Screen of anti-migraine active compounds from Duijinsan by spectrum-effect relationship analysis and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Duijinsan (DJS) is a famous Chinese medicine prescription composed of Radix scutellariae (RS) and Rhei Radix (RRR), which has been mainly used for treating migraine. AIM OF THE STUDY: This study aimed to uncover the anti-migraine active compounds from DJS and preliminary predicted the pharmacological mechanism by evaluating the spectrum-effect relationship between high-performance liquid chromatography (HPLC) fingerprints and anti-migraine effects of Duijinsan (DJS) extract combined with molecular docking. MATERIALS AND METHODS: HPLC and LC-MS were applied for chemical analyses of DJS extracts in different proportions. Inhibition of DJS extracts on trigeminal nerve cell releasing calcitonin gene related peptide (CGRP) experiment was performed. The active compounds were screened by spectrum-effect relationship analysis and confirmed by molecular docking and the activities of major predicted compounds were validated in vitro. RESULTS: Twenty-six common peaks were assigned and identified from the fingerprints of different proportions DJS extracts. In vitro experimental results showed that DJS extracts inhibited inflammation and release of CGRP from trigeminal nerve cells. Five predicted active compounds, Chrysin 6-C-arabinoside 8-C-glucoside, Chrysin 6-C-glucoside 8-C-arabinoside, baicalin, Chrysin-7-O-Beta-D-glucoronide and Oroxylin A 7-O-glucuronide were sorted out according to spectrum-effect relationship analysis and molecular docking comprehensively. In vitro validation experiments showed that all the predicted compounds inhibited the CGRP releasing and the activation of TRPV1 channel. Baicalin, chrysin-7-O-ß-D-glucuronide and Oroxylin A-7-glucoronide significantly inhibited the activation of TRPV1 channel. CONCLUSION: Chrysin 6-C-arabinoside 8-C-glucoside, Chrysin 6-C-glucoside 8-C-arabinoside, baicalin, Chrysin-7-O-Beta-D-glucoronide and Oroxylin A 7-O-glucuronide which can inhibit the CGRP releasing and the activation of TRPV1 channel were screened as the anti-migraine active compounds by spectrum-effect relationship analysis and molecular docking.

Negative Modulation of TRPM8 Channel Function by Protein Kinase C in Trigeminal Cold Thermoreceptor Neurons.

TRPM8 is the main molecular entity responsible for cold sensing. This polymodal ion channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. In corneal cold thermoreceptor neurons (CTNs), TRPM8 expression determines not only their sensitivity to cold, but also their role as neural detectors of ocular surface wetness. Several reports suggest that Protein Kinase C (PKC) activation impacts on TRPM8 function; however, the molecular bases of this functional modulation are still poorly understood. We explored PKC-dependent regulation of TRPM8 using Phorbol 12-Myristate 13-Acetate to activate this kinase. Consistently, recombinant TRPM8 channels, cultured trigeminal neurons, and free nerve endings of corneal CTNs revealed a robust reduction of TRPM8-dependent responses under PKC activation. In corneal CTNs, PKC activation decreased ongoing activity, a key parameter in the role of TRPM8-expressing neurons as humidity detectors, and also the maximal cold-evoked response, which were validated by mathematical modeling. Biophysical analysis indicated that PKC-dependent downregulation of TRPM8 is mainly due to a decreased maximal conductance value, and complementary noise analysis revealed a reduced number of functional channels at the cell surface, providing important clues to understanding the molecular mechanisms of how PKC activity modulates TRPM8 channels in CTNs.

Oral thermosensing by murine trigeminal neurons: modulation by capsaicin, menthol and mustard oil.

KEY POINTS: Orosensory thermal trigeminal afferent neurons respond to cool, warm, and nociceptive hot temperatures with the majority activated in the cool range. Many of these thermosensitive trigeminal orosensory afferent neurons also respond to capsaicin, menthol, and/or mustard oil (allyl isothiocyanate) at concentrations found in foods and spices. There is significant but incomplete overlap between afferent trigeminal neurons that respond to oral thermal stimulation and to the above chemesthetic compounds. Capsaicin sensitizes warm trigeminal thermoreceptors and orosensory nociceptors; menthol attenuates cool thermoresponses. ABSTRACT: When consumed with foods, mint, mustard, and chili peppers generate pronounced oral thermosensations. Here we recorded responses in mouse trigeminal ganglion neurons to investigate interactions between thermal sensing and the active ingredients of these plants - menthol, allyl isothiocyanate (AITC), and capsaicin, respectively - at concentrations found in foods and commercial hygiene products. We carried out in vivo confocal calcium imaging of trigeminal ganglia in which neurons express GCaMP3 or GCAMP6s and recorded their responses to oral stimulation with thermal and the above chemesthetic stimuli. In the V3 (oral sensory) region of the ganglion, thermoreceptive neurons accounted for ∼10% of imaged neurons. We categorized them into three distinct classes: cool-responsive and warm-responsive thermosensors, and nociceptors (responsive only to temperatures ≥43-45 °C). Menthol, AITC, and capsaicin also elicited robust calcium responses that differed markedly in their latencies and durations. Most of the neurons that responded to these chemesthetic stimuli were also thermosensitive. Capsaicin and AITC increased the numbers of warm-responding neurons and shifted the nociceptor threshold to lower temperatures. Menthol attenuated the responses in all classes of thermoreceptors. Our data show that while individual neurons may respond to a narrow temperature range (or even bimodally), taken collectively, the population is able to report on graded changes of temperature. Our findings also substantiate an explanation for the thermal sensations experienced when one consumes pungent spices or mint.

Response characteristics of pruriceptive and nociceptive trigeminoparabrachial tract neurons in the rat.

We tested the possibility that the trigeminoparabrachial tract (VcPbT), a projection thought to be importantly involved in nociception, might also contribute to sensation of itch. In anesthetized rats, 47 antidromically identified VcPbT neurons with receptive fields involving the cheek were characterized for their responses to graded mechanical and thermal stimuli and intradermal injections of pruritogens (serotonin, chloroquine, and ß-alanine), partial pruritogens (histamine and capsaicin), and an algogen (mustard oil). All pruriceptive VcPbT neurons were responsive to mechanical stimuli, and more than half were additionally responsive to thermal stimuli. The majority of VcPbT neurons were activated by injections of serotonin, histamine, capsaicin, and/or mustard oil. A subset of neurons were inhibited by injection of chloroquine. The large majority of VcPbT neurons projected to the ipsilateral and/or contralateral external lateral parabrachial and Kölliker-Fuse nuclei, as evidenced by antidromic mapping techniques. Analyses of mean responses and spike-timing dynamics of VcPbT neurons suggested clear differences in firing rates between responses to noxious and pruritic stimuli. Comparisons between the present data and those previously obtained from trigeminothalamic tract (VcTT) neurons demonstrated several differences in responses to some pruritogens. For example, responses of VcPbT neurons to injection of serotonin often endured for nearly an hour and showed a delayed peak in discharge rate. In contrast, responses of VcTT neurons endured for roughly 20 min and no delayed peak of firing was noted. Thus the longer duration responses to 5-HT and the delay in peak firing of VcPbT neurons better matched behavioral responses to stimulation in awake rats than did those of VcTT neurons. The results indicate that VcPbT neurons may have important roles in the signaling of itch as well as pain.

Perception of specific trigeminal chemosensory agonists.

The intranasal trigeminal system is a third chemical sense in addition to olfaction and gustation. As opposed to smell and taste, we still lack knowledge on the relationship between receptor binding and perception for the trigeminal system. We therefore investigated the sensitivity of the intranasal trigeminal system towards agonists of the trigeminal receptors TRPM8 and TRPA1 by assessing subjects' ability to identify which nostril has been stimulated in a monorhinal stimulation design. We summed the number of correct identifications resulting in a lateralization score. Stimuli were menthol (activating TRPM8 receptors), eucalyptol (TRPM8), mustard oil (TRPA1) and two mixtures thereof (menthol/eucalyptol and menthol/mustard oil). In addition, we examined the relationship between intensity and lateralization scores and investigated whether intensity evaluation and lateralization scores of the mixtures show additive effects. All stimuli were correctly lateralized significantly above chance. Across subjects the lateralization scores for single compounds activating the same receptor showed a stronger correlation than stimuli activating different receptors. Although single compounds were isointense, the mixture of menthol and eucalyptol (activating only TRPM8) was perceived as weaker and was lateralized less accurately than the mixture of menthol and mustard oil (activating both TRPM8 and TRPA1) suggesting suppression effects in the former mixture. In conclusion, sensitivity of different subpopulations of trigeminal sensory neurons seems to be related, but only to a certain degree. The large coherence in sensitivity between various intranasal trigeminal stimuli suggests that measuring sensitivity to one single trigeminal chemical stimulus may be sufficient to generally assess the trigeminal system's chemosensitivity. Further, for stimuli activating the same receptor a mixture suppression effect appears to occur similar to that observed in the other chemosensory systems.

Coexpression of VGLUT1 and VGLUT2 in trigeminothalamic projection neurons in the principal sensory trigeminal nucleus of the rat.

VGLUT1 and VGLUT2 have been reported to show complementary distributions in most brain regions and have been assumed to define distinct functional elements. In the present study, we first investigated the expression of VGLUT1 and VGLUT2 in the trigeminal sensory nuclear complex of the rat by dual-fluorescence in situ hybridization. Although VGLUT1 and/or VGLUT2 mRNA signals were detected in all the nuclei, colocalization was found only in the principal sensory trigeminal nucleus (Vp). About 64% of glutamatergic Vp neurons coexpressed VGLUT1 and VGLUT2, and the others expressed either VGLUT1 or VGLUT2, indicating that Vp neurons might be divided into three groups. We then injected retrograde tracer into the thalamic regions, including the posteromedial ventral nucleus (VPM) and posterior nuclei (Po), and observed that the majority of both VGLUT1- and VGLUT2-expressing Vp neurons were retrogradely labeled with the tracer. We further performed anterograde labeling of Vp neurons and observed immunoreactivies for anterograde tracer, VGLUT1, and VGLUT2 in the VPM and Po. Most anterogradely labeled axon terminals showed immunoreactivities for both VGLUT1 and VGLUT2 in the VPM and made asymmetric synapses with dendritic profiles of VPM neurons. On the other hand, in the Po, only a few axon terminals were labeled with anterograde tracer, and they were positive only for VGLUT2. The results indicated that Vp neurons expressing VGLUT1 and VGLUT2 project to the VPM, but not to the Po, although the functional differences of three distinct populations of Vp neurons, VGLUT1-, VGLUT2-, and VGLUT1/VGLUT2-expressing ones, remain unsettled.

JNK1 regulates histone acetylation in trigeminal neurons following chemical stimulation.

Trigeminal nerve fibers in nasal and oral cavities are sensitive to various environmental hazardous stimuli, which trigger many neurotoxic problems such as chronic migraine headache and trigeminal irritated disorders. However, the role of JNK kinase cascade and its epigenetic modulation of histone remodeling in trigeminal ganglion (TG) neurons activated by environmental neurotoxins remains unknown. Here we investigated the role of JNK/c-Jun cascade in the regulation of acetylation of H3 histone in TG neurons following in vitro stimulation by a neuro-inflammatory agent, mustard oil (MO). We found that MO stimulation elicited JNK/c-Jun pathway significantly by enhancing phospho-JNK1, phospho-c-Jun expression, and c-Jun activity, which were correlated with an elevated acetylated H3 histone in TG neurons. However, increases in phospho-c-Jun and c-Jun activity were significantly blocked by a JNK inhibitor, SP600125. We also found that altered H3 histone remodeling, assessed by H3 acetylation in triggered TG neurons, was reduced by SP600125. The study suggests that the activated JNK signaling in regulation of histone remodeling may contribute to neuro-epigentic changes in peripheral sensory neurons following environmental neurotoxic exposure.

Layer V neurons in mouse cortex projecting to different targets have distinct physiological properties.

Layer V pyramidal neurons are anatomically and physiologically heterogeneous and project to multiple intracortical and subcortical targets. However, because most physiological studies of layer V pyramidal neurons have been carried out on unidentified cells, we know little about how anatomical and physiological properties relate to subcortical projection site. Here we combine neuroanatomical tract tracing with whole cell recordings in mouse somatosensory cortex to test whether neurons with the same projection target form discrete subpopulations and whether they have stereotyped physiological properties. Our findings indicate that corticothalamic and -trigeminal neurons are two largely nonoverlapping subpopulations, whereas callosal and corticostriatal neurons overlap extensively. The morphology as well as the intrinsic membrane and firing properties of corticothalamic and corticotrigeminal neurons differ from those of callosal and corticostriatal neurons. In addition, we find that each class of projection neuron exhibits a unique compliment of hyperpolarizing and depolarizing afterpotentials that further suggests that cortical neurons with different subcortical targets are distinct from one another.

Smelling of odorous sex hormone-like compounds causes sex-differentiated hypothalamic activations in humans.

The anatomical pathways for processing of odorous stimuli include the olfactory nerve projection to the olfactory bulb, the trigeminal nerve projection to somatosensory and insular cortex, and the projection from the accessory olfactory bulb to the hypothalamus. In the majority of tetrapods, the sex-specific effects of pheromones on reproductive behavior is mediated via the hypothalamic projection. However, the existence of this projection in humans has been regarded as improbable because humans lack a discernable accessory olfactory bulb. Here, we show that women smelling an androgen-like compound activate the hypothalamus, with the center of gravity in the preoptic and ventromedial nuclei. Men, in contrast, activate the hypothalamus (center of gravity in paraventricular and dorsomedial nuclei) when smelling an estrogen-like substance. This sex-dissociated hypothalamic activation suggests a potential physiological substrate for a sex-differentiated behavioral response in humans.

Organization of cortical projections to the medullary subnucleus reticularis dorsalis: a retrograde and anterograde tracing study in the rat.

The distribution and organization of cortical projections to the subnucleus reticularis dorsalis (SRD), the neighboring cuneate nucleus (Cu), and trigeminal nucleus caudalis (Sp5C) were studied in the rat using microinjections of wheat germ agglutinin-apo horseradish peroxidase-gold and Biotin-Dextran. Cortical cells projecting to the caudal medulla were confined to the contralateral layer V with their descending axons crossing the midline at the level of pyramidal decussation. Cortical afferents to Sp5C originated from cells located mainly in the primary somatosensory cortex (S1) and the insular cortex, whereas cortical projections to the Cu originated mainly from the primary motor cortex (M1), the primary and secondary somatosensory cortex (S1 and S2). The SRD received dense cortical afferents from larger, widespread cortical areas: M1, M2, S1, S2, and the insular cortex. The existence of dense cortico-SRD connections supports the possibility of a pyramidal influence over SRD neurons, which might modify nociceptive information ascending to the cortex itself. This proposal is consistent with the fact that SRD efferents terminate densely in thalamic areas that influence sensorimotor cortical regions which in turn project to the SRD. Moreover, these corticofugal mechanisms could allow the cortex to select its own input by suppressing or augmenting transmission of signals through SRD-hindbrain/forebrain pathways or by coordinating activities in spino-SRD-spinal circuits and thus selecting the relevant information produced by the noxious stimulus.

Aromatase in developing sensory systems of the rat brain.

Sex differences in the rat brain are dependent, in part, on oestrogen exposure during specific developmental perinatal periods. The availability of oestrogen requires precursor androgen and the presence of intraneuronal aromatase. To examine sites of oestrogen formation and action in the brain, immunocytochemical and biochemical localization of aromatase in the rat brain were determined between embryonic day 14 and postnatal day 20. Aromatase-immunolabelled neuronal profiles were present in hypothalamic, cortical and limbic regions. Surprisingly, aromatase immunoreactivity was also observed in non-limbic regions of the immature brain where it was previously unsuspected. Among these regions, aromatase staining was robust in developing sensory systems, including primary afferents of the olfactory, trigeminal, vestibulocochlear, and visual systems. To determine whether this aromatase is functional in these systems, i.e. converts testosterone to estradiol, the trigeminal nerve was dissected from the hindbrain of perinatal animals and studied for enzyme activity by the tritium release method. The dpm/mg protein/h tritium release in these tissues equalled that of hypothalamic or limbic controls, indicating that these sensory areas are sites of in-situ estradiol synthesis. Our data suggests that aromatase (estradiol)-dependent mechanisms may play a role in the differentiation and maturation of sensory pathways, which, in turn, may contribute to sex differences in the activity of these systems.

Transient developmental expression of monoamine transporters in the rodent forebrain.

Neurons in first-order sensory thalamic nuclei have been shown to express functional plasma membrane serotonin (SERT) and vesicular monoamine (VMAT2) transporters during early postnatal development. In the present study, we provide an extensive description of the spatial and the temporal patterns of VMAT2 and SERT expression, during early embryonic development and postnatal life, by using in situ hybridization and immunocytochemistry. VMAT2 and SERT genes are transiently expressed in a wide population of non-monoaminergic neurons in the central and peripheral nervous system with a large overlap in the temporal and spatial pattern of expression of both genes. A selective pattern of expression of both genes was observed in the thalamus with expression limited to the dorsal thalamus and more particularly to primary sensory relay nuclei that convey point to point projection maps. Transient expression of the transporters was also observed in sensory cranial nerves, in the hippocampus, cerebral cortex, septum, and amygdala. VMAT2 and SERT gene expression was not necessarily linked, as some neural populations expressed only VMAT2, while others only contained SERT. Since VMAT2 serves to transport catecholamines besides serotonin, we examined the developmental expression of the plasma membrane dopamine and norepinephrine transporters but found no transient expression of these genes. Despite minor temporal disparities, VMAT2 and SERT extinguished almost simultaneously during the second and third weeks of post-natal life. These expressions did not seem to be dependent on peripheral neural inputs, since monocular enucleations and infraorbital nerve cuts effected on the day of birth, did not modify the period of transporter expression or of extinction.

Morphological development of afferent segregation and onset of synaptic transmission in the trigeminothalamic pathway of the wallaby (Macropus eugenii).

A light and electron microscopic study has been made of the time of formation of whisker-related patterns in trigeminothalamic afferents and the onset of synapse formation between afferents and cells in the ventroposteromedial nucleus (VPM) of the marsupial mammal, the wallaby, by labelling afferents with a carbocyanine dye. A parallel in vitro study was made of the functional development of the trigeminothalamic pathway to the VPM. Evoked synaptic responses could be recorded in the VPM from the time that afferents first reached the VPM at postnatal day 15 (P15). At all stages, the excitatory response comprised both N-methyl-D-aspartate- and non-N-methyl-D-aspartate-mediated components. At P40, the response decreased markedly in duration, coinciding with the onset of synaptogenesis. This implies that transmission is occurring prior to synapse formation, probably through transmitter release from growth cones. At P50, synaptic responses became dominated by a fast, non-N-methyl-D-aspartate potential, and this coincided with the first appearance of whisker-related patterns in the VPM. A gamma-aminobutyric acid (subtype A)-mediated, inhibitory component was also present from the time of afferent arrival. These findings support the idea that functional interactions between afferents and their targets may play a role in pattern formation in the somatosensory thalamus.

Distribution of nociceptive neurons in the ventrobasal complex of macaque thalamus.

In urethane-chloralose anesthetized Japanese macaques, the distribution of nociceptive neurons within the thalamic ventrobasal (VB) complex was studied. Nociceptive specific (NS) and wide dynamic range (WDR) neurons were found in the periphery of the contralateral integument compartment of the VB complex. Thus, they formed a shell at the perimeter of this compartment with a somatotopic organization. The compartment consisted of large parts of nucleus ventralis posteromedialis (VPM) and nucleus ventralis posterolateralis, pars caudalis (VPLc). NS neurons were located more caudally than WDR neurons. In the NS zone of VPM, the forehead was represented caudally, and oral structures rostrally. In the ventral NS zone of VPM, there was a sequential representation of the tongue, gum and mandibular skin from the medial to the lateral edge. The hand was represented medially in the NS zone of VPLc, and its representation dominated in the rostral NS zone. There was a sequential representation of the cervical, thoracic, lumbar, sacral and caudal segments mediolaterally along the dorsal VPLc. In the medial half of ventral NS zone of VPLc, the upper body half was represented, and in the lateral half, the lower body half. The foot was represented at or near the medial edge of lateral half. In the rostral WDR zone, the trunk and peripheral face were represented.

Lateralization and functional organization of the locus coeruleus projection to the trigeminal somatosensory pathway in rat.

The primary goals of this study were to (1) examine the distribution of locus coeruleus (LC) neurons, which project to cortical and subcortical sites along the trigeminal somatosensory pathway in rats, and (2) determine the extent to which different regions within this ascending sensory system receive collateral projections from the same LC neuron. Long-Evans hooded rats received unilateral pressure injections of different combinations of retrograde fluorescent tracers into whisker-related regions of primary (SI) and secondary (SII) somatosensory cortices, the ventrobasal (VB) and posterior group (POm) nuclei of the thalamus, and the principalis nucleus of the trigeminal complex (PrV). Coronal sections (40-100 microm) through the LC were examined by fluorescence microscopy, and the distribution of retrogradely labeled cells was recorded. The major finding was that whisker-related regions of the cortex receive efferent projections from neurons concentrated in the caudal portion of the ipsilateral LC, whereas subcortical trigeminal somatosensory structures receive bilateral input from both LC nuclei. Despite the bilateral nature of the LC projection to subcortical sites, the majority of LC efferents to VB and POm thalamus originate in the ipsilateral LC nucleus, whereas projections to PrV originate primarily from the contralateral LC. An additional finding was that a relatively large proportion of LC cells, which project to a single somatosensory structure, also send axon collaterals to other relay sites along the same ascending somatosensory pathway. Taken together, these results suggest that the LC-noradrenergic system maintains a more selective relationship with functionally related efferent targets than has been previously appreciated.

Spontaneous discharge and peripherally evoked orofacial responses of trigemino-thalamic tract neurons during wakefulness and sleep.

In the present study, ongoing and evoked activity of antidromically identified trigemino-thalamic tract (TGT) neurons was examined over the sleep-wake cycle in cats. There was no difference in the mean spike discharge rate of TGT neurons when quiet sleep (QS) and active sleep (AS) were compared with wakefulness (W). However, tooth pulp-evoked responses of TGT neurons were decreased during AS when compared to W. Conversely, the responses of TGT neurons to air puff activation of facial hair mechanoreceptors reciprocally increased during AS when compared to W. The present data demonstrate that ascending sensory information emanating from distinct orofacial areas is differentially modified during the behavioral state of AS. Specifically, the results obtained suggest that during AS, sensory information arising from hair mechanoreceptors is enhanced, whereas information arising from tooth pulp afferents is suppressed. These data may provide functional evidence for an AS-related gate control mechanism of sensory outflow to higher brain centers.

Effects of cardiac vagal afferent electrostimulation on the responses of trigeminal and trigeminothalamic neurons to noxious orofacial stimulation.

We have previously reported that electrical stimulation of cardiac vagal afferents produces an inhibition of the feline's digastric reflex evoked by tooth-pulp stimulation. In the present study, we evaluated whether cardiac vagal afferent stimulation (CVAS) alters the responses of trigeminal sensory neurons to noxious orofacial stimulation in alpha-chloralose-anesthetized cats. A total of 37 trigeminal and trigeminothalamic neurons were recorded from trigeminal nucleus caudalis and trigeminal nucleus oralis. Thirty-five of these 37 neurons were classified as wide-dynamic-range (WDR) neurons because they had cutaneous receptive fields and responded to both noxious heat and non-noxious tactile stimuli. The effects of continuous CVAS (5 Hz, 3 msec, 2 mA) on heat-evoked responses (6 sec 50 degrees C heat pulse) were examined on 32 WDR neurons. CVAS inhibited (21 of 32 neurons), facilitated (5 of 32 neurons) or did not affect neuronal responses (6 of 32 neurons) to noxious heat. The effects of CVAS on heat-evoked responses of trigeminal and trigeminothalamic neurons were equivalent. The effects of intermittent CVAS (7 pulses at 333 Hz, 5 mA, delivered 200 msec prior to the test stimulus) on the responses to electrical test stimuli delivered to the center of a neuron's cutaneous receptive field or to the tooth pulp were also examined. Intermittent CVAS inhibited (15 of 24 neurons), facilitated (4 of 24 neurons) or had no effect (5 of 24 neurons) on A delta-mediated responses evoked by the electrical stimulation of facial skin. Intermittent CVAS either inhibited (8 of 12 neurons) or had no effect (4 of 12 neurons) on C-fiber-mediated responses evoked by electrical stimulation of the facial skin. Eight cells were recorded that received tooth-pulp input. Six of these 8 cells also received afferent input from facial skin, the remaining 2 cells responded only to tooth-pulp stimulation. Intermittent CVAS either inhibited (7 of 8 neurons) or had no effect (1 of 8 neurons) on A delta-mediated responses evoked by electrical stimulation of the tooth pulp. The modulatory actions of intermittent CVAS on trigeminal and trigeminothalamic neuronal responses to convergent afferent input from both skin and tooth pulp were equivalent. The outcomes of this study provide additional evidence that cardiopulmonary vagal afferent stimulation modulates neuronal responses to noxious stimulation and suggest that alterations in cardiopulmonary dynamics may modulate nociception.

Timecourse of development of the wallaby trigeminal pathway. II. Brainstem to thalamus and the emergence of cellular aggregations.

This paper is the second in a series which makes use of the protracted postnatal maturation of the wallaby to study the development of the trigeminal sensory system. Previous work has established similarities in the organisation of the trigeminal sensory system in the wallaby and in rodents. This study describes the structure and development of the ventroposteromedial nucleus in the wallaby in relationship to the arrival of afferents from the trigeminal nuclei, the formation of neuronal aggregations and naturally occurring cell death. Enzyme histochemistry, Nissl and myelin stains were used. Pathway development was followed using carbocyanine dyes. In the adult wallaby the nucleus demonstrates evidence of a parcellated organisation. Cells are arranged in dorsoventrally aligned bands resembling fingers. In the horizontal plane, these appear as circular clusters which are encircled by fine myelinated bundles. The clusters of cells are believed to correspond to the mystacial vibrissae. The first afferents from the principal trigeminal nucleus arrive between 10 and 15 days postnatal. This is more than two weeks prior to the time at which the borders of the nucleus can be discerned cytoarchitecturally. The first hints of segmentation are visible around day 50, and discrete aggregations form over the ensuing 3-4 weeks. Coincident with the aggregation of the neurons is an increase in their level of reactivity for acetylcholinesterase. A high level of acetylcholinesterase reactivity is maintained for at least 4 months, but has disappeared in adult animals. The peak of cell death occurs subsequent to the appearance of aggregations in the thalamus, but coincident with the appearance of vibrissae related patches in the cortex at day 85 (Waite et al. [1991] Dev. Brain Res. 58:35-41). The timing of the appearance of the neuronal aggregations supports the hypothesis that pattern formation occurs sequentially at successive levels of the pathway, and suggests the importance of target maturation in pattern formation.

Vibrissal stimulation affects glucose utilization in the trigeminal/somatosensory system of normal rats and rats prenatally exposed to ethanol.

The effect of gestational ethanol exposure on stimulus-induced sensory activity in the trigeminal/somatosensory system was determined. The mature offspring of mothers fed an ethanol-containing diet (Et) or pair-fed a nutritionally matched control diet (Ct) were examined. The C-row mystacial whiskers were stimulated. Glucose utilization in the principal sensory nucleus of the trigeminal nerve (PSN), ventrobasal thalamus, and somatosensory cortex was determined with [14C]2-deoxyglucose autoradiography. In Ct- and Et-treated rats, whisker stimulation increased glucose utilization in C-row barrel(oid)s in the left PSN, the right ventrobasal thalamus, and the right somatosensory cortex. The rate of glucose utilization in the C-row barrel(oid)s and in nonstimulated regions was lower in the Et-treated rats than in controls. In the cortices of Ct-treated rats, the activity in the C-row barrels on the right side was greater than in the right nonbarrel somatosensory cortex. Et-treated rats also exhibited an increase in glucose utilization, albeit smaller than that in the Ct-treated rats. In contrast, the glucose utilization in the left B- and C-row barrels of Ct-treated rats was decreased. No such decrease was evident in the left cortices of Et-treated rats. Thus, stroking whiskers stimulates the activity of sites in the trigeminal/somatosensory system. In cortex, the definition of these sites is emphasized by depressed activity, i.e., "surround" inhibition, in sites connected via callosal or corticocortical projections. Prenatal exposure to ethanol depresses the metabolic activity regardless of the physiological state; however, the "surround" inhibition of cortical activity is eliminated by prenatal exposure to ethanol through an exuberant projection.

Immunoreactive calcium-binding protein (calbindin-D28k) in interneurons and trigeminothalamic neurons of the rat nucleus caudalis localized with peroxidase and immunogold methods.

Calbindin-D28k is a highly abundant protein found in neurons in selected brain regions, including cells in sensory systems of the brainstem. Because of its capacity to bind cytosolic Ca++, calbindin-D28k is thought to contribute to the regulation of compartmental Ca++ concentrations in neurons. In this study of the rat spinal trigeminal nucleus, calbindin-D28k was localized with immunoperoxidase and immunogold methods. Results showed that immunoreactive calbindin-D28k neurons were widely distributed to all regions of the nucleus, but were particularly numerous in the substantia gelatinosa. Some trigemino-thalamic neurons that were identified by retrograde labeling of a conjugated wheat-germ agglutinin with horseradish peroxidase also contained calbindin-D28k immunoreactivity. Most of the calbindin-D28k labeling was found in cell bodies and dendrites. Axon terminals were rarely stained. More discrete labeling with a gold-conjugated second antibody showed that the predominant site of calbindin-D28k was the matrix of the cytoplasm. Gold label was also heavily associated with euchromatin within nuclei. These findings show that immunoreactive calbindin-D28k is localized to both interneurons and projecting neurons of the spinal trigeminal nucleus. Many of these cells are likely to receive glutamatergic afferent inputs, which may act in part by increasing Ca++ flux into the neurons. Calbindin-D28k has a high capacity for buffering Ca++ and under some conditions may protect neurons against glutamate-induced excitotoxicity. We speculate that calbindin-D28k may function to regulate calcium concentrations in spinal trigeminal neurons.