellular current injection protocols consisting of membrane hyperpolarization followed by depolarization to separate the ISN neurons into different groups based on their repetitive firing characteristics. A number of other investigators have used this protocol to define different neuron groups based on biophysical characteristics (Bradley and Sweazey 1992; Champagnat et al. 1986; Dekin and Getting 1987; Dekin et al. 1987; Manis 1990; Yarom et al. 1985).
Neuron reconstruction
After completion of recording and cellular labeling, slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and stored for 24 h. Slices were then rinsed in this buffer for 30 min, embedded in agar (4% in distilled water) and cut into 100-µm-thick sections on a Vibratome. The sections were mounted on slides, dried overnight, dehydrated by graded alcohols, cleared by xylene and put on coverslips.
Lucifer-yellow-filled neurons were visualized with Bio-Rad MRC-600 laser scanning confocal microscope. Stacked serial, 1-µm optical confocal images were obtained. Morphometric reconstruction of stacked images was performed using the Neurolucida program (MicroBrightfield). For each neuron, morphometric measures included soma area, soma form factor (4
x area/perimeter2; resulting values vary between 0 and 1, where 1 indicates a circular profile and values close to 0 indicate a fusiform shape), total number of primary dendrites, total number of dendritic segments, and total dendritic length. Only one neuron per slice was recorded and 67 neurons were satisfactorily filled to allow characterization and reconstruction.
Data analysis
All analysis was conducted using the SPSS statistical package. Comparisons of different neuron groups were analyzed with one-way ANOVA with Tukey post hoc tests, Student’s paired t-test or 
2 test. Correlations were investigated by Pearson’s correlation coefficient and the line fitted by linear regression. Data are presented as means ± SE, and statistical significance was reached at P 
0.05.
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RESULTS |
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100 rat pups. Whole cell recordings were made from over 140 neurons in the ISN. Of these neurons 138 were selected because they had a stable resting membrane potential more negative than –40 mV, a spike overshoot of 20 mV, and an input resistance >100 M. Biophysical properties of ISN neurons
BIOPHYSICAL PROPERTIES OF ISN NEURONS INNERVATING THE PAROTID SALIVARY GLAND.
Whole cell recordings were made from 55 labeled neurons. Resting membrane potentials ranged from –41 to –73 mV with a mean of –58 ± 1.0 mV (Table 1). Input resistance ranged from 104 to 807 M with a mean of 373 ± 23.4 M. Time constants were determined from the time course of the voltage response to a small hyperpolarizing current step that lay within the linear portion of the current-voltage relationship, and the mean value was 30 ± 1.5 ms. Action potential amplitude ranged from 76 to 120 mV with a mean of 95 ± 1.2 mV, and spike half-width was 1.6 ± 0.1 ms.
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BIOPHYSICAL PROPERTIES OF ISN NEURON INNERVATING THE VON EBNER SALIVARY GLANDS.
Whole cell recordings were made from 83 labeled neurons, and data are summarized in Table 2. Resting membrane potentials ranged from –40 to –73 mV with a mean of –56 ± 0.7 mV. Input resistance ranged from 275 to 928 M with a mean of 608 ± 15 M, and membrane time constant was 46 ± 1.6 ms. Action potential amplitude ranged from 68 to 117 mV with a mean of 93 ± 1.1 mV. The mean spike half-width was 2.4 ± 0.15 ms.
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2 test: P < 0.05). Comparison of the morphological and neurophysiological properties of ISN neurons
Reconstructions were made from 37 parotid and 30 von Ebner gland neurons that were completely filled with Lucifer yellow. Parotid gland neurons had a mean soma area of 207.4 ± 9.6 µm2, a form factor of 0.69 ± 0.08, and total dendritic length of 660.1 ± 60.5 µm. On average, they had 4.3 ± 0.23 primary dendrites and 9.2 ± 0.56 dendritic segments. Von Ebner gland neurons had a mean soma area of 129.1 ± 7.4 µm2, a form factor of 0.73 ± 0.09, and total dendritic length of 498 ± 39.1 µm. On average, they had 3.7 ± 0.15 primary dendrites and 5.9 ± 0.3 dendritic segment. With the exception of the form factor measurement, neurons innervating the parotid salivary glands had significantly larger soma and more and longer dendrites than von Ebner gland neurons (Student’s t-test: P < 0.05; Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; McCormick and Prince 1987) because the recordings were made from relatively young animals.
A surprising finding was the size difference between neurons innervating the parotid and von Ebner salivary glands. ISN neurons innervating the parotid gland were significantly larger than those innervating the von Ebner salivary glands. This size difference is accompanied by differences in membrane properties. The mean input resistance, time constant, and spike half-width of parotid gland neurons was significantly lower than in von Ebner gland neurons. It is possible that these morphological and electrophysiological dissimilarities represent differences in function. Although it is true that activation of the ISN neurons innervating the parotid and von Ebner glands results in secretion of saliva, according to the size principle the smaller cells are more excitable (Clamann and Henneman 1976; Henneman et al. 1965; Luscher et al. 1979). Neurons with smaller cell bodies have a lower threshold for synaptic activation and will therefore respond to weaker synaptic input. Thus ISN neurons innervating the von Ebner salivary glands would potentially be activated with less afferent input than the ISN neurons innervating the parotid gland. Because the von Ebner glands are intimately involved in the delivery and removal of taste stimuli from the clefts of the circumvallate and foliate papillae, rapid reflex coordination between sensory input and secretion would be important in the function of taste receptors situated deeply in the clefts of the papillae. In contrast, parotid glands contribute saliva to the entire mouth and coordination between salivary secretion and afferent stimulation is less critical.
A further difference between neurons controlling the parotid and von Ebner salivary glands is the proportion of neurons that express anomalous rectification. More than 50% of the von Ebner neurons were characterized by having anomalous rectification as compared with less than 15% of neurons innervating the parotid glands. This membrane property results from a cation conductance that is activated by membrane hyperpolarization and serves a number of functions including pacemaker activity (Pape 1996). Thus it is possible that the resting secretion of saliva that occurs in the absence of any apparent sensory input (Emmelin 1972) could result from oscillatory activity of the ISN neurons that transmit a low level of efferent activity to the glands. A basal level of secretion provided by the von Ebner gland into the clefts of the circumvallate and foliate papillae would be especially important in preventing build up of food debris in the clefts and could account for the larger percentage of neurons innervating these glands that have anomalous rectification. Unfortunately there is no evidence from in vivo studies that these neurons have any oscillatory activity and these neurons are not spontaneously active in the brain slices.
Parasympathetic neurons in the superior salivatory nucleus have repetitive discharge patterns that are similar to those in the ISN (Matsuo and Kang 1998). Most neurons were of the delayed excitation pattern and the rest of the long first ISI pattern. By use of specific blockers under voltage clamp, the current responsible for this pattern was shown to be the fast transient outward potassium current, IA. Most parasympathetic neurons in the dorsal motor nucleus of the vagus nerve also have IA currents as well, so this appears to be a common characteristic of brain stem parasympathetic neurons (Browning et al. 1999).
The parasympathetic cell column is closely associated with the NST, which is the termination site of the sensory input conveyed in the facial, glossopharyngeal, and vagus nerves. Examination of the repetitive discharge characteristics of both the rostral and caudal NST neurons has shown that neurons of the NST have similar response patterns to neurons of the parasympathetic cell column. NST neurons have been characterized as having delayed excitation, long first ISI, tonic, and burst patterns as well as expressing IA (Bradley and Sweazey 1992; Dekin and Getting 1987; Dekin et al. 1987; Tell and Bradley 1994).
Despite the fact that similar repetitive discharge patterns have been reported in different brain stem areas, the hypothesized functional role of these discharge patterns differs between each of these areas. It is sometimes possible to correlate the patterns defined in vitro to patterns of spike activity recorded extracellularly in vivo. In systems such as the dorsal cochlear nucleus in which in vitro patterns of activity have been documented, the temporal patterns observed in brain slices have similarities to the in vivo patterns (Manis 1990). Unfortunately the response patterns of ISN neurons observed in vitro cannot be compared with patterns recorded in vivo because the only examples of in vivo recordings from ISN neurons result from experiments designed to measure response latency using electrical stimulation of presumed afferent input (Ishizuka and Murakami 1986). Therefore it is difficult to speculate whether these discharge patterns represent patterns that would occur during normal brain stem activity. Moreover, it is also possible by using different temporal patterns of current injection, to demonstrate several repetitive discharge patterns in the same neuron as demonstrated for dorsal cochlear nucleus neurons (Manis 1990).
Efferent output initiated by neurons of the ISN leads to secretion of saliva. Moreover, secretion of saliva is generally elicited only in response to stimulation of the innervation of the glands (Emmelin 1972; Schneyer et al. 1972). Thus afferent input to ISN neurons is required for the initiation and maintenance of secretion. Two types of peripheral stimuli have been shown to be important in initiating secretion. Input originating from stimulation of taste and mechanoreceptors results in the secretion of saliva (Anderson et al. 1985; Kawamura and Yamamoto 1978). In addition the magnitude of the afferent stimulus modulates salivary flow rate (Kawamura and Yamamoto 1978). Recordings from the efferent supply to the submandibular gland reveal a linear relationship between impulse frequency and concentration of taste stimuli (Yamamoto and Kawamura 1977).
Details of the brain stem circuits responsible for the reflex secretion of saliva are unclear. Anatomical evidence suggests that there is no monosynaptic input between the afferent fibers and ISN neurons (Whitehead and Frank 1983). Thus afferent fibers first synapse with NST neurons, which then synapse with the ISN neurons directly or via other interneurons in the NST. The afferent input to the NST is excitatory, but inhibitory synaptic activity also occurs within the NST (Bradley and Grabauskas 1998; Li and Smith 1997). Combinations of excitation and inhibition would alter the pattern of response in the ISN neurons as demonstrated by the hyperpolarizing/depolarizing current injection protocols. Thus in some circumstances, the output would be of the tonic pattern, whereas in other circumstances, the output would resemble the delayed excitation pattern of discharge. This could account for the different salivary flow rates resulting from sour (acid) stimulation of the tongue when compared with the low flow rates resulting from sweet stimuli (Kawamura and Yamamoto 1978). Because primary afferent taste fibers are grouped into fibers responding best to the different taste modalities (Frank et al. 1983), these fibers conceivably make different synaptic connections to the ISN neurons to control modality specific flow rates.
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GRANTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. M. Bradley, Dept. of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078 (E-mail: rmbrad{at}umich.edu)
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REFERENCES |
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