GluN2A- and GluN2B-immunoreactive type I cells attached to vesicular glutamate transporter 2-immunoreactive afferent nerve terminals of the rat carotid body.

We previously reported the immunoreactivity for the vesicular glutamate transporter 2 (VGLUT2) in afferent nerve terminals attached to chemoreceptor type I cells of the carotid body (CB), suggesting that glutamate is released from afferent terminals to stimulate these cells. In the present study, we examined the immunoreactivity for the glutamate-binding subunits of N-methyl-D-aspartate (NMDA) receptors, GluN2A and GluN2B in the rat CB, and the immunohistochemical relationships between these subunits and VGLUT2. Immunoreactivities for GluN2A and GluN2B were predominant in a subpopulation of tyrosine hydroxylase-immunoreactive type I cells rather than those of dopamine beta-hydroxylase-immunoreactive cells. Punctate VGLUT2-immunoreactive products were attached to GluN2A- and GluN2B-immunoreactive type I cells. Bassoon-immunoreactive products were localized between VGLUT2-immunoreactive puncta and type I cells immunoreactive for GluN2A and GluN2B. These results suggest that afferent nerve terminals release glutamate by exocytosis to modulate chemosensory activity of a subpopulation of type I cells via GluN2A- and GluN2B subunits-containing NMDA receptors.

Functional glutamate transporters are expressed in the carotid chemoreceptor.

BACKGROUND: The carotid body (CB) plays a critical role in cyclic intermittent hypoxia (CIH)-induced chemosensitivity; however, the underlying mechanism remains uncertain. We have demonstrated the presence of multiple inotropic glutamate receptors (iGluRs) in CB, and that CIH exposure alters the level of some iGluRs in CB. This result implicates glutamatergic signaling in the CB response to hypoxia. The glutamatergic neurotransmission is not only dependent on glutamate and glutamate receptors, but is also dependent on glutamate transporters, including vesicular glutamate transporters (VGluTs) and excitatory amino acid transporters (EAATs). Here, we have further assessed the expression and distribution of VGluTs and EAATs in human and rat CB and the effect of CIH exposure on glutamate transporters expression. METHODS: The mRNA of VGluTs and EAATs in the human CB were detected by RT-PCR. The protein expression of VGluTs and EAATs in the human and rat CB were detected by Western blot. The distribution of VGluT3, EAAT2 and EAAT3 were observed by immunohistochemistry staining and immunofluorescence staining. Male Sprague-Dawley (SD) rats were exposed to CIH (FIO2 10-21%, 3 min/3 min for 8 h per day) for 2 weeks. The unpaired Student's t-test was performed. RESULTS: Here, we report on the presence of mRNAs for VGluT1-3 and EAAT1-3 in human CB, which is consistent with our previous results in rat CB. The proteins of VGluT1 and 3, EAAT2 and 3, but not VGluT2 and EAAT1, were detected with diverse levels in human and rat CB. Immunostaining showed that VGluT3, the major type of VGluTs in CB, was co-localized with tyrosine hydroxylase (TH) in type I cells. EAAT2 and EAAT3 were distributed not only in type I cells, but also in glial fibrillary acidic protein (GFAP) positive type II cells. Moreover, we found that exposure of SD rats to CIH enhanced the protein level of EAAT3 as well as TH, but attenuated the levels of VGluT3 and EAAT2 in CB. CONCLUSIONS: Our study suggests that glutamate transporters are expressed in the CB, and that glutamate transporters may contribute to glutamatergic signaling-dependent carotid chemoreflex to CIH.

Carotid body chemoreceptors, sympathetic neural activation, and cardiometabolic disease.

The carotid body (CB) is the main peripheral chemoreceptor that senses the arterial PO2, PCO2 and pH. In response to hypoxemia, hypercapnia and acidosis, carotid chemosensory discharge elicits reflex respiratory, autonomic and cardiovascular adjustments. The classical construct considers the CB as the main peripheral oxygen sensor, triggering reflex physiological responses to acute hypoxemia and facilitating the ventilatory acclimation to chronic hypoxemia at high altitude. However, a growing body of experimental evidence supports the novel concept that an abnormally enhanced CB chemosensory input to the brainstem contributes to overactivation of the sympathetic nervous system, and consequent pathology. Indeed, the CB has been implicated in several diseases associated with increases in central sympathetic outflow. These include hypertension, heart failure, sleep apnea, chronic obstructive pulmonary disease and metabolic syndrome. Indeed, ablation of the CB has been proposed for the treatment of severe and resistant hypertension in humans. In this review, we will analyze and discuss new evidence supporting an important role for the CB chemoreceptor in the progression of autonomic and cardiorespiratory alterations induced by heart failure, obstructive sleep apnea, chronic obstructive pulmonary disease and metabolic syndrome.

Coexpression of Galanin and Nestin in the Chemoreceptor Cells of the Human Carotid Body.

The carotid body is a highly specialized chemoreceptive organ of neural crest origin whose role is to detect changes in arterial oxygen content. The sensory units are the chemoreceptor cells, which are neuronal-like cells, surrounded by sustentacular or glial-like cells. It is suggested that the carotid body contains self-renewing multipotent stem cells, which are putatively represented by glial-like sustentacular cells. The mechanisms of renewal of neuronal-like cells are unclear. Recently, we have demonstrated the expression of galanin, a peptide promoting neurogenesis, in chemoreceptor cells in the human CB. Thus, in the present study we seek to determine whether galanin expression in chemoreceptor cells could be matched with that of nestin, a peptide that is a marker of multipotent neural stem cells, or rather with the glial fibrillary acidic protein (GFAP), a marker for glial cells. The latter would underscore the pluasibly essential role of sustentacular cells in the self-renewal capability of chemorecetors. We found that galanin expression is matched with nestin in chemoreceptor cells of the human carotid body, but not with that of GFAP. Thus, galanin expression in chemoreceptor cells could provide a signal for neurogenesis and chemoreceptor cell differentiation in the carotid body.

Selective Expression of Galanin in Neuronal-Like Cells of the Human Carotid Body.

The carotid body is a neural-crest-derived organ devoted to respiratory homeostasis through sensing changes in blood oxygen levels. The sensory units are the glomeruli composed of clusters of neuronal-like (type I) cells surrounded by glial-like (type II) cells. During chronic hypoxia, the carotid body shows growth, with increasing neuronal-like cell numbers. We are interested in the signals involved in the mechanisms that underlie such response, because they are not well understood and described. Considering that, in literature, galanin is involved in neurotrophic or neuroprotective role in cell proliferation and is expressed in animal carotid body, we investigated its expression in human. Here, we have shown the expression and localisation of galanin in the human carotid body.

Neurotrophic Properties, Chemosensory Responses and Neurogenic Niche of the Human Carotid Body.

The carotid body (CB) is a polymodal chemoreceptor that triggers the hyperventilatory response to hypoxia necessary for the maintenance of O(2) homeostasis essential for the survival of organs such as the brain or heart. Glomus cells, the sensory elements in the CB, are also sensitive to hypercapnia, acidosis and, although less generally accepted, hypoglycemia. Current knowledge on CB function is mainly based on studies performed on lower mammals, but the information on the human CB is scant. Here we describe the structure, neurotrophic properties, and cellular responses to hypoxia and hypoglycemia of CBs dissected from human cadavers. The adult CB parenchyma contains clusters of chemosensitive glomus (type I) and sustentacular (type II) cells as well as nestin-positive progenitor cells. This organ also expresses high levels of the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF). GDNF production and the number of progenitor and glomus cells were preserved in the CBs of human subjects of advanced age. As reported for other mammalian species, glomus cells responded to hypoxia by external Ca(2+)-dependent increase of cytosolic [Ca(2+)] and quantal catecholamine release. Human glomus cells are also responsive to hypoglycemia and together the two stimuli, hypoxia and hypoglycemia, can potentiate each other's effects. The chemo-sensory responses of glomus cells are also preserved at an advanced age. Interestingly, a neurogenic niche similar to that recently described in rodents is also preserved in the adult human CB. These new data on the cellular and molecular physiology of the CB pave the way for future pathophysiological studies involving this organ in humans.

Using redox-sensitive fluorescent probes to record real-time reactive oxygen species production in cells from mouse carotid body slices.

Reactive oxygen species (ROS) are important signaling molecules for physiologic processes such as acute response to hypoxia. However, reliable real-time ROS measurement in cells has been a long-standing methodological challenge. Here, we present a protocol to record acute changes in ROS production in sensory cells from mouse carotid body (CB) slices using redox-sensitive green fluorescent protein probes and microfluorimetry. This protocol provides sensitive and reproducible quantification of ROS during acute hypoxia in different subcellular compartments of CB glomus cells. For complete details on the use and execution of this protocol, please refer to Fernández-Agüera et al. (2015) and Arias-Mayenco et al. (2018).

The role of testosterone in the respiratory and thermal responses to hypoxia and hypercapnia in rats.

Many diseases of the respiratory system occur differently in males and females, indicating a possible role of gonadal hormones in respiratory control. We hypothesized that testosterone (T) is important for the ventilatory chemosensitivity responses in males. To test this hypothesis, we evaluated ventilation (V̇ E), metabolic rate and body temperature (Tb) under normoxia/normocapnia, hypercapnia and hypoxia in orchiectomized (ORX), ORX with testosterone replacement (ORX + T) or flutamide (FL, androgen receptor blocker)-treated rats. We also performed immunohistochemistry to evaluate the presence of androgen receptor (AR) in the carotid body (CB) of intact males. Orchiectomy promoted a reduction V̇ E and ventilatory equivalent (V̇ E /V̇ O2) under room-air conditions, which was restored with testosterone treatment. Moreover, during hypoxia or hypercapnia, animals that received testosterone replacement had a higher V̇ E and V̇ E /V̇ O2 than control and ORX, without changes in metabolic and thermal variables. Flutamide decreased the hypoxic ventilatory response without changing the CO2-drive to breathe, suggesting that the testosterone effect on hypercapnic hyperventilation does not appear to involve the AR. We also determined the presence of AR in the CB of intact animals. Our findings demonstrate that testosterone seems to be important for maintaining resting V̇ E in males. In addition, the influence of testosterone on V̇ E, either during resting conditions or under hypoxia and hypercapnia, seems to be a direct and specific effect, as no changes in metabolic rate or Tb were observed during any treatment. Finally, a putative site of testosterone action during hypoxia is the CB, since we detected the presence of AR in this structure.

Amperometric ATP microbiosensors for the analysis of chemosensitivity at rat carotid bodies.

The physiological application of amperometric adenosine triphosphate (ATP) microbiosensors for characterizing the stimulus-response at rat carotid bodies superfused with high potassium concentrations, during normoxic hypercapnia, and during hypoxia is demonstrated using the peripheral arterial chemoreceptors in the carotid body of rats as a model system. Amperometric microbiosensors based on glucose oxidase (GOD) and hexokinase (HEX) immobilized within a polymer matrix at the surface of Pt disk microelectrodes (diameter: 25 microm) are positioned at a distance of approximately 100 microm above the carotid body surface for detecting extracellular ATP. A linear calibration function of ATP microbiosensors in the physiologically relevant concentration range of 0-40 microM ATP enables quantitative detection of ATP released at the carotid body surface in response to physiological stimuli. It is shown that these stimuli induce extracellular ATP release from the carotid body at levels of 4-10 microM. Other electroactive neurotransmitters such as, e.g., catecholamines are coreleased by the carotid body at hypercapnic, hypoxic and high-potassium stimulus, are simultaneously detected utilizing a dual-electrode assembly with an ATP microbiosensor and a second bare channel providing a colocalized reference measurement for ATP quantification.

Bilateral carotid body tumor: the role of fine-needle aspiration biopsy in the preoperative diagnosis.

Carotid body (CB) is a round to ovoid or flattened structure situated within the adventitia of the common carotid artery bifurcation on both sides of the neck. CB contains two basic types of cells: chief cells (or glomus type 1) and sustentacular cells (glomus type 2). Carotid body tumor (CBT) or paraganglioma arises from the chief cells of the carotid body. The diagnosis of CBT is typically made with radiological studies. Fine needle aspiration biopsy (FNAB) is seldom requested for this purpose due to rare but dreadful reported complications such as hemorrhage and damage to the carotid artery. In this report we discuss the cytological findings of a malignant CBT diagnosed by FNAB in a 22 year-old female.

Specific localization of manserin peptide in the rat carotid body.

The carotid body, located at the bifurcation of the common carotid artery, is a small sensory organ that detects changes in oxygen concentration and plays a vital role in controlling respiration. Although several molecules, such as neurotransmitters and neuropeptides, are involved in the regulation of the respiratory system, their detailed mechanisms have not been established yet. This study identifies that the presence of manserin, a neuropeptide, in the carotid body may play a crucial role in regulating respiration. The carotid bodies of adult Wistar rats were perfused with paraformaldehyde, and the frozen sections were subjected to immunohistochemical analyses. The carotid body comprises two distinct types of cells, neuron-like glomus cells and glial-like sustentacular cells. We used specific antibodies to distinguish the specific location of manserin in the carotid body, which included a tyrosine hydroxylase-positive antibody for glomus cells and an S100 protein antibody for sustentacular cells. Immunofluorescence analysis revealed that while tiny, round signals were exclusively observed in the cytoplasm of glomus cells, no signals were observed in sustentacular cells. Because manserin is believed to be secreted from precursor proteins by the endoproteolytic processing of a large precursor protein called secretogranin II, manserin secretion systems may exist in the carotid body, and thus, behave as potential regulators of respiration in the carotid body.

Immunohistochemical localization of angiotensin AT1 receptors in the rat carotid body.

The carotid body (CB) is a major peripheral arterial chemoreceptor that initiates respiratory and cardiovascular adjustments to maintain homeostasis. Recent evidence suggests that circulating or locally produced hormones like angiotensin II acting via AT1 receptors modulate its activity in a paracrine-autocrine manner. The aim of this study was to examine the immunohistochemical localization of AT1 receptor in the CB of adult rats and to compare its expression in vehicle-treated animals, and after the long-term application of its selective blocker losartan. Immunohistochemistry revealed that a subset of CB glomeruli and the vast majority of neurons in the adjacent superior cervical ganglion (SCG) were strongly AT1 receptor-immunoreactive. In the CB immunostaining was observed in the chemosensory glomus cells typically aggregated in cell clusters while the nerve fibers in-between and large capillaries around them were immunonegative. Exogenous administration of losartan for a prolonged time significantly reduces the intensity of AT1 receptor immunostaining in the CB glomus cells and SCG neurons. Our results show that AT1 receptors are largely expressed in the rat CB under physiological conditions, and their expression is down-regulated by losartan treatment.

Arginine-vasopressin mediates central and peripheral glucose regulation in response to carotid body receptor stimulation with Na-cyanide.

Hypoxic stimulation of the carotid body receptors (CBR) results in a rapid hyperglycemia with an increase in brain glucose retention. Previous work indicates that neurohypophysectomy inhibits this hyperglycemic response. Here, we show that systemic arginine vasopressin (AVP) induced a transient, but significant, increase in blood glucose levels and increased brain glucose retention, a response similar to that observed after CBR stimulation. Comparable results were obtained after intracerebral infusion of AVP. Systemic AVP-induced changes were maintained in hypophysectomized rats but were not observed after adrenalectomy. Glycemic changes after CBR stimulation were inhibited by pharmacological blockage of AVP V1a receptors with a V1a-selective receptor antagonist ([beta-Mercapto-beta,beta-cyclopentamethylenepropionyl1,O-me-Tyr2, Arg8]-vasopressin). Importantly, local application of micro-doses of this antagonist to the liver was sufficient to abolish the hyperglycemic response after CBR stimulation. These results suggest that AVP is a mediator of the hyperglycemic reflex and cerebral glucose retention following CBR stimulation. We propose that hepatic activation of AVP V1a receptors is essential for this hyperglycemic response.

Adrenomedullin immunoreactivity in the human carotid body.

We studied by immunocytochemistry the expression of AM in human carotid bodies, sampled at autopsy from 16 adult subjects (mean age+/-S.D.: 44.3+/-3.4 years) and from six fetuses (mean gestational age+/-S.D.: 167+/-11 days). No AM immunoreactivity was visible in the type II cells of both series. The percentage of immunoreactive type I cells was higher in the adult subjects (32.3+/-7.7%) with respect to the fetuses (11.8+/-2.7%, P < 0.001). Dark cells showed a higher percentage of positive immunoreaction with respect to light cells, both in adult subjects (61.7+/-13.4% versus 19.2+/-5.2%) and in fetuses (25.3+/-4.4% versus 6.2+/-2.0%). AM may play a role in the regulation of chemoreceptor discharge through paracrine releasing action and/or vasodilator effect. The low expression of AM in fetuses may be ascribed to the absence of pulmonary respiration with lack of regulatory role of the carotid body during the prenatal period.

Pivotal role of nucleotide P2X2 receptor subunit of the ATP-gated ion channel mediating ventilatory responses to hypoxia.

In mammals, the ventilatory response to decreased oxygen tension in the arterial blood is initiated by excitation of specialized O2-sensitive chemoreceptor cells in the carotid body that release neurotransmitters to activate endings of the sinus nerve afferent fibers. We investigated the role of ATP acting via ionotropic P2X receptors in the carotid body function and ventilatory response to hypoxia in mice. Mice deficient in P2X2 receptor subunit showed a markedly attenuated ventilatory response to hypoxia, whereas the response to hypoxia in P2X3-deficient mice was comparable with that seen in wild-type controls. P2X2 and P2X3 receptor subunit deficiency did not affect the ventilatory responses to hypercapnia. P2X2 subunit deficiency resulted in a dramatic reduction in the responses of the carotid sinus nerve to hypoxia in the in vitro carotid body-sinus nerve preparation. ATP and its stable analog alpha,beta-methyleneATP both evoked rapid excitation of sinus nerve afferents, and the P2 receptor antagonist PPADS (pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid) (100 microm) blocked hypoxia-induced increase in sinus nerve discharge. Immunoreactivities for P2X2 and P2X3 subunits were both detected on afferent terminals surrounding clusters of glomus cells in the wild-type animals but were absent in mice deficient in P2X2 and P2X3 receptor subunits. These observations provide the first definitive evidence that, in the carotid body, ATP is a key transmitter released by chemoreceptor cells to activate endings of the sinus nerve afferent fibers. We conclude that P2X receptors containing the P2X2 subunit play a pivotal role in carotid body function and in mediating ventilatory responses to hypoxia.

Ultrastructural localization of neuropeptide Y and expression of its mRNA in the glomus cells distributed in the wall of the common carotid artery of the chicken.

In the chicken, glomus cells are widely distributed in the carotid body and in the wall of the common carotid artery and around its branches. The cells located in the wall of the common carotid artery express intense immunoreactivity for neuropeptide Y (NPY). They contain abundant dense-cored vesicles ranging from 70 to 220 nm in diameter. In this study, we examined ultrastructural localization of NPY in the glomus cells by using the postembedding immunogold method. Gold particles representing immunoreactivity for NPY were specifically localized on the dense-cored vesicles of the glomus cells. In addition, the localization of NPY mRNA in the glomus cells was examined by in situ hybridization with digoxigenin-labeled chicken NPY cRNA probe. A strong hybridization signal for NPY mRNA was detected in the glomus cells located in the wall of the common carotid artery. Few glomus cells of the carotid body, however, displayed labeling for NPY mRNA. Northern blot analysis with the chicken NPY exon 4 probe demonstrated that a single band for NPY mRNA was present in the poly (A) + RNA isolated from the common carotid artery where the glomus cells were distributed. Furthermore, the expression of NPY mRNA in the common carotid artery was confirmed by the reverse transcription-polymerase chain reaction. These results indicate that the chicken glomus cells are able to produce NPY but that the level of its translation varies according to the location of the cells.

Ultrastructure of calcitonin gene-related peptide-immunoreactive, unmyelinated afferents to the cat carotid body: a case of volume transmission.

To relate the ultrastructure of unmyelinated afferents to the cat carotid body with the known electrophysiological properties of cat chemosensory C-fibers, we took advantage of the fact that the calcitonin gene-related peptide is exclusively present in a population of sparsely branched afferents to the carotid body. They have a morphology identical to the afferents originating from carotid sinus nerve unmyelinated axons. Immunoreactive axons were stained using pre-embedding protocols and horseradish peroxidase-labeled secondary antibody. Labeling was present only in unmyelinated axons and boutons distributed in the interstitial and parenchymal tissue. The varicosities had an average diameter of 0.7 micron, and contained both small, clear vesicles and larger dense-core vesicles. No labeled axons were ever seen to contact glomus cells, but could be observed as close as 0.2 micron to a glomus cell, always with an interposed glial process. With a very sensitive protocol, that used tungstate-stabilized tetramethylbenzidine as the chromogen, amorphous deposits of reaction product were often detected in the extracellular space around a labeled bouton. We interpret these findings as indicating that the reciprocal chemical transmission between the oxygen-sensitive glomus cells and the unmyelinated afferents takes place through non-synaptic transmission, via the rather large extracellular space of the carotid body. In addition, the larger distances between glomus cells and unmyelinated afferents could explain the lowered sensitivity and sluggishness of chemosensory C-fibers, compared to the A-fibers.

Adrenomedullin expression and function in the rat carotid body.

Adrenomedullin (AM) immunoreactivity has been found in granules of the glomus (type I) cells of the carotid bodies in rats. The identity of these cells was ascertained by colocalization of immunoreactivities for AM and tyrosine hydroxylase in their cytoplasm. Exposure of freshly isolated carotid bodies to synthetic AM resulted in a concentration- and time-dependent degranulation of glomus cells as measured by dopamine (DA) release. DA release reached a zenith 30 min after exposure to AM (94.2% over untreated controls). At this time-point, the response to AM was similar to the one elicited by 5 min of exposure to 100 mM K+. Nevertheless, injection of 1 micro l 60 nM AM/g body weight into the tail vein of the rats did not induce statistical differences in DA release from the carotid bodies. Exposure of the oxygen-sensitive cell line PC-12 to hypoxia elicited an increase in AM mRNA expression and peptide secretion into serum-free conditioned medium. Previous data have shown that elevation of AM expression under hypoxia is mediated through hypoxia-inducible factor-1, and that exposure of chromaffin cells to AM results in degranulation. All these data suggest that AM is an important autocrine regulator of carotid body function.

Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies.

Substance P-immunoreactive (SP-1) structures in the carotid bodies of rats and cats were examined with the light and electron microscopes. In both species SP-I varicose nerve fibers were located singly in the interstitial connective tissue in close association with blood vessels. They were small unmyelinated fibers enveloped in a common Schwann cell sheath with other SP-negative fibers. Some of SP-I fibers contained large dense-cored granules and small clear vesicles in addition to microtubules and mitochondria and probably represented nerve fiber varicosities. The latter often were found incompletely invested by Schwann cell sheaths. SP-fibers were found occasionally in the envelopes of supporting cells at the periphery of parenchymal cell groups. However, none of the nerve terminals making synaptic contacts with glomus cells exhibited SP-like immunoreactivity. In cat carotid bodies some glomus cells showed moderate to intense SP-like immunoreactivity. The intense SP-I glomus cells displayed numerous dense-cored vesicles of 85 to 140 nm in diameter and frequently showed synaptic contacts with SP-negative nerve terminals. In rat carotid bodies we were unable to detect consistent SP-immunoreactivity in glomus cells. Our results do not favor the hypothesis that SP is a neurotransmitter/modulator in the chemoreceptor afferents synapsing on glomus cells in either the cat or rat carotid body. However our results support the hypothesis that SP in cat glomus cells may play a role in the modulation of chemoreceptor activity.

Glutamate in the glomus cells of the cat carotid body: immunocytochemistry and in vitro release.

The identity of the postulated excitatory transmitter released by glomus cells is not known. Since our preliminary work on paraffin sections of the cat carotid body indicated that most glomus cells were intensely immunoreactive to glutamate, we decided to investigate whether glutamate might be such a transmitter, using two approaches. One approach was to make a quantitative immunogold analysis of ultrathin sections to assess the level of glutamate immunoreactivity of glomus cells relative to glia and to afferent axon terminals. The other approach was to measure the potassium-induced release of glutamate from carotid bodies superfused in vitro. We consistently found that glomus cell profiles had 50% more immunogold particles per unit of area than glial cell or axonal profiles. However, the levels of glutamate immunoreactivity of glomus cells were lower than those expected for glutamatergic terminals. We also found that glutamate was not released from in vitro carotid bodies stimulated with high concentrations of potassium. These findings indicate that the oxygen-sensitive glomus cells have a high concentration of glutamate, which is not released by superfusion with high potassium. Thus, glutamate is not the excitatory transmitter released by glomus cells. We speculate that the high concentrations of glutamate might instead be related to the known dependence of the "in vitro" chemosensory activity on metabolic substrates.