Immunohistochemical Characteristics of the Human Carotid Body in the Antenatal and Postnatal Periods of Development

The evolutionary and ontogenetic development of the carotid body is still understudied. Research aimed at studying the comparative morphology of the organ at different periods in the individual development of various animal species should play a crucial role in understanding the physiology of the carotid body. However, despite more than two centuries of study, the human carotid body remains poorly understood. There are many knowledge gaps in particular related to the antenatal development of this structure. The aim of our work is to study the morphological and immunohistochemical characteristics of the human carotid body in the antenatal and postnatal periods of development. We investigated the human carotid bodies from 1 embryo, 20 fetuses and 13 adults of different ages using samples obtained at autopsy. Immunohistochemistry revealed expression of βIII-tubulin and tyrosine hydroxylase in the type I cells and nerve fibers at all periods of ontogenesis; synaptophysin and PGP9.5 in the type I cells in some of the antenatal cases and all of the postnatal cases; 200 kDa neurofilaments in nerve fibers in some of the antenatal cases and all of the postnatal cases; and GFAP and S100 in the type II cells and Schwann cells in some of the antenatal cases and all of the postnatal cases. A high level of tyrosine hydroxylase in the type I cells was a distinctive feature of the antenatal carotid bodies. On the contrary, in the type I cells of adults, the expression of tyrosine hydroxylase was significantly lower. Our data suggest that the human carotid body may perform an endocrine function in the antenatal period, while in the postnatal period of development, it loses this function and becomes a chemosensory organ.

Carotid Body Chemoreceptors: Physiology, Pathology, and Implications for Health and Disease

The carotid body (CB) is the main peripheral chemoreceptor for arterial respiratory gases O2 and CO2, and pH, eliciting reflex ventilatory, cardiovascular and humoral responses to maintain homeostasis. This review examines the fundamental biology underlying CB chemoreceptor function, its contribution to integrated physiologic responses, and its role in maintaining health and potentiating disease. Emphasis will be placed on: i) Transduction mechanisms in chemoreceptor (type I) cells, highlighting the role played by the hypoxic inhibition of O2-dependent K+ channels and mitochondrial oxidative metabolism, and their modification by intracellular molecules and other ionic channels; ii) Synaptic mechanisms linking type I cells and petrosal nerve terminals, focusing on the role played by the main proposed transmitters and modulatory gases, and the participation of glial cells in regulation of the chemosensory process; iii) Integrated reflex responses to CB activation, emphasizing that the responses differ dramatically depending on the nature of the physiological, pathological or environmental challenges, and the interactions of the chemoreceptor reflex with other reflexes in optimizing oxygen delivery to the tissues; and iv) The contribution of enhanced CB chemosensory discharge to autonomic and cardiorespiratory pathophysiology in obstructive sleep apnea, congestive heart failure, resistant hypertension and metabolic diseases, and how modulation of enhanced CB reactivity in disease conditions may attenuate pathophysiology.

GAD65Cre drives reporter expression in multiple taste cell types

ABSTRACTIn taste buds, Type I cells represent the majority of cells (50-60%) and primarily have a glial-like function in taste buds. However, recent studies suggest that they have additional sensory and signaling functions including amiloride-sensitive salt transduction, oxytocin modulation of taste, and substance P mediated GABA release. Nonetheless, the overall function of Type I cells in transduction and signaling remains unclear, primarily because of the lack of a reliable reporter for this cell type. GAD65 expression is specific to Type I taste cells and GAD65 has been used as a Cre driver to study Type I cells in salt taste transduction. To test the specificity of transgene-driven expression, we crossed GAD65Cre mice with floxed tdTomato and Channelrhodopsin (ChR2) lines and examined the progeny with immunochemistry, chorda tympani recording, and calcium imaging. We report that while many tdTomato+ taste cells express NTPDase2, a specific marker of Type I cells, we see expression of tdTomato in both Gustducin and SNAP25 positive taste cells. We also see ChR2 in cells just outside the fungiform taste buds. Chorda tympani recordings in the GAD65Cre/ChR2 mice show large responses to blue light, larger than any response to standard taste stimuli. Further, several isolated tdTomato positive taste cells responded to KCl depolarization with increases in intracellular calcium, indicating the presence of voltage-gated calcium channels. Taken together, these data suggest that GAD65Cre mice drive expression in multiple taste cell types and thus cannot be considered a reliable reporter of Type I cell function.

Growth Factors in the Carotid Body—An Update

The carotid body may undergo plasticity changes during development/ageing and in response to environmental (hypoxia and hyperoxia), metabolic, and inflammatory stimuli. The different cell types of the carotid body express a wide series of growth factors and corresponding receptors, which play a role in the modulation of carotid body function and plasticity. In particular, type I cells express nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, insulin-like-growth factor-I and -II, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-α and -β, interleukin-1β and -6, tumor necrosis factor-α, vascular endothelial growth factor, and endothelin-1. Many specific growth factor receptors have been identified in type I cells, indicating autocrine/paracrine effects. Type II cells may also produce growth factors and express corresponding receptors. Future research will have to consider growth factors in further experimental models of cardiovascular, metabolic, and inflammatory diseases and in human (normal and pathologic) samples. From a methodological point of view, microarray and/or proteomic approaches would permit contemporary analyses of large groups of growth factors. The eventual identification of physical interactions between receptors of different growth factors and/or neuromodulators could also add insights regarding functional interactions between different trophic mechanisms.

Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells

Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O2 and CO2/H+ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 μM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 μM), an effect opposed by the D2/3R antagonist, sulpiride (1–10 μM). As expected, acute hypercapnia (10% CO2; pH 7.4), or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.

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 hours per day) for 2 weeks. The 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 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 level 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.