Responses of cultured rat trigeminal ganglion neurons to bitter tastants

The initial steps in taste and olfaction result from the activation by chemical stimuli of taste receptor cells (TRCs) and olfactory receptor neurons (ORNs). In parallel with these two pathways is the chemosensitive trigeminal pathway whose neurons terminate in the oral and nasal cavities and which are activated by many of the same chemical stimuli that activate TRCs and ORNs. In a recent single unit study we investigated the responses of rat chorda tympani and glossopharnygeal neurons to a variety of bitter-tasting alkaloids, including nicotine, yohimbine, quinine, strychnine and caffeine, as well as capsaicin, the pungent ingredient in hot pepper. Here we apply many of these same compounds to cultured rat trigeminal ganglion (TG) neurons and measure changes in intracellular calcium [Ca2+]i to determine whether TG neurons will respond to these same compounds. Of the 89 neurons tested, 34% responded to 1 mM nicotine, 7% to 1 mM caffeine, 5% to 1 mM denatonium benzoate, 22% to 1 mM quinine hydrochloride, 18% to 1 mM strychnine and 55% to 1 microM capsaicin. These data suggest that neurons from the TG respond to the same bitter-tasting chemical stimuli as do TRCs and are likely to contribute information sent to the higher CNS regarding the perception of bitter/irritating chemical stimuli.      

Quinine and citric acid elicit distinctive Fos-like immunoreactivity in the rat nucleus of the solitary tract

The present experiment investigated Fos-like immunoreactivity (FLI) in the nucleus of the solitary tract (NST) after intraoral infusions of 0.1 M citric acid, 0.3 M NaCl, and 0.3-30 mM quinine monohydrochloride (QHCl) in awake, behaving rats. Increases in QHCl concentration produced increases in the numbers of FLI-labeled neurons in the rostral part of the intermediate (i(r)) and rostral (r) NST, but the topographic distribution of FLI was consistent across QHCl concentrations and distinctive compared with effects of citric acid. Quinine elicited FLI concentrated in the medial third of the nucleus; acid elicited more broadly distributed FLI concentrated farther laterally. Surprisingly, in contrast to QHCl and citric acid, NaCl produced FLI that was indistinguishable from that produced by water. Although the functional significance of these patterns is unknown, citric acid and QHCl are nonpreferred stimuli but produced different oromotor behaviors. QHCl (30 mM) elicited approximately 3.2 times as many gapes as citric acid (0.1 M), and acid elicited more ingestive responses. Parallel differences in FLI expression suggest that different NST regions may have distinctive roles in triggering oromotor behaviors.     

Nociceptive afferents to the premotor neurons that send axons simultaneously to the facial and hypoglossal motoneurons by means of axon collaterals

It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP) was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals.     

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fos-positive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.     

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fospositive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.     

Diverse bitter stimuli elicit highly similar patterns of Fos-like immunoreactivity in the nucleus of the solitary tract

Previous studies have demonstrated that oral stimulation with quinine elicits Fos-like immunoreactivity in the first-order gustatory nucleus, the NST, with a different topographic distribution than sucrose or citric acid. However, it is unknown whether the quinine pattern is unique to this alkaloid or common across bitter stimuli with different chemical structures. Indeed, recent physiological experiments suggest that taste receptor cells and primary afferent neurons may exhibit selectivity for various bitter tastants. The present investigation compared the distribution of FLI in NST following stimulation with three bitter chemicals: QHCl, denatonium and propylthiouracil, stimuli that evoked Ca(2+) currents in almost entirely different sets of receptor cells. The results demonstrate that the quinine pattern is not idiosyncratic but instead generalizes to the other two tastants. Although it remains possible that intermingled but different NST neurons are activated by these stimuli, these data suggest that a specialized region in the NST is preferentially involved in processing a common aspect of bitter tastants. In contrast to citric acid, quinine, denatonium and propylthiouracil all elicited vigorous oromotor rejection responses, consistent with our earlier hypothesis that the medial third of the NST may be an afferent trigger zone for oromotor rejection.     

Brainstem neuronal populations activated in the model of ovalbumine induced allergic rhinitis in guinea pigs — the c-Fos study

Central neuronal interactions may contribute to up regulation of cough in subjects with rhinitis. Previously we have shown that noxious stimulation of the nose induces considerable Fos-like immunoreactivity (FLI) in the solitary nuclei and the region of ventral respiratory group and these neurons are involved in the cough pattern generator. Recent study addressed the question, which additional groups are activated in model of trigeminal hyperresponsiveness and whether some of them might also cooperate with cough pattern gating areas. 24 guinea pigs were sensitized with intraperitoneal ovalbumin (OVA) and later were once weekly challenged with intranasal OVA to develop the neural hyperresponsiveness, 12 animals were left unsensitized. The nasal symptom score was evaluated after each challenge. Finally, animals were anaesthetized and the latest challenges with nasal OVA, capsaicin and saline were applied to induce c-Fos expression in designed groups. Following the survival time animals were deeply anaesthetized, exsanguinated, and transcardially perfused with heparinised saline (200 ml) and paraformaldehyde fixation (200 ml). The brainstems were removed, postfixed, and brainstem slices were processed immunohistochemically (c-Fos, Calbiochem, SR). FLI at the level of obex and areas relative to the obex was analyzed. In all groups (excluding the saline group) the FLI was detected bilaterally in the trigeminal complex, nuclei of solitary tract, lateral reticular and nucleus ambiguus. There were no differences between the OVA and capsaicin groups. Count of Fos-positive neurons within the trigeminal complex does not correlate with the magnitude of clinical symptoms, which gradually increased each week in OVA induced model of hyperresponsiveness. Whereas trigeminal hyperresponsiveness contributes to the up-regulation of cough in animal models, it does not induce any additional neuronal FLI at the middle medulla than observed in naive animals.     

窦内压升高和灌流腺苷激活颈动脉窦压力感受器时延髓内c-fos蛋白的表达

在14 只隔离灌流颈动脉窦区的大鼠, 观察了窦内压(ISP) 升高和灌流腺苷 (adenosine, Ado) 激活压力感受器时延髓内c-fos蛋白的表达.结果显示: 在孤束核、最后区、延髓腹外侧头端区和中缝苍白核可见Fos蛋白样免疫阳性反应(FLI)神经元分布, 且其数量随ISP升高而增多.在给定ISP下, 颈动脉窦内灌流Ado, 可使上述区域中FLI表达明显增多.根据以上结果, 得出如下结论: c-fos在压力感受器反射延髓通路中的表达, 可由ISP增高和灌流Ado而增强, 表明Ado对压力感受器反射有易化作用.     

Taste responses to amino acids in the southern leopard frog, Rana sphenocephala

Integrated taste recordings of the glossopharyngeal (IX) nerve innervating the tongue of the southern leopard frog were studied in response to various amino acids and quinine hydrochloride. Amino acids and quinine hydrochloride elicited primarily phasic taste responses. Acidic (L-aspartic and L-glutamic) and basic (L-lysine and L-arginine) amino acids, adjusted to pH8, were effective taste stimuli. All glossopharyngeal nerve twigs that responded to amino acid stimuli also responded to quinine; however, not all quinine-sensitive IX nerve bundles were responsive to amino acids. Electrophysiological thresholds for amino acids were estimated to be 2.5-10 mM, whereas threshold for quinine hydrochloride averaged approximately 10 microM.     

Neurochemical features of enkephalinergic neurons in the mouse trigeminal subnucleus caudalis

Enkephalinergic (ENKergic) neurons have been proposed to play crucial roles in pain modulation in the trigeminal subnucleus caudalis (Vc). To assist an advance in the research of ENKergic neurons, here we used preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mice, in which all ENKergic neurons were fluorescent. We first performed fluorescent in situ hybridization combined with immunofluorescent histochemistry to confirm the specificity of this transgenic mouse and its advantages in showing ENKergic neurons in the Vc. Then based on this useful transgenic mouse, we examined the phenotypic diversity of PPE-GFP neurons by immunostaining for several markers that characterize ENKergic neuron subtypes. About 25.9±1.9% of GFP-positive neurons were regarded as immunoreactive for glutamic acid decarboxylase (GAD)(67) mRNA and 14.7±1.4% of GFP-positive neurons were positive for γ-aminobutyric acid. The proportions of calbindin-, calretinin-positive cells among the ENKergic neurons were 8.4±1.2% and 7.3±1.7%, respectively. Only 1.1±0.1% of GFP-positive neurons colocalized with parvalbumin and no GFP-positive neurons were found to co-express neuronal nitric oxide synthase. We then injected retrograde tracer into the thalamic regions and observed that a small number of ENKergic neurons in the Vc were retrogradely labeled with the tracer. The present results provide a detailed morphological evidence of the neurochemical features of ENKergic neurons. These results have broad implications for understanding the functional roles of ENKergic neurotransmission in the Vc.     

大鼠三叉神经尾侧亚核内SPR阳性神经元与初级传入和下行投射纤维之间的突触联系

用追踪和免疫电镜技术研究三叉神经尾侧亚核（Ｖｃ）内Ｐ物质受体（ＳＰＲ）阳性神经元与初级传入和下行投射之间的突触联系。光镜观察发现,在Ｖｃ浅层,ＳＰＲ阳性神经元的分布与ＲＭｇ下行投射终末的分布有重叠。电镜观察发现,三叉初级传入终末和ＳＰＲ阳性神经元树突形成非对称性轴树突触;ＲＭｇ下行投射终末与ＳＰＲ阳性神经元树突也形成非对称性轴树突触,提示ＲＭｇ下行投射纤维可能通过直接作用于丘脑投射神经元对三叉初级传入的伤害性信息进行调控。     

心外膜应用腺苷时c—fos在脊髓延髓和丘脑中的表达

在１２只切断两侧缓冲神经和迷走神经的麻醉大鼠,观察了心外膜应用腺苷对脊髓,延髓和丘脑ｃ－ｆｏｓ原部基因表达的影响。结果显示：心外膜应用腺苷组大鼠,动脉血压和心率无明显变化;脊髓Ｔ３节段背角,延髓巨细胞旁外侧核以及丘脑的腹后外侧核,后核,中央外侧核和束旁核等部位Ｆｏｓ蛋白样免疫阳性反应神经元显著增加;而在溶剂对照组大鼠,仅见少数ＦＬＩ细胞。     

Novel menthol-derived cooling compounds activate primary and second-order trigeminal sensory neurons and modulate lingual thermosensitivity

We presently investigated 2 novel menthol derivatives GIV1 and GIV2, which exhibit strong cooling effects. In previous human psychophysical studies, GIV1 delivered in a toothpaste medium elicited a cooling sensation that was longer lasting compared with GIV2 and menthol carboxamide (WS-3). In the current study, we investigated the molecular and cellular effects of these cooling agents. In calcium flux studies of TRPM8 expressed in HEK cells, both GIV1 and GIV2 were approximately 40- to 200-fold more potent than menthol and WS-3. GIV1 and GIV2 also activated TRPA1 but at levels that were 400 times greater than those required for TRPM8 activation. In calcium imaging studies, subpopulations of cultured rat trigeminal ganglion and dorsal root ganglion cells responded to GIV1 and/or GIV2; the majority of these were also activated by menthol and some were additionally activated by the TRPA1 agonist cinnamaldehyde and/or the TRPV1 agonist capsaicin. We also made in vivo single-unit recordings from cold-sensitive neurons in rat trigeminal subnucleus caudalis (Vc). GIV 1 and GIV2 directly excited some Vc neurons, GIV1 significantly enhanced their responses to cooling, and both GIV1 and GIV2 reduced responses to noxious heat. These novel cooling compounds provide additional molecular tools to investigate the neural processes of cold sensation.     

Stimulation of bitterness by capsaicin and menthol: differences between lingual areas innervated by the glossopharyngeal and chorda tympani nerves

Capsaicin is viewed as a purely chemesthetic stimulus that selectively stimulates the somatosensory system. Here we show that when applied to small areas of the tongue, capsaicin can produce a bitter taste as well as sensory irritation. In experiment 1, individuals were screened for the ability to perceive bitterness from capsaicin on the circumvallate papillae. Fifteen of 25 subjects who reported at least weak bitterness rated the intensity of taste, irritation and coolness produced by 100-320 microM capsaicin and 100-320 mM menthol applied via cotton swabs to the tip (fungiform region), the posterior edge (foliate region), and the dorsal posterior surface (circumvallate region) of the tongue. Sucrose, citric acid, sodium chloride and quinine hydrochloride were applied to the same areas to assess tastes responsiveness. On average, capsaicin and menthol produced "moderate" bitterness (and no other significant taste qualities) in the circumvallate region, and weaker bitterness on the side and tip of the tongue. Sensory irritation from capsaicin was rated significantly higher at the tongue tip, whereas menthol coolness was rated higher in the circumvallate region. In experiment 2 we applied sucrose and quinine hydrochloride together with capsaicin to investigate the effects other taste stimuli might have on capsaicin's reported bitterness. As expected, adding quinine produced stronger bitterness in the circumvallate and fungiform regions, and adding sucrose significantly reduced the bitterness of capsaicin in the circumvallate region. Overall, the results suggest that capsaicin and menthol are capable of stimulating a subset of taste neurons that respond to bitter substances, perhaps via receptor-gated ion channels like those recently found in capsaicin- and menthol-sensitive trigeminal ganglion neurons, and that the glossopharyngeal nerve may contain more such neurons than the chorda tympani nerve. That some people fail to perceive bitterness from capsaicin further implies that the incidence of capsaicin-sensitive taste neurons varies across people as well as between gustatory nerves.     

Medullary CO2 chemoreceptor neuron identification by c-fos immunocytochemistry.

In a search for CO2 chemoreceptor neurons in the brain stem, we used immunocytochemistry to monitor the expression of neuronal c-fos, a marker of increased activity, after 1 h of exposure to CO2 in five groups of Sprague-Dawley rats (294 +/- 20 g): five air breathing controls, three breathing 10% CO2, three breathing 13% CO2, three breathing 15% CO2, and three breathing 15% CO2 and treated with morphine (10 mg/kg sc). After exposure the rats were anesthetized with pentobarbital sodium and perfused intracardially with 4% paraformaldehyde. The brain stem was removed and cryoprotected, and then 50-microns frozen sections were cut and immunostained for the fos protein. Brain stem fos-immunoreactive neurons were plotted and counted in the superficial 0.5 mm of the ventral medullary surface. Thirteen to 15% CO2 evoked fos-like immunoreactivity (FLI) in 321 +/- 146 neurons/rat. Significant CO2-induced labeling was confined within the superficial 150 microns: 67% of identified cells were less than 50 microns below the surface, greater than 90% between 1.0 and 3.0 mm from the midline, and approximately 60% in the rostral half of the medulla. Thirteen to 15% CO2 also evoked FLI in the area of the nucleus tractus solitarius but not in other medullary regions. Morphine (10 mg/kg sc) did not suppress high CO2-evoked FLI in either the ventral medullary surface or the nucleus tractus solitarius, although it eliminated excitement and hyperventilation. We suggest that respiratory CO2 chemoreceptor neurons can be identified in rats by their expression of c-fos after 1 h of hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monosodium glutamate but not linoleic acid differentially activates gustatory neurons in the rat geniculate ganglion

To date, only one study has examined responses to monosodium glutamate (MSG) from gustatory neurons in the rat geniculate ganglion and none to free fatty acids. Accordingly, we recorded single-cell responses from geniculate ganglion gustatory neurons in anesthetized male rats to MSG and linoleic acid (LA), as well as to sucrose, NaCl, citric acid, and quinine hydrochloride. None of the 52 neurons responded to any LA concentration. In contrast, both narrowly tuned groups of gustatory neurons (sucrose specialists and NaCl specialists) responded to MSG, as did 2 of the broadly tuned groups (NaCl generalist(I) and acid generalists). NaCl-generalist(II) neurons responded only to the highest MSG concentration and only at low rates. No neuron type responded best to MSG; rather, responses to 0.1 M MSG were significantly less than those to NaCl for Na(+) -sensitive neurons and to sucrose for sucrose specialists. Interestingly, most Na(+) -sensitive neurons responded to 0.3 M MSG at levels comparable with those to 0.1 M NaCl, whereas sucrose specialists responded to 0.1 M MSG despite being unresponsive to NaCl. These results suggest that the stimulatory effect of MSG involves activation of sweet- or salt-sensitive receptors. We propose that glutamate underlies the MSG response of sucrose specialists, whereas Na(+) -sensitive neurons respond to the sodium cation. For the latter neuron groups, the large glutamate anion may reduce the driving force for sodium through epithelial channels on taste cell membranes. The observed concentration-dependent responses are consistent with this idea, as are cross-adaptation studies using 0.1 M concentrations of MSG and NaCl in subsets of these Na(+) -sensitive neurons.     

电针刺激可在大鼠脊髓诱发Fos样蛋白的生成

本研究利用Fos蛋白的免疫组织化学方法首次报道电针“三阴交”穴位可在大鼠脊髓诱发原癌基因c-fos的表达。电针后大量Fos免疫反应(FLI)细胞出现在脊髓腰膨大的背、腹角,但标记最密集区为背角Ⅲ,Ⅳ层。在动物足部注射福尔马林产生的伤害性刺激亦可在脊髓腰膨大背、腹角诱发大量FLI细胞,但以背角Ⅰ,Ⅱ层标记最为密集。因此电针和伤害性刺激引起的脊髓c-fos表达在分布上是不同的。电针诱发的Foc蛋白可能参与针刺镇痛。     

Neurochemical properties of the synapses in the pathways of orofacial nociceptive reflexes

The brainstem premotor neurons of the facial nucleus (VII) and hypoglossal (XII) nucleus can integrate orofacial nociceptive input from the caudal spinal trigeminal nucleus (Vc) and coordinate orofacial nociceptive reflex (ONR) responses. However, the synaptoarchitectures of the ONR pathways are still unknown. In the current study, we examined the distribution of GABAergic premotor neurons in the brainstem local ONR pathways, their connections with the Vc projections joining the brainstem ONR pathways and the neurochemical properties of these connections. Retrograde tracer fluoro-gold (FG) was injected into the VII or XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the Vc. Immunofluorescence histochemical labeling for inhibitory/excitatory neurotransmitters combined with BDA/FG tracing showed that GABAergic premotor neurons were mainly distributed bilaterally in the ponto-medullary reticular formation with an ipsilateral dominance. Some GABAergic premotor neurons made close appositions to the BDA-labeled fibers coming from the Vc, and these appostions were mainly distributed in the parvicellular reticular formation (PCRt), dorsal medullary reticular formation (MdD), and supratrigeminal nucleus (Vsup). We further examined the synaptic relationships between the Vc projecting fibers and premotor neurons in the VII or XII under the confocal laser-scanning microscope and electron microscope, and found that the BDA-labeled axonal terminals that made asymmetric synapses on premotor neurons showed vesicular glutamate transporter 2 (VGluT2) like immunoreactivity. These results indicate that the GABAergic premotor neurons receive excitatory neurotransmission from the Vc and may contribute to modulating the generation of the tonic ONR.     

Effects on breathing of rostral pons glutamate injection during opossum development

To determine whether pathways from the rostral pons, capable of influencing breathing, were present in immature mammals, the excitatory amino acid glutamate (sodium salt) was pressure injected in very small volumes into the rostral pons of suckling and adult opossums. The youngest animals tested were approximately 3 wk old (1.5-2.9 g). Animals were anesthetized with the thiobarbituric acid derivative, Inactin, and the electromyogram of the diaphragm was used to assess changes in breathing rhythm and ventilatory output. Glutamate concentrations of 50, 150, and 1,000 mM were injected into the rostral pons. Active sites were generally located between parabrachial and either lateral lemniscal or trigeminal nuclei. Effects of glutamate in opossums of all ages included changes in diaphragm activity and respiratory timing over several breaths. In the youngest animals, a very high incidence of apnea occurred as an initial response (17 of 20 sites) at the 1,000 mM concentration. The high incidence of apneic response in the youngest animals suggests that strong activation of rostral pontine neurons can more easily disrupt respiratory output; a physiological circumstance of such activation might include a diving response stimulated by trigeminal afferents.     

Locally released small (non-protein) ninhydrin-reacting molecules underlie developmental differences of cultured medullary versus spinal dorsal horn neurons

Neurons located in the trigeminal subnucleus caudalis (Vc) play crucial roles in pain and sensorimotor functions in the orofacial region. Because of many anatomical and functional similarities with the spinal dorsal horn (SDH), Vc has been termed the medullary dorsal horn--analogous to the SDH. Here, we report that when compared with embryonic SDH neurons in culture, neurons isolated from the Vc region showed significantly slower growth, lower glutamate receptor activity, and more cells undergoing cell death. SDH neuron development was inhibited in co-cultures of SDH and Vc tissues while Vc neuron development was promoted by co-culture with SDH tissues. Furthermore, we identified that small (non-protein) ninhydrin-reacting molecules purified from either embryonic or post-natal Vc-conditioned medium inhibited neuronal growth whereas ninhydrin-reacting molecules from SDH-conditioned medium promoted neuronal growth. These findings suggest the involvement of locally released factors in the region-specific regulation of neuronal development in Vc and SDH, central nervous system regions playing critical roles in pain, and point to novel avenues for investigating central nervous system regionalization and for designing therapeutic approaches to manage neurodegenerative diseases and pain.