RESUMO
Elevated sugar consumption is associated with an increased risk for metabolic diseases. Whereas evidence from humans, rodents, and insects suggests that dietary sucrose modifies sweet taste sensation, understanding of peripheral nerve or taste bud alterations is sparse. To address this, male rats were given access to 30% liquid sucrose for 4 weeks (sucrose rats). Neurophysiological responses of the chorda tympani (CT) nerve to lingual stimulation with sugars, other taste qualities, touch, and cold were then compared with controls (access to water only). Morphological and immunohistochemical analyses of fungiform papillae and taste buds were also conducted. Sucrose rats had substantially decreased CT responses to 0.15-2.0 M sucrose compared with controls. In contrast, effects were not observed for glucose, fructose, maltose, Na saccharin, NaCl, organic acid, or umami, touch, or cold stimuli. Whereas taste bud number, size, and innervation volume were unaffected, the number of PLCß2+ taste bud cells in the fungiform papilla was reduced in sucrose rats. Notably, the replacement of sucrose with water resulted in a complete recovery of all phenotypes over 4 weeks. The work reveals the selective and modality-specific effects of sucrose consumption on peripheral taste nerve responses and taste bud cells, with implications for nutrition and metabolic disease risk. VIDEO ABSTRACT.
Assuntos
Sacarina , Paladar , Animais , Dieta , Sacarose Alimentar , Frutose , Glucose , Humanos , Masculino , Maltose , Ratos , Cloreto de Sódio , Paladar/fisiologia , ÁguaRESUMO
The fungiform papilla (FP) is a gustatory and somatosensory structure incorporating chorda tympani (CT) nerve fibers that innervate taste buds (TB) and also contain somatosensory endings for touch and temperature. Hedgehog (HH) pathway inhibition eliminates TB, but CT innervation remains in the FP. Importantly, after HH inhibition, CT neurophysiological responses to taste stimuli are eliminated, but tactile responses remain. To examine CT fibers that respond to tactile stimuli in the absence of TB, we used Phox2b-Cre; Rosa26LSL-TdTomato reporter mice to selectively label CT fibers with TdTomato. Normally CT fibers project in a compact bundle directly into TB, but after HH pathway inhibition, CT fibers reorganize and expand just under the FP epithelium where TB were. This widened expanse of CT fibers coexpresses Synapsin-1, ß-tubulin, S100, and neurofilaments. Further, GAP43 expression in these fibers suggests they are actively remodeling. Interestingly, CT fibers have complex terminals within the apical FP epithelium and in perigemmal locations in the FP apex. These extragemmal fibers remain after HH pathway inhibition. To identify tactile end organs in FP, we used a K20 antibody to label Merkel cells. In control mice, K20 was expressed in TB cells and at the base of epithelial ridges outside of FP. After HH pathway inhibition, K20 + cells remained in epithelial ridges but were eliminated in the apical FP without TB. These data suggest that the complex, extragemmal nerve endings within and disbursed under the apical FP are the mechanosensitive nerve endings of the CT that remain after HH pathway inhibition.
Assuntos
Proteínas Hedgehog , Papilas Gustativas , Animais , Nervo da Corda do Tímpano/metabolismo , Proteínas Hedgehog/metabolismo , Camundongos , Terminações Nervosas/metabolismo , Paladar/fisiologia , Papilas Gustativas/metabolismo , LínguaRESUMO
When solid or liquid stimuli contact the tongue tip during eating, the sensations of taste, touch and temperature are immediately evoked, and tongue function relies on these simultaneous multimodal responses. We focus on the fungiform papilla of the anterior tongue as a complex organ for taste, tactile and thermal modalities, all via chorda tympani nerve innervation from the geniculate ganglion. Rather than a review, our aim is to revise the classic archetype of the fungiform as predominantly a taste bud residence only and instead emphasize an amended concept of the papilla as a multimodal organ. Neurophysiological maps of fungiform papillae in functional receptive fields demonstrate responses to chemical, stroking and cold lingual stimuli. Roles are predicted for elaborate extragemmal nerve endings in tactile and temperature sensations, and potential functions for keratinocytes in noncanonical sensory signaling. The fungiform papilla is presented as a polymodal lingual organ, not solely a gustatory papilla.
RESUMO
During development of the peripheral taste system, oral sensory neurons of the geniculate ganglion project via the chorda tympani nerve to innervate taste buds in fungiform papillae. Germline deletion of the p75 neurotrophin receptor causes dramatic axon guidance and branching deficits, leading to a loss of geniculate neurons. To determine whether the developmental functions of p75 in geniculate neurons are cell autonomous, we deleted p75 specifically in Phox2b + oral sensory neurons (Phox2b-Cre; p75fx/fx) or in neural crest-derived cells (P0-Cre; p75fx/fx) and examined geniculate neuron development. In germline p75-/- mice half of all geniculate neurons were lost. The proportion of Phox2b + neurons, as compared to Phox2b-pinna-projecting neurons, was not altered, indicating that both populations were affected similarly. Chorda tympani nerve recordings demonstrated that p75-/- mice exhibit profound deficits in responses to taste and tactile stimuli. In contrast to p75-/- mice, there was no loss of geniculate neurons in either Phox2b-Cre; p75fx/fx or P0-Cre; p75fx/fx mice. Electrophysiological analyses demonstrated that Phox2b-Cre; p75fx/fx mice had normal taste and oral tactile responses. There was a modest but significant loss of fungiform taste buds in Phox2b-Cre; p75fx/fx mice, although there was not a loss of chemosensory innervation of the remaining fungiform taste buds. Overall, these data suggest that the developmental functions of p75 are largely cell non-autonomous and require p75 expression in other cell types of the chorda tympani circuit.
Assuntos
Gânglio Geniculado/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Células Receptoras Sensoriais/metabolismo , Alelos , Animais , Biomarcadores , Nervo da Corda do Tímpano/metabolismo , Imunofluorescência , Genótipo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de Fator de Crescimento Neural/genética , Paladar/fisiologia , TatoRESUMO
The development of the taste system relies on the coordinated regulation of cues that direct the simultaneous development of both peripheral taste organs and innervating sensory ganglia, but the underlying mechanisms remain poorly understood. In this study, we describe a novel, biphasic function for glial cell line-derived neurotrophic factor (GDNF) in the development and subsequent diversification of chemosensory neurons within the geniculate ganglion (GG). GDNF, acting through the receptor tyrosine kinase Ret, regulates the expression of the chemosensory fate determinant Phox2b early in GG development. Ret-/- mice, but not Retfx/fx ; Phox2b-Cre mice, display a profound loss of Phox2b expression with subsequent chemosensory innervation deficits, indicating that Ret is required for the initial amplification of Phox2b expression but not its maintenance. Ret expression is extinguished perinatally but reemerges postnatally in a subpopulation of large-diameter GG neurons expressing the mechanoreceptor marker NF200 and the GDNF coreceptor GFRα1. Intriguingly, we observed that ablation of these neurons in adult Ret-Cre/ERT2; Rosa26LSL-DTA mice caused a specific loss of tactile, but not chemical or thermal, electrophysiological responses. Overall, the GDNF-Ret pathway exerts two critical and distinct functions in the peripheral taste system: embryonic chemosensory cell fate determination and the specification of lingual mechanoreceptors.
Assuntos
Diferenciação Celular/fisiologia , Células Quimiorreceptoras/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Paladar/fisiologia , Animais , Gânglio Geniculado , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transdução de Sinais , Tamoxifeno , Temperatura , Língua/inervação , Tato , Fator de Transcrição Brn-3A , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Afferent chorda tympani (CT) fibers innervating anterior tongue fungiform papillae have neuron cell bodies in the geniculate ganglion (GG). To characterize electrophysiological and receptive field properties, we recorded extracellular responses from single GG neurons to lingual application with chemical, thermal and mechanical stimuli. Receptive field size was mapped by electrical stimulation of individual fungiform papillae. Responses of GG neurons to room temperature chemical stimuli representing five taste qualities, and distilled water at 4⯰C and mechanical stimulation were used. Based on responses to these stimuli, GG neurons were divided into CHEMICAL, CHEMICAL/THERMAL, THERMAL and TACTILE groups. Neurons in the CHEMICAL group responded to taste stimuli but not to either cold water or stroking stimuli. CHEMICAL/THERMAL neurons responded to both taste stimuli and cold water. THERMAL neurons responded only to cold water and TACTILE neurons responded only to light stroking stimuli. The receptive field sizes for CHEMICAL, and CHEMICAL/THERMAL neurons averaged five papillae exceeding the field size of THERMAL and TACTILE neurons which averaged about two papillae. Detailed analysis of the receptive field of CHEMICAL/THERMAL neurons revealed that within one field only a subset of the fungiform papillae making up the receptive field responded to the cold stimuli, whereas the other papillae responded only to chemical stimuli. These finding demonstrate that fungiform papilla are complex sensory organs with a multisensory function suggesting a unique role in detecting and sampling food components prior to ingestion.
Assuntos
Potenciais de Ação/fisiologia , Gânglio Geniculado/citologia , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/fisiologia , Língua/inervação , Animais , Biofísica , Feminino , Fibras Nervosas/fisiologia , Técnicas de Patch-Clamp , Estimulação Física/efeitos adversos , Ratos , Ratos Sprague-Dawley , Estimulação Química , Temperatura , Tato/fisiologiaRESUMO
Striking taste disturbances are reported in cancer patients treated with Hedgehog (HH)-pathway inhibitor drugs, including sonidegib (LDE225), which block the HH pathway effector Smoothened (SMO). We tested the potential for molecular, cellular, and functional recovery in mice from the severe disruption of taste-organ biology and taste sensation that follows HH/SMO signaling inhibition. Sonidegib treatment led to rapid loss of taste buds (TB) in both fungiform and circumvallate papillae, including disruption of TB progenitor-cell proliferation and differentiation. Effects were selective, sparing nontaste papillae. To confirm that taste-organ effects of sonidegib treatment result from HH/SMO signaling inhibition, we studied mice with conditional global or epithelium-specific Smo deletions and observed similar effects. During sonidegib treatment, chorda tympani nerve responses to lingual chemical stimulation were maintained at 10 d but were eliminated after 16 d, associated with nearly complete TB loss. Notably, responses to tactile or cold stimulus modalities were retained. Further, innervation, which was maintained in the papilla core throughout treatment, was not sufficient to sustain TB during HH/SMO inhibition. Importantly, treatment cessation led to rapid and complete restoration of taste responses within 14 d associated with morphologic recovery in about 55% of TB. However, although taste nerve responses were sustained, TB were not restored in all fungiform papillae even with prolonged recovery for several months. This study establishes a physiologic, selective requirement for HH/SMO signaling in taste homeostasis that includes potential for sensory restoration and can explain the temporal recovery after taste dysgeusia in patients treated with HH/SMO inhibitors.
Assuntos
Antineoplásicos/efeitos adversos , Compostos de Bifenilo/efeitos adversos , Disgeusia/fisiopatologia , Piridinas/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Paladar/efeitos dos fármacos , Língua/fisiopatologia , Animais , Carcinoma Basocelular/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nervo da Corda do Tímpano/efeitos dos fármacos , Nervo da Corda do Tímpano/fisiopatologia , Modelos Animais de Doenças , Disgeusia/induzido quimicamente , Disgeusia/patologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recuperação de Função Fisiológica , Neoplasias Cutâneas/tratamento farmacológico , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Células-Tronco/efeitos dos fármacos , Paladar/fisiologia , Papilas Gustativas/citologia , Papilas Gustativas/efeitos dos fármacos , Papilas Gustativas/patologia , Papilas Gustativas/fisiopatologia , Língua/efeitos dos fármacos , Língua/inervaçãoRESUMO
Afferent chorda tympani (CT) fibers innervating taste and somatosensory receptors in fungiform papillae have neuron cell bodies in the geniculate ganglion (GG). The GG/CT fibers branch in the tongue to innervate taste buds in several fungiform papillae. To investigate receptive field characteristics of GG/CT neurons, we recorded extracellular responses from GG cells to application of chemical and thermal stimuli. Receptive field size was mapped by electrical stimulation of individual fungiform papillae. Response latency to electrical stimulation was used to determine fiber conduction velocity. Responses of GG neurons to lingual application of stimuli representing four taste qualities, and water at 4°C, were used to classify neuron response properties. Neurons classified as SALT, responding only to NaCl and NH4Cl, had a mean receptive field size of six papillae. Neurons classified as OTHER responded to salts and other chemical stimuli and had smaller mean receptive fields of four papillae. Neurons that responded to salts and cold stimuli, classified as SALT/THERMAL, and neurons responding to salts, other chemical stimuli and cold, classified as OTHER/THERMAL, had mean receptive field sizes of six and five papillae, respectively. Neurons responding only to cold stimuli, categorized as THERMAL, had receptive fields of one to two papillae located at the tongue tip. Based on conduction velocity most of the neurons were classified as C fibers. Neurons with large receptive fields had higher conduction velocities than neurons with small receptive fields. These results demonstrate that GG neurons can be distinguished by receptive field size, response properties and afferent fiber conduction velocity derived from convergent input of multiple taste organs.
Assuntos
Nervo da Corda do Tímpano/fisiologia , Gânglio Geniculado/citologia , Condução Nervosa/fisiologia , Neurônios/fisiologia , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação/fisiologia , Cloreto de Amônio/farmacologia , Análise de Variância , Animais , Estimulação Elétrica , Feminino , Condução Nervosa/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tempo de Reação , Células Receptoras Sensoriais/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Estimulação Química , Paladar/fisiologia , Papilas Gustativas/fisiologiaRESUMO
Taste sensation on the anterior tongue requires chorda tympani nerve function and connections with continuously renewing taste receptor cells. However, it is unclear which signaling pathways regulate the receptor cells to maintain chorda tympani sensation. Hedgehog (HH) signaling controls cell proliferation and differentiation in numerous tissues and is active in taste papillae and taste buds. In contrast, uncontrolled HH signaling drives tumorigenesis, including the common skin cancer, basal cell carcinoma. Systemic HH pathway inhibitors (HPIs) lead to basal cell carcinoma regression, but these drugs cause severe taste disturbances. We tested the hypothesis that taste disruption by HPIs reflects a direct requirement for HH signaling in maintaining taste organs and gustatory sensation. In mice treated with the HPI LDE225 up to 28 days, HH-responding cells were lost in fungiform papilla epithelium, and papillae acquired a conical apex. Taste buds were either absent or severely reduced in size in more than 90% of aberrant papillae. Taste bud remnants expressed the taste cell marker keratin 8, and papillae retained expression of nerve markers, neurofilament and P2X3. Chorda tympani nerve responses to taste stimuli were markedly reduced or absent in LDE225-treated mice. Responses to touch were retained, however, whereas cold responses were retained after 16 days of treatment but lost after 28 days. These data identify a critical, modality-specific requirement for HH signaling in maintaining taste papillae, taste buds and neurophysiological taste function, supporting the proposition that taste disturbances in HPI-treated patients are an on-target response to HH pathway blockade in taste organs.
Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Proteínas Hedgehog/metabolismo , Piridinas/farmacologia , Papilas Gustativas/efeitos dos fármacos , Paladar , Animais , Feminino , Camundongos , Transdução de Sinais , Papilas Gustativas/metabolismo , Papilas Gustativas/fisiologia , TatoRESUMO
The rostral nucleus of the solitary tract (rNST) receives orosensory information from taste bud cells in the tongue and palate via cranial nerves VII and IX. These nerves enter the brainstem, form the solitary tract (ST) and synapse with neurons in the rNST, which then relay incoming sensory information to other brain areas to process external gustatory stimuli. Factors that direct or regulate the trajectory of the developing ST are largely unknown. We used 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) to identify ST projections originating from cells in the geniculate ganglia of embryonic rats from embryonic day 14 through 18 (E14-E18). After identifying the ST fibers, immunolabeling for and protein expression analysis of the axon guidance molecules neuropilin-1 (Npn-1) and neuropilin-2 (Npn-2) and their binding partners, semaphorin-3A (Sema-3A) and semaphorin-3F (Sema-3F) were performed. The results detail the formation of ST projections into the gustatory brainstem and their relationship to developing rNST neurons. DiI-labeled ST fibers were present in the brainstem as early as E14. Npn-1 was expressed in the ST and in the trigeminal tract at E14, but levels of the protein declined through E18. The expression levels of the binding partner of Npn-1, Sema-3A, increased from E14 to E18. Npn-2 was expressed in the ST and, additionally, in radially oriented, tuft-like structures within the brainstem at E14. Expression levels of Npn-2 also declined through E18, in contrast to the expression levels of its binding partner, Sema-3F, which increased during this time period. For the first time, the time course and particular molecular components involved in development of the ST have been identified. These results indicate that the neuropilin and semaphorin families of axon guidance molecules are potential molecular participants in ST formation.
Assuntos
Neurogênese/fisiologia , Neuropilinas/metabolismo , Núcleo Solitário/embriologia , Núcleo Solitário/metabolismo , Animais , Western Blotting , Imunofluorescência , Ratos , Ratos Sprague-Dawley , Semaforinas/metabolismoRESUMO
The rostral nucleus of the solitary tract (rNTS), the first-order relay of gustatory information, not only transmits sensory information to more rostral brain areas but also connects to various brain stem sites responsible for orofacial reflex activities. While much is known regarding ascending projections to the parabrachial nucleus, intramedullary projections to the reticular formation (which regulate oromotor reflexive behaviors) remain relatively unstudied. The present study examined the intrinsic firing properties of these neurons as well as their morphological properties and synaptic connectivity with primary sensory afferents. Using in vitro whole cell patch-clamp recording, we found that intramedullary projection neurons respond to depolarizing current injection with either tonic or bursting action potential trains and subsets of these groups of neurons express A-type potassium, H-like, and postinhibitory rebound currents. Approximately half of the intramedullary projection neurons tested received monosynaptic innervation from primary afferents, while the rest received polysynaptic innervation, indicating that at least a subpopulation of these neurons can be directly activated by incoming sensory information. Neuron morphological reconstructions revealed that many of these neurons possessed numerous dendritic spines and that neurons receiving monosynaptic primary afferent input have a greater spine density than those receiving polysynaptic primary afferent input. These results reveal that intramedullary projection neurons represent a heterogeneous class of rNTS neurons and, through both intrinsic voltage-gated ion channels and local circuit interactions, transform incoming gustatory information into signals governing oromotor reflexive behaviors.
Assuntos
Neurônios/fisiologia , Formação Reticular/citologia , Núcleo Solitário/citologia , Núcleo Solitário/fisiologia , Vias Aferentes/fisiologia , Animais , Potenciais Evocados , Feminino , Técnicas In Vitro , Vias Neurais , Ratos , Ratos Sprague-Dawley , Potenciais SinápticosRESUMO
The embryonic development of synapses in the rostral nucleus of the solitary tract (rNST) was investigated in rat to determine when synapses begin to function. Using a brain slice preparation we studied appearance of synaptic receptors on second order rNST neurons and investigated the development of postsynaptic responses elicited by afferent nerve stimulation. Prenatal excitatory and inhibitory synaptic responses were recorded as early as E14. Glutamatergic and GABAergic postsynaptic responses were detected as early as E16. Both NMDA and AMPA receptors contributed to glutamatergic postsynaptic responses. GABAergic postsynaptic responses resulted primarily from activation of GABA(A) receptors. However, functional GABA(C) receptors were also demonstrated. A glycinergic postsynaptic response was not found although functional glycine receptors were demonstrated at E16. Solitary tract (ST) stimulation-evoked EPSCs, first detected at E16, were eliminated by glutamate receptor antagonists. ST-evoked IPSPs, also detected at E16, were eliminated by GABA(A) receptor antagonist. Thus, considerable prenatal development of rNST synaptic connections occurs and this will ensure postnatal function of central taste processing circuits.
Assuntos
Núcleo Solitário/embriologia , Núcleo Solitário/fisiologia , Sinapses/fisiologia , Animais , Tronco Encefálico/citologia , Tronco Encefálico/embriologia , Tronco Encefálico/fisiologia , Calbindinas , Interpretação Estatística de Dados , Estimulação Elétrica , Desenvolvimento Embrionário , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Potenciais da Membrana/fisiologia , Proteínas de Neurofilamentos/metabolismo , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Glicina/agonistas , Proteína G de Ligação ao Cálcio S100/metabolismo , Núcleo Solitário/citologia , Paladar/fisiologiaRESUMO
Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract. We have used whole cell recording to investigate the characteristics of the Na(+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. GG neurons expressed two classes of Na(+) channels, TTX sensitive (TTX-S) and TTX resistant (TTX-R). The majority of GG neurons expressed TTX-R currents of different amplitudes. TTX-R currents were relatively small in 60% of the neurons but were large in 12% of the sampled population. In a further 28% of the neurons, TTX completely abolished all Na(+) currents. Application of TTX completely inhibited action potential generation in all CT and PA neurons but had little effect on the generation of action potentials in 40% of GSP neurons. Most CT, GSP, and PA neurons stained positively with IB(4), and 27% of the GSP neurons were capsaicin sensitive. The majority of IB(4)-positive GSP neurons with large TTX-R Na(+) currents responded to capsaicin, whereas IB(4)-positive GSP neurons with small TTX-R Na(+) currents were capsaicin insensitive. These data demonstrate the heterogeneity of GG neurons and indicate the existence of a subset of GSP neurons sensitive to capsaicin, usually associated with nociceptors. Since there are no reports of nociceptors in the GSP receptive field, the role of these capsaicin-sensitive neurons is not clear.
Assuntos
Potenciais de Ação/fisiologia , Gânglio Geniculado/citologia , Neurônios/fisiologia , Canais de Sódio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Biofísica , Capsaicina/farmacologia , Estimulação Elétrica , Lectinas/metabolismo , Masculino , Neurônios/classificação , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Fármacos do Sistema Sensorial/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologiaRESUMO
There is little known about the prenatal development of the rostral nucleus of the solitary tract (rNST) neurons in rodents or the factors that influence circuit formation. With morphological and electrophysiological techniques in vitro, we investigated differences in the biophysical properties of rNST neurons in pre- and postnatal rats from embryonic day 14 (E14) through postnatal day 20. Developmental changes in passive membrane and action potential (AP) properties and the emergence and maturation of ion channels important in neuron function were characterized. Morphological maturation of rNST neurons parallels changes in passive membrane properties. Mean soma size, dendritic branch points, neurite endings, and neurite length all increase prenatally. whereas neuron resting membrane potential, input resistance, and time constant decrease. Dendritic spines, on the other hand, develop after birth. AP discharge patterns alter in pre- and postnatal stages. At E14, neurons generated a single TTX-sensitive, voltage-gated Na(+) AP when depolarized; a higher discharge rate appeared at older stages. AP amplitude, half-width, and rise and fall times all change during development. Responses to current injection revealed a number of voltage-gated conductances in embryonic rNST, including a hyperpolarization-activated inward current and a low-threshold Ca(2+) current that initiated Ca(2+) spikes. A hyperpolarization-activated, transient outward potassium current was also present in the developing neurons. Although the properties of these channels change during development, they are present before synapses form and therefore, can contribute to initial establishment of neural circuits, as well as to the changing electrophysiological properties in developing rNST neurons.
Assuntos
Potenciais de Ação/fisiologia , Canais Iônicos/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Núcleo Solitário , Fatores Etários , Animais , Canais de Cálcio/fisiologia , Forma Celular/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Feminino , Idade Gestacional , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Potenciais da Membrana/fisiologia , Neuritos/fisiologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Gravidez , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/embriologia , Núcleo Solitário/crescimento & desenvolvimento , Núcleo Solitário/fisiologia , Paladar/fisiologiaRESUMO
Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract (rNST). We used whole cell recording to study the characteristics of the Ca(2+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. PA neurons were significantly larger than CT and GSP neurons, and CT neurons could be further subdivided based on soma diameter. Although all GG neurons possess both low voltage-activated (LVA) "T-type" and high voltage-activated (HVA) Ca(2+) currents, CT, GSP, and PA neurons have distinctly different Ca(2+) current expression patterns. Of GG neurons that express T-type currents, the CT and GSP neurons had moderate and PA neurons had larger amplitude T-type currents. HVA Ca(2+) currents in the GG neurons were separated into several groups using specific Ca(2+) channel blockers. Sequential applications of L, N, and P/Q-type channel antagonists inhibited portions of Ca(2+) current in all CT, GSP, and PA neurons to a different extent in each neuron group. No difference was observed in the percentage of L- and N-type Ca(2+) currents reduced by the antagonists in CT, GSP, and PA neurons. Action potentials in GG neurons are followed by a Ca(2+) current initiated after depolarization (ADP) that may influence intrinsic firing patterns. These results show that based on Ca(2+) channel expression the GG contains a heterogeneous population of sensory neurons possibly related to the type of sensory information they relay to the rNST.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Gânglio Geniculado/fisiologia , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação/fisiologia , Animais , Nervo da Corda do Tímpano/citologia , Nervo da Corda do Tímpano/fisiologia , Pavilhão Auricular/inervação , Gânglio Geniculado/citologia , Masculino , Potenciais da Membrana/fisiologia , Modelos Animais , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
The rostral nucleus of the solitary tract (rNST) plays a pivotal role in taste processing. The rNST contains projection neurons and interneurons that differ in morphology and intrinsic membrane properties. Although characteristics of the projection neurons have been detailed, similar information is lacking on the interneurons. We determined the intrinsic properties of the rNST GABAergic interneurons using a transgenic mouse model that expresses enhanced green fluorescent protein under the control of a GAD67 promoter. Glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) neurons were distributed throughout the rNST but were concentrated in the ventral subdivision with minimal interaction with the terminal field of the afferent input. Furthermore, the density of the GAD67-GFP neurons decreased in more rostral areas of rNST. In whole cell recordings, GAD67-GFP neurons responded with either an initial burst (73%), tonic (18%), or irregular (9%) discharge pattern of action potentials (APs) in response to membrane depolarization. These three groups also differed in passive and AP characteristics. Initial burst neurons had small ovoid or fusiform cell bodies, whereas tonic firing neurons had large multipolar or fusiform cell bodies. Irregular firing neurons had larger spherical soma. Some of the initial burst and tonic firing neurons were also spontaneously active. The GAD67-GFP neurons could also be categorized in subgroups based on colocalization with somatostatin and parvalbumin immunolabeling. Initial burst neurons would transmit the early dynamic portion of the encoded sensory stimuli, whereas tonic firing neurons could respond to both dynamic and static components of the sensory input, suggesting different roles for GAD67-GFP neurons in taste processing.
Assuntos
Potenciais de Ação/fisiologia , Interneurônios/fisiologia , Núcleo Solitário/citologia , Ácido gama-Aminobutírico/metabolismo , Análise de Variância , Animais , Estimulação Elétrica/métodos , Corantes Fluorescentes/metabolismo , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Interneurônios/classificação , Camundongos , Camundongos Transgênicos , Modelos Neurológicos , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp/métodos , Fosfopiruvato Hidratase/metabolismo , Somatostatina/metabolismoRESUMO
Chorda tympani (CT) and glossopharyngeal (IXth) nerves relay taste information from anterior and posterior tongue to brainstem where they synapse with second order neurons in the rostral nucleus of solitary tract (rNST). rNST neurons monosynaptically connected to afferent gustatory input were identified both by anatomical labeling and synaptic latency measures. Anterograde tracing was used to label the CT and IXth terminal fields, and neurons surrounded by fluorescent neural profiles visualized with differential interference contrast (DIC) optics in horizontal brainstem slices. Anatomically identified neurons were patch-clamped and excitatory postsynaptic currents (EPSCs) evoked by electrically stimulating the solitary tract (ST) under GABA(A) receptor blockade. Monosynaptic connections were confirmed by measures of the standard deviation of synaptic latency (jitter). rNST neurons responded to ST stimulation with either all-or-none or graded amplitude EPSCs. Most (70%) of the rNST neurons with CT input and 30% with IX input responded with all-or-none EPSCs. The remainder of the neurons with CT and IX input responded with increasing EPSC amplitudes to greater intensity stimulus shocks. EPSCs evoked in rNST neurons by increasing shock frequency to both CT and IXth nerves resulted in reduced amplitude EPSCs characteristic of frequency-dependent synaptic depression. Our results suggest that the second order rNST neurons respond to afferent input with different patterns of EPSCs that potentially influence transmission of gustatory information. Frequency-dependent synaptic depression would act as a low pass filter important in the initial processing of gustatory derived sensory messages.
Assuntos
Nervo da Corda do Tímpano/fisiologia , Nervo Glossofaríngeo/fisiologia , Células Receptoras Sensoriais/fisiologia , Núcleo Solitário/fisiologia , Paladar/fisiologia , Fibras Aferentes Viscerais/fisiologia , Animais , Nervo da Corda do Tímpano/citologia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Corantes Fluorescentes , Nervo Glossofaríngeo/citologia , Inibição Neural/fisiologia , Técnicas de Rastreamento Neuroanatômico/métodos , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/citologia , Núcleo Solitário/citologia , Coloração e Rotulagem/métodos , Transmissão Sináptica/fisiologia , Fibras Aferentes Viscerais/citologiaRESUMO
Afferent information derived from oral chemoreceptors is transmitted to second-order neurons in the rostral solitary tract nucleus (rNST) and then relayed to other CNS locations responsible for complex sensory and motor behaviors. Here we investigate the characteristics of rNST neurons sending information rostrally to the parabrachial nucleus (PBN). Afferent connections to these rNST-PBN projection neurons were identified by anterograde labeling of the chorda tympani (CT), glossopharyngeal (IX), and lingual (LV) nerves. We used voltage- and current-clamp recordings in brain slices to characterize the expression of both the transient A-type potassium current, IKA and the hyperpolarization-activated inward current, Ih, important determinants of neuronal repetitive discharge characteristics. The majority of rNST-PBN neurons express IKA, and these IKA-expressing neurons predominate in CT and IX terminal fields but were expressed in approximately half of the neurons in the LV field. rNST-PBN neurons expressing Ih were evenly distributed among CT, IX and LV terminal fields. However, expression patterns of IKA and Ih differed among CT, IX, and LV fields. IKA-expressing neurons frequently coexpress Ih in CT and IX terminal fields, whereas neurons in LV terminal field often express only Ih. After GABAA receptor block all rNST-PBN neurons responded to afferent stimulation with all-or-none excitatory synaptic responses. rNST-PBN neurons had either multipolar or elongate morphologies and were distributed throughout the rNST, but multipolar neurons were more often encountered in CT and IX terminal fields. No correlation was found between the biophysical and morphological characteristics of the rNST-PBN projection neurons in each terminal field.
Assuntos
Neurônios/fisiologia , Ponte/fisiologia , Núcleo Solitário/citologia , 4-Aminopiridina/farmacologia , Vias Aferentes/fisiologia , Aminoácidos/metabolismo , Análise de Variância , Animais , Bicuculina/farmacologia , Fenômenos Biofísicos , Toxina da Cólera/metabolismo , Nervo da Corda do Tímpano/fisiologia , Estimulação Elétrica , Antagonistas GABAérgicos/farmacologia , Nervo Glossofaríngeo/fisiologia , Técnicas In Vitro , Nervo Lingual/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Ponte/citologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/fisiologia , Potenciais Sinápticos/efeitos dos fármacos , Potenciais Sinápticos/fisiologia , Tetraetilamônio/farmacologiaRESUMO
Salivary secretion results from reflex stimulation of autonomic neurons via afferent sensory information relayed to neurons in the rostral nucleus of the solitary tract (rNST), which synapse with autonomic neurons of the salivatory nuclei. We investigated the synaptic properties of the afferent sensory connection to neurons in the inferior salivatory nucleus (ISN) controlling the parotid and von Ebner salivary glands. Mean synaptic latency recorded from parotid gland neurons was significantly shorter than von Ebner gland neurons. Superfusion of GABA and glycine resulted in a concentration-dependent membrane hyperpolarization. Use of glutamate receptor antagonists indicated that both AMPA and N-methyl-D-aspartate (NMDA) receptors are involved in the evoked excitatory postsynaptic potentials (EPSPs). Inhibitory postsynaptic potential (IPSP) amplitude increased with higher intensity ST stimulation. Addition of the glycine antagonist strychnine did not affect the amplitude of the IPSPs significantly. The GABA(A) receptor antagonist, bicuculline (BMI) or mixture of strychnine and BMI abolished the IPSPs in all neurons. IPSP latency was longer than EPSP latency, suggesting that more than one synapse is involved in the inhibitory pathway. Results show that ISN neurons receive both excitatory and inhibitory afferent input mediated by glutamate and GABA respectively. The ISN neuron response to glycine probably derives from descending connections. Difference in the synaptic characteristics of ISN neurons controlling the parotid and von Ebner glands may relate to the different function of these two glands.