Quantifying gene network connectivity in silico: scalability and accuracy of a modular approach.

Large, complex data sets that are generated from microarray experiments, create a need for systematic analysis techniques to unravel the underlying connectivity of gene regulatory networks. A modular approach, previously proposed by Kholodenko and co-workers, helps to scale down the network complexity into more computationally manageable entities called modules. A functional module includes a gene's mRNA, promoter and resulting products, thus encompassing a large set of interacting states. The essential elements of this approach are described in detail for a three-gene model network and later extended to a ten-gene model network, demonstrating scalability. The network architecture is identified by analysing in silico steady-state changes in the activities of only the module outputs, communicating intermediates, that result from specific perturbations applied to the network modules one at a time. These steady-state changes form the system response matrix, which is used to compute the network connectivity or network interaction map. By employing a known biochemical network, the accuracy of the modular approach and its sensitivity to key assumptions are evaluated.

Scattered-light imaging in vivo tracks fast and slow processes of neurophysiological activation.

We imaged fast optical changes associated with evoked neural activation in the dorsal brainstem of anesthetized rats, using a novel imaging device. The imager consisted of a gradient-index (GRIN) lens, a microscope objective, and a miniature charged-coupled device (CCD) video camera. We placed the probe in contact with tissue above cardiorespiratory areas of the nucleus of the solitary tract and illuminated the tissue with 780-nm light through flexible fibers around the probe perimeter. The focus depth was adjusted by moving the camera and microscope objective relative to the fixed GRIN lens. Back-scattered light images were relayed through the GRIN lens to the CCD camera. Video frames were digitized at 100 frames per second, along with tracheal pressure, arterial blood pressure, and electrocardiogram signals recorded at 1 kHz per channel. A macroelectrode placed under the GRIN lens recorded field potentials from the imaged area. Aortic, vagal, and superior laryngeal nerves were dissected free of surrounding tissue within the neck. Separate shocks to each dissected nerve elicited evoked electrical responses and caused localized optical activity patterns. The optical response was modeled by four distinct temporal components corresponding to putative physical mechanisms underlying scattered light changes. Region-of-interest analysis revealed image areas which were dominated by one or more of the different time-course components, some of which were also optimally recorded at different tissue depths. Two slow optical components appear to correspond to hemodynamic responses to metabolic demand associated with activation. Two fast optical components paralleled electrical evoked responses.

Analysis and neuronal modeling of the nonlinear characteristics of a local cardiac reflex in the rat.

Previous experimental results have suggested the existence of a local cardiac reflex in the rat. In this study, the putative role of such a local reflex in cardiovascular regulation is quantitatively analyzed. A model for the local reflex is developed from anatomical experimental results and physiological data in the literature. Using this model, a systems-level analysis is conducted. Simulation results indicate that the neuromodulatory mechanism of the local reflex attenuates the nonlinearity of the relationship between cardiac vagal drive and arterial pressure. This behavior is characterized through coherence analysis. Furthermore, the modulation of phase-related characteristics of the cardiovascular system is suggested as a plausible mechanism for the nonlinear attenuation. Based on these results, it is plausible that the functional role of the local reflex is highly robust nonlinear compensation at the heart, which results in less complex dynamics in other parts of the reflex.

Response properties of baroreceptive NTS neurons.

Neurons in the nucleus of the solitary tract (NTS) responding to activation of arterial baroreceptors were recorded intracellularly using patch pipettes in an in situ arterially perfused working heart-brain stem preparation of rat. Seven of 15 (i.e., 46%) of NTS neurons showed adaptive (nonlinear) excitatory synaptic response patterns during baroreceptor stimulation followed by an "evoked hyperpolarization." This evoked hyperpolarization was stimulus intensity dependent and capable of shunting out a subsequent baroreceptor input. We suggest that this adaptive response behavior may be mediated, in part, by calcium-dependent potassium currents (IKCa) since neurons showed spike frequency adaptation during step depolarizations and an after-hyperpolarization after repetitive firing. Furthermore, in in vivo anesthetized rats, NTS microinjections of either charybdotoxin (225 fmol) or apamin (4.5 pmol) to block IKCa increased the baroreceptor reflex gain. Our data purport that the responsiveness of baroreceptive NTS neurons can be regulated by intrinsic membrane conductances such as IKCa. Modulation of such conductances during either physiological (exercise) or pathophysiological (essential hypertension) conditions may lead to changes in both the operating point and gain of the baroreceptor reflex.

Information theoretic analysis of pulmonary stretch receptor spike trains.

Primary afferent neurons transduce physical, continuous stimuli into discrete spike trains. Investigators have long been interested in interpreting the meaning of the number or pattern of action potentials in attempts to decode the spike train back into stimulus parameters. Pulmonary stretch receptors (PSRs) are visceral mechanoreceptors that respond to deformation of the lungs and pulmonary tree. They provide the brain stem with feedback that is used by cardiorespiratory control circuits. In anesthetized, paralyzed, artificially ventilated rabbits, we recorded the action potential trains of individual PSRs while continuously manipulating ventilator rate and volume. We describe an information theoretic-based analytical method for evaluating continuous stimulus and spike train data that is of general applicability to any continuous, dynamic system. After adjusting spike times for conduction velocity, we used a sliding window to discretize the stimulus (average tracheal pressure) and response (number of spikes), and constructed co-occurrence matrices. We systematically varied the number of categories into which the stimulus and response were evenly divided at 26 different sliding window widths (5, 10, 20, 30,..., 230, 240, 250 ms). Using the probability distributions defined by the co-occurrence matrices, we estimated associated stimulus, response, joint, and conditional entropies, from which we calculated information transmitted as a fraction of the maximum possible, as well as encoding and decoding efficiencies. We found that, in general, information increases rapidly as the sliding window width increases from 5 to approximately 50 ms and then saturates as observation time increases. In addition, the information measures suggest that individual PSRs transmit more "when" than "what" type of information about the stimulus, based on the finding that the maximum information at a given window width was obtained when the stimulus was divided into just a few (usually <6) categories. Our results indicate that PSRs provide quite reliable information about tracheal pressure, with each PSR conveying about 31% of the maximum possible information about the dynamic stimulus, given our analytical parameters. When the stimulus and response are divided into more categories, slightly less information is transmitted, and this quantity also saturates as a function of observation time. We consider and discuss the importance of information contained in window widths on the time scales of an excitatory postsynaptic potential and Hering-Breuer reflex central delay.

Fas-mediated apoptosis eliminates B cells that acquire self-reactivity during the germinal center response to NP.

C57Bl/6 mice with the lpr mutation of Fas (CD95) were tested for deviation from the genetically restricted antibody response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). lambda1+ germinal centers (GC) with the canonical v186.2 V(H) gene element develop in lpr/lpr mice with the same time course as in wild-type (+/+) mice. In contrast to +/+ mice, however, lambda1+ GC persist in the spleens of lpr/lpr mice 25 days after immunization. Virtually all of the lambda1+ GC are reactive with NP 10 days after immunization. Sixteen days after immunization, however, many of the lambda1+ GC are not reactive with NP, and few of the lambda1+ GC are reactive with NP 25 days after immunization. The V(H) gene elements of three lambda1+NP- GC 25 days after immunization are derived by somatic mutation of v186.2, but have lost reactivity with NP. The mutated VDJs from these GC react with cells in spleen sections from +/+ and lpr/lpr mice, indicating that they represented secondary antibody responses induced by self antigens that are available as presented antigen. These data indicate that Fas-mediated apoptosis serves to eliminate a (limited) population of B cells that acquire reactivity to "self antigens" by somatic mutation of VDJs in the GC.

Engineering a living cell to desired metabolite concentrations and fluxes: pathways with multifunctional enzymes.

With molecular genetics enabling modulation of the concentrations of cellular enzymes, metabolic engineering becomes limited by the question of which modulations of the enzyme concentrations are required to bring about a desired pattern of cellular metabolism. In an earlier paper (Kholodenko et al. (1998). Biotechnol. Bioeng. 59, 239-247) we derived a method to determine the required modulations. This method, however, cannot be immediately applied to cellular pathways with enzymes catalyzing more than one step in metabolism (multifunctional enzymes). In the present paper we show to which extent the presence of multifunctional enzymes limits biotechological ambitions, which one might otherwise pursue in vain. In particular, it is impossible to change the concentration of a single intermediate and leave the rest of metabolism unperturbed if that intermediate interacts directly with a multifunctional enzyme. The analytical machinery of Metabolic Control Analysis is used to relate the desired and ensuing changes in the metabolic pattern. An explicit solution to this problem of engineering metabolism is then given in the form of a single matrix equation.

Computational modeling of the baroreflex arc: nucleus tractus solitarius.

In this study, we examine the utility of computational modeling in understanding nervous system function. We start by examining the reasons for, and major approaches to, computational modeling. We then chose a modeling approach and applied different variations to understanding nucleus tractus solitarius (NTS) neuronal responses to various baroreceptive stimuli. We examine the results in light of our objectives and with regard to the known parameters of the system under investigation. Our results demonstrate that modeling can be a useful tool in analysis of (and examination of underlying mechanisms for) NTS behavior on many levels.

BTK mutations in patients with X-linked agammaglobulinemia: lack of correlation between presence of peripheral B lymphocytes and specific mutations.

X-linked agammaglobulinemia (XLA) is a human antibody deficiency that results from mutation of the tyrosine kinase btk. We tested the hypothesis that XLA patients who varied from the classic phenotype of XLA by presence of normal or near normal number of peripheral B lymphocytes would have a set of mutations of BTK that is different from the mutations found in patients without peripheral B lymphocytes. The mutations of BTK we found in two patients with normal numbers of peripheral B lymphocytes have been previously identified in patients without peripheral B lymphocytes. A third patient, without peripheral B cells, was found to express normal levels of wild type btk. Exmination of the mutations of the BTK gene in patients in the BTKbase who were identified as having peripheral B lymphocytes found that these same mutations, or mutations of the same protein domains, were also present in patients identified as lacking peripheral B lymphocytes. Analysis of mutations in BTK has previously led to the conclusion that severity of disease in XLA cannot be predicted from the specific mutation of BTK. The results of this study suggest that whether an XLA patient will develop peripheral B lymphocytes cannot be predicted from the specific mutation of BTK.

Projections of the dorsal motor nucleus of the vagus to cardiac ganglia of rat atria: an anterograde tracing study.

We injected the anterograde fluorescent tracer 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the dorsal motor nucleus of the vagus (DmnX), counterstained the cardiac ganglia with Fluorogold (FG), and used confocal microscopy to examine the distributions and different types of DmnX fibers in wholemounts of the atria. We also quantified the number of DmnX cardiac axons and the number of innervated cardiac principal neurons (PNs). Rats with unilateral DiI injections were used in three different experiments, including unilateral FG soaking of cervical vagal trunks, intracranially rhizotomizing the vagal afferent roots, or contralaterally sectioning the cervical vagus. These manipulations indicated that DiI-labeled cardiac fibers were exclusively from the DmnX. Our observations established that: (1) three major ganglionic plexuses were localized in the epicardium; (2) both sides of the DmnX supplied significant fibers to each of the plexuses; (3) these cardiac efferents formed dense basket terminals around individual PNs; (4) collaterals of individual DmnX fibers diverged, producing calyx endings on multiple PNs; (5) small intensely fluorescent (SIF) cells in the cardiac plexuses were innervated pericellularly; (6) individual axons could innervate both PNs and SIF cells; (7) the total number of DmnX fibers were in the range of [68, 96; left] and [67, 115; right]; (8) these fibers innervated 709 (left) and 494 (right), or at least 18% and 12%, of the PNs, respectively; and (9) vagal preganglionics exhibited a degree of lateralization: Significantly more PNs were contacted by fiber varicosities in the sinoatrial plexus than in the atrioventricular plexus after right DmnX injections. In summary, the present observations suggest that the DmnX plays a significant role(s) in controlling the heart.

Metabolic design: how to engineer a living cell to desired metabolite concentrations and fluxes.

A biotechnological aim of genetic engineering is to increase the intracellular concentration or secretion of valuable compounds, while making the other concentrations and fluxes optimal for viability and productivity. Efforts to accomplish this based on over-expression of the enzyme, catalyzing the so-called "rate-limiting step," have not been successful. Here we develop a method to determine the enzyme concentrations that are required to achieve such an aim. This method is called Metabolic Design Analysis and is based on the perturbation method and the modular ("top-down") approach-formalisms that were first developed for the analysis of biochemical regulation such as, Metabolic Control Analysis. Contrary to earlier methods, the desired alterations of cellular metabolism need not be small or confined to a single metabolite or flux. The limits to the alterations of fluxes and metabolite concentrations are identified. To employ Metabolic Design Analysis, only limited kinetic information concerning the pathway enzymes is needed.

A neuro-mimetic dynamic scheduling algorithm for control: analysis and applications.

A simple neuronal network model of the baroreceptor reflex is analyzed. From a control perspective, the analysis suggests a dynamic scheduled control mechanisms by which the baroreflex may perform regulation of the blood pressure. The main objectives of this work are to investigate the static and dynamic response characteristics of the single neurons and the network, to analyze the neuromimetic dynamic scheduled control function of the model, and to apply the algorithm to nonlinear process control problems. The dynamic scheduling activity of the network is exploited in two control architectures. Control structure I is drawn directly from the present model of the baroreceptor reflex. An application of this structure for level control in a conical tank is described. Control structure II employs an explicit set point to determine the feedback error. The performance of this control structure is illustrated on a nonlinear continuous stirred tank reactor with van de Vusse kinetics. The two case studies validate the dynamic scheduled control approach for nonlinear process control applications.

Vagal afferent innervation of the atria of the rat heart reconstructed with confocal microscopy.

We have used confocal microscopy to analyze the vagal afferent innervation of the rat heart. Afferents were labeled by injecting 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the nodose ganglia of animals with prior supranodose de-efferentations, autonomic ganglia were stained with Fluoro-gold, and tissues were examined in whole mounts. Distinctively different fiber specializations were observed in the epi-, myo-, and endocardium: Afferents to the epicardium formed complexes associated with cardiac ganglia. These ganglia consisted of four major ganglionated plexuses, two on each atrium, at junctions of the major vessels with the atria. Ganglionic locations and sizes (left > right) were consistent across animals. In addition to principal neurons (PNs), significant numbers of small intensely fluorescent (SIF) cells were located in each of these plexuses, and vagal afferents provided dense pericellular varicose endings around the SIF cells in each ganglionic plexus, with few if any terminations on PNs. In the myocardium, vagal afferents formed close contacts with cardiac muscles, including conduction fibers. In the endocardium, vagal fibers formed "flower-spray" and "end-net" terminals in connective tissue. With three-dimensional reconstruction of confocal optical sections, a novel polymorphism was seen: Some fibers had one or more collaterals ending as endocardial flower sprays and other collaterals ending as myocardial intramuscular endings. Some unipolar or pseudounipolar neurons within each cardiac ganglionic plexus were retrogradely labeled from the nodose ganglia. In conclusion, vagal afferents form a heterogeneity of differentiated endings in the heart, including structured elements which may mediate chemoreceptor function, stretch reception, and local cardiac reflexes.

Modeling neural mechanisms for genesis of respiratory rhythm and pattern. II. Network models of the central respiratory pattern generator.

The present paper describes several models of the central respiratory pattern generator (CRPG) developed employing experimental data and current hypotheses for respiratory rhythmogenesis. Each CRPG model includes a network of respiratory neuron types (e.g., early inspiratory; ramp inspiratory; late inspiratory; decrementing expiratory; postinspiratory; stage II expiratory; stage II constant firing expiratory; preinspiratory) and simplified models of lung and pulmonary stretch receptors (PSR), which provide feedback to the respiratory network. The used models of single respiratory neurons were developed in the Hodgkin-Huxley style as described in the previous paper. The mechanism for termination of inspiration (the inspiratory off-switch) in all models operates via late-I neuron, which is considered to be the inspiratory off-switching neuron. Several two- and three-phase CRPG models have been developed using different accepted hypotheses of the mechanism for termination of expiration. The key elements in the two-phase models are the early-I and dec-E neurons. The expiratory off-switch mechanism in these models is based on the mutual inhibitory connections between early-I and dec-E and adaptive properties of the dec-E neuron. The difference between the two-phase models concerns the mechanism for ramp firing patterns of E2 neurons resulting either from the intrinsic neuronal properties of the E2 neuron or from disinhibition from the adapting dec-E neuron. The key element of the three-phase models is the pre-I neuron, which acts as the expiratory off-switching neuron. The three-phase models differ by the mechanisms used for termination of expiration and for the ramp firing patterns of E2 neurons. Additional CRPG models were developed employing a dual switching neuron that generates two bursts per respiratory cycle to terminate both inspiration and expiration. Although distinctly different each model generates a stable respiratory rhythm and shows physiologically plausible firing patterns of respiratory neurons with and without PSR feedback. Using our models, we analyze the roles of different respiratory neuron types and their interconnections for the respiratory rhythm and pattern generation. We also investigate the possible roles of intrinsic biophysical properties of different respiratory neurons in controlling the duration of respiratory phases and timing of switching between them. We show that intrinsic membrane properties of respiratory neurons are integrated with network properties of the CRPG at three hierarchical levels: at the cellular level to provide the specific firing patterns of respiratory neurons (e.g., ramp firing patterns); at the network level to provide switching between the respiratory phases; and at the systems level to control the duration of inspiration and expiration under different conditions (e.g., lack of PSR feedback).

Modeling neural mechanisms for genesis of respiratory rhythm and pattern. I. Models of respiratory neurons.

The general objectives of our research, presented in this series of papers, were to develop a computational model of the brain stem respiratory neural network and to explore possible neural mechanisms that provide the genesis of respiratory oscillations and the specific firing patterns of respiratory neurons. The present paper describes models of single respiratory neurons that have been used as the elements in our network models of the central respiratory pattern generator presented in subsequent papers. The models of respiratory neurons were developed in the Hodgkin-Huxley style employing both physiological and biophysical data obtained from brain stem neurons in mammals. Two single respiratory neuron models were developed to match the two distinct firing behaviors of respiratory neurons described in vivo: neuron type I shows an adapting firing pattern in response to synaptic excitation, and neuron type II shows a ramp firing pattern during membrane depolarization after a period of synaptic inhibition. We found that a frequency ramp firing pattern can result from intrinsic membrane properties, specifically from the combined influence of calcium-dependent K(AHP)(Ca), low-threshold Ca(T) and K(A) channels. The neuron models with these ionic channels (type II) demonstrated ramp firing patterns similar to those recorded from respiratory neurons in vivo. Our simulations show that K(AHP)(Ca) channels in combination with high-threshold Ca(L) channels produce spike frequency adaptation during synaptic excitation. However, in combination with low-threshold Ca(T) channels, they cause a frequency ramp firing response after release from inhibition. This promotes a testable hypothesis that the main difference between the respiratory neurons that adapt (for example, early inspiratory, postinspiratory, and decrementing expiratory) and those that show ramp firing patterns (for example, ramp inspiratory and augmenting expiratory) consists of a ratio between the two types of calcium channels: Ca(L) channels predominate in the former and Ca(T) channels in the latter respiratory neuron types. We have analyzed the dependence of adapting and ramp firing patterns on maximal conductances of different ionic channels and values of synaptic drive. The effect of adjusting specific membrane conductances and synaptic interactions revealed plausible neuronal mechanisms that may underlie modulatory effects on respiratory neuron firing patterns and network performances. The results of computer simulation provide useful insight into functional significance of specific intrinsic membrane properties and their interactions with phasic synaptic inputs for a better understanding of respiratory neuron firing behavior.

Modeling neural mechanisms for genesis of respiratory rhythm and pattern. III. Comparison of model performances during afferent nerve stimulation.

The goal of the present study was to evaluate the relative plausibility of the models of the central respiratory pattern generator (CRPG) proposed in our previous paper. To test the models, we compared changes in generated patterns with the experimentally observed alterations of the respiratory pattern induced by various stimuli applied to superior laryngeal (SLN), vagus and carotid sinus (CS) nerves. In all models, short-duration SLN simulation caused phase-resetting behavior consistent with experimental data. Relatively weak sustained SLN stimulation elicited a two-phase rhythm comprising inspiration and postinspiration whereas a stronger stimulation stopped oscillations in the postinspiratory phase ("postinspiratory apnea"). In all models, sustained vagus nerve stimulation produced postinspiratory apnea. A short vagal stimulus delivered during inspiration terminated this phase. The threshold for inspiratory termination decreased during the course of the inspiratory phase. The effects of short-duration vagal stimulation applied during expiration were different in different models. In model 1, stimuli delivered in the postinspiratory phase prolonged expiration whereas the late expiratory phase was insensitive to vagal stimulation. No insensitive period was found in model 2 because vagal stimuli delivered at any time during expiration prolonged this phase. Model 3 demonstrated a short period insensitive to vagal stimulation at the very end of expiration. When phasic CS nerve stimulation was applied during inspiration or the first half of expiration, the performances of all models were similar and consistent with experimental data: stimuli delivered at the beginning inspiration shortened this phase whereas stimuli applied in the middle or at the end of inspiration prolonged it and stimuli delivered in the first half of expiration prolonged the expiratory interval. Behavior of the models were different when CS stimuli were delivered during the late expiratory phase. In model 1, these stimuli were ineffective or shortened expiration initiating the next inspiration. Alternatively, in models 2 and 3, they caused a prolongation of expiration. Although all CRPG models demonstrated a number of plausible alterations in the respiratory pattern elicited by afferent nerve stimulation, the behavior of model 1 was most consistent with experimental data. Taking into account differences in the model architectures and employed neural mechanisms, we suggest that the concept of respiratory rhythmogenesis based on the essential role of postinspiratory neurons is more plausible than the concept employing specific functional properties of decrementing expiratory (dec-E) neurons and that the ramp firing pattern of the late expiratory neuron is more likely to reflect intrinsic properties than disinhibition from the dec-E neurons.

Transcription of germline VH gene elements by normal human fetal liver.

Transcription of the gene elements that form the variable region of immunoglobulin heavy chains has been proposed to represent the process that controls access for the recombination enzymes in their sequential steps of catalysis. Evidence for germline transcription of VH gene elements, as part of VH to DJH recombination, has been limited to transcripts of only a few gene elements. We have examined normal fetal liver mRNA by Northern blotting and present evidence for germline transcripts from six human VH gene families. The candidate VH4 transcripts have been confirmed as germline transcripts by hybridization with 3' flanking sequences that would have been removed by recombination from mature VHDJH genes. The candidate transcripts for VH1, VH3, VH4 and VH6 have been confirmed by polymerase chain reaction amplification with primers from the 3' flanking sequences of these gene families and determination of the sequence of these products. Determination of sequence from two clones of VH1, VH3 and VH4 indicates that more than one gene from each of these families is transcribed. PCR amplification of VH4 and VH6 with primers specific for the leader sequence (exon 1) and 3' flanking sequence indicate that these transcripts are spliced, representing RNA processing. Germline transcripts from these families are also present in normal human bone marrow. These results indicate that transcriptional activation of germline VH gene elements is a general phenomenon in tissues undergoing V to DJ recombination.

Esophageal achalasia and adenocarcinoma in Barrett's esophagus: a report of two cases and a review of the literature.

Two cases of a rare combination of conditions, achalasia and adenocarcinoma in Barrett's esophagus are reported. Cancer developed 26 years after the onset of gastroesophageal reflux in one and 30 years after esophagomyotomy in the other. Twenty-one cases of Barrett's esophagus and achalasia have now been reported; adenocarcinoma developed in six patients. Only one has survived more than five years after treatment. Long-term surveillance of patients with achalasia is recommended.

A laser confocal microscopic study of vagal afferent innervation of rat aortic arch: chemoreceptors as well as baroreceptors.

Although the aortic nerves contain vagal afferents that terminate in both the wall of the aortic arch (putative baroreceptors) and its associated glomus tissue (putative chemoreceptors) in most mammalian species, the aortic nerves of the rat have been widely assumed to contain only baro- or pressor afferents. The present study reconsidered this anomaly by characterizing vagal afferent endings and their targets in the aortic arch region of the rat, both qualitatively and quantitatively. Eight Sprague-Dawley rats received intracranial vagal motor rhizotomy unilaterally to eliminate efferents in the nerve and then, two weeks later, injections of the tracer DiI (1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate) into the ipsilateral nodose ganglion. The aortic arch and its surrounding tissue, with the common carotid and subclavian arteries attached, were examined with both conventional epifluorescence and confocal microscopes. Consistent with earlier observations, vagal afferents formed both flower-spray and end-net terminals rather diffusely within the wall of the aortic arch. More interestingly, vagal afferents also innervated glomus or SIF (i.e., small intensely fluorescent) cell bodies at the junction areas of the common carotid and subclavian arteries. To identify the course of these fibers, six additional animals received DiI injection into the nodose unilaterally after a complete cervical vagotomy caudal to the nodose; in these animals, the aortic nerve had been separated from the vagal trunk and kept intact. There were no marked differences in innervation patterns between the nonvagotomized and the cervically vagotomized animals, indicating that the vagal axons innervating the walls of the blood vessels and the SIF cells in the aortic arch region travel through the aortic nerves. Using a stereological method, we estimated the relative number of chemo- and baroreceptor afferents innervating the aortic arch. About 16.4% (left) and 13.1% (right) of fibers in the aortic nerves innervate SIF cells. These findings challenge the general consensus that the aortic nerves of rats contain exclusively baroreceptor fibers.