Analgesic and Neuroprotective Effects of Electroacupuncture in a Dental Pulp Injury Model-A Basic Research.

Irreversible pulpitis is an extremely painful condition and its consequence in the central nervous system (CNS) remains unclear. A mouse model of dental pulp injury (DPI) resembles the irreversible pulpitis profile in humans. This study sought to determine whether pain induced by DPI activates microglia and astrocytes in the trigeminal subnucleus caudalis (Vc), as well as increases levels of proinflammatory cytokines, and whether electroacupuncture (EA) can be a potential analgesic and neuroprotective therapy following DPI. Pain behavior was measured via head-withdrawal threshold (HWT) and burrowing behavior at days 1, 3, 7, 14 and 21 after DPI. A marked decrease in HWT and burrowing activity was observed from day 1 to 14 after DPI and no changes were seen on day 21. Microglial and astrocytes activation; along with high cytokine (TNFα, IL-1ß, and IL-6) levels, were observed in the Vc at 21 days after DPI. These effects were attenuated by verum (local and distal) EA, as well as oral ibuprofen administration. The results suggest that DPI-induced pain and glial activations in the Vc and EA exert analgesic efficacy at both local and distal acupoints. Furthermore, verum (local and distal) EA might be associated with the modulations of microglial and astrocytes activation.

Differential expression of VGLUT1 or VGLUT2 in the trigeminothalamic or trigeminocerebellar projection neurons in the rat.

The vesicular glutamate transporters, VGLUT1 and VGLUT2, reportedly display complementary distribution in the rat brain. However, co-expression of them in single neurons has been reported in some brain areas. We previously found co-expression of VGLUT1 and VGLUT2 mRNAs in a number of single neurons in the principal sensory trigeminal nucleus (Vp) of the adult rat; the majority of these neurons sent their axons to the thalamic regions around the posteromedial ventral nucleus (VPM) and the posterior nuclei (Po). It is well known that trigeminothalamic (T-T) projection fibers arise not only from the Vp but also from the spinal trigeminal nucleus (Vsp), and that trigeminocerebellar (T-C) projection fibers take their origins from both of the Vp and Vsp. Thus, in the present study, we examined the expression of VGLUT1 and VGLUT2 in Vp and Vsp neurons that sent their axons to the VPM/Po regions or the cortical regions of the cerebellum. For this purpose, we combined fluorescence in situ hybridization (FISH) histochemistry with retrograde tract-tracing; immunofluorescence histochemistry was also combined with anterograde tract-tracing. The results indicate that glutamatergic Vsp neurons sending their axons to the cerebellar cortical regions mainly express VGLUT1, whereas glutamatergic Vsp neurons sending their axons to the thalamic regions express VGLUT2. The present data, in combination with those of our previous study, indicate that glutamatergic Vp neurons projecting to the cerebellar cortical regions express mainly VGLUT1, whereas the majority of glutamatergic Vp neurons projecting to the thalamus co-express VGLUT1 and VGLUT2.

GABA-mimetic actions of Withania somnifera on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice.

The plant Withania somnifera (WS), also known as Ashwagandha, has been used widely in traditional medicine systems in India and Nepal (Ayurveda), and has been accepted to cure various ailments. In this study, the whole-cell patch clamp technique was performed to examine the mechanism of action of WS on the SG neurons of the Vc from mouse brainstem slices. In whole-cell patch clamp mode, methanol extract of Withania somnifera (mWS) induced short-lived and repeatable inward currents in all SG neurons tested (31.3 ± 8.51 pA, n = 7) using a high chloride pipette solution. The mWS-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated Na (+) channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, AP5, an NMDA receptor antagonist and strychnine, a glycine receptor antagonist. The mWS induced currents were blocked by picrotoxin, a GABAA receptor antagonist. These results show that mWS has an inhibitory effects on SG neurons of the Vc through GABAA receptor-mediated activation of chloride ion channels, indicating that mWS contains compounds with sedative effects on the central nervous system. These results also suggest that mWS may be a potential target for modulating orofacial pain processing.

Itch and analgesia resulting from intrathecal application of morphine: contrasting effects on different populations of trigeminothalamic tract neurons.

Intrathecal application of morphine is among the most powerful methods used to treat severe chronic pain. However, this approach commonly produces itch sufficiently severe that patients are forced to choose between relief of pain or itch. The neuronal populations responsible for processing and transmitting information underlying itch caused by intrathecal application of morphine have not been identified and characterized. We describe two populations of antidromically identified trigeminothalamic tract (VTT) neurons in anesthetized rats that are differentially affected by morphine and explain several aspects of opioid-induced itch and analgesia. We found that intrathecal application of morphine increased ongoing activity of itch-responsive VTT neurons. In addition, intrathecal application of morphine increased responses to pruritogens injected into the skin and greatly heightened responses to innocuous mechanical stimuli. In contrast, the ongoing activity and responses to noxious pinches in nociceptive VTT neurons were frequently inhibited by the same dose of morphine. These results reveal that i.t. application of morphine affects specific subpopulations of VTT neurons in ways that may produce itch, hyperknesis, alloknesis, and analgesia.

A translational in vivo model of trigeminal autonomic cephalalgias: therapeutic characterization.

Trigeminal autonomic cephalalgias are highly disabling primary headache disorders, characterized by severe unilateral head pain and associated ipsilateral cranial autonomic features. There is limited understanding of their pathophysiology and how and where treatments act to reduce symptoms; this is significantly hindered by a lack of animal models. We have developed the first animal model to explore trigeminal autonomic cephalalgias, using stimulation within the brainstem, at the level of the superior salivatory nucleus, to activate the trigeminal autonomic reflex arc. Using electrophysiological recording of neurons of the trigeminocervical complex and laser Doppler blood flow changes around the ipsilateral lacrimal duct, superior salivatory nucleus stimulation exhibited both neuronal trigeminovascular and cranial autonomic manifestations. These responses were specifically inhibited by the autonomic ganglion blocker hexamethonium bromide. These data demonstrate that brainstem activation may be the driver of both sensory and autonomic symptoms in these disorders, and part of this activation may be via the parasympathetic outflow to the cranial vasculature. Additionally, both sensory and autonomic manifestations were significantly inhibited by highly effective treatments for trigeminal autonomic cephalalgias, such as oxygen, indomethacin and triptans, and some part of their therapeutic action appears to be specifically on the parasympathetic outflow to the cranial vasculature. Treatments more used to migraine, such as naproxen and a calcitonin gene-related peptide receptor inhibitor, olcegepant, were less effective in this model. This is the first model to represent the phenotype of trigeminal autonomic cephalalgias and their response to therapies, and indicates the parasympathetic pathway may be uniquely involved in their pathophysiology and targeted to relieve symptoms.

The possible additional role of the cystic fibrosis transmembrane regulator to motoneuron inhibition produced by glycine effects.

In the present work we study the contribution of the chloride channel of the Cystic Fibrosis Transmembrane Regulator (CFTR) in the postsynaptic inhibition of somatic motoneurons during rapid-eye-movement (REM) sleep atonia. Postsynaptic inhibition of motoneurons is partially responsible for the atonia that occurs during REM sleep. Disfacilitation is an additional mechanism that lowers motoneuron excitability in this state. Postsynaptic inhibition is mediated by the release of glycine from synaptic terminals on motoneurons, and by GABA that plays a complementary role to that of glycine. In this work we look in brain stem motoneurons of neonatal rats at a mechanism unrelated to the actions of glycine, GABA or to disfacilitation which depends on the chloride channel of the CFTR. We studied the presence of CFTR by immunocytochemistry. In electrophysiological experiments utilizing whole cell recordings in in vitro slices we examined the consequences of blocking this chloride channel. The effects on motoneurons of the application of glycine, of the application of glibenclamide (a CFTR blocker) and again of glycine during the effects of glibenclamide were studied. Glycine produced an hyperpolarization, a decrease in motoneuron excitability and a decrease in input resistance, all characteristic changes of the postsynaptic inhibition produced by this neurotransmitter. Glibenclamide produced an increase in input resistance and in motoneurons' repetitive discharge as well as a shift in the equilibrium potential for chloride ions as indicated by the displacement of the reversal potential for glycinergic actions. In motoneurons treated with glibenclamide, glycine produced postsynaptic inhibition but this effect was smaller when compared to that elicited by glycine in control conditions. The fact that blocking of the CFTR-chloride channel in brain stem motoneurons influences glycinergic inhibition suggests that this channel may play a complementary role in the glycinergic inhibition that occurs during REM sleep.

The transcription factor, Lmx1b, is necessary for the development of the principal trigeminal nucleus-based lemniscal pathway.

Little is known of transcriptional mechanisms underlying the development of the trigeminal (V) principal sensory nucleus (PrV), the brainstem nucleus responsible for the development of the whisker-to-barrel cortex pathway. Lmx1b, a LIM homeodomain transcription factor, is expressed in embryonic PrV. In Lmx1b knockout ((-)(/)(-)) mice, V primary afferent projections to PrV are normal, albeit reduced in number, whereas the PrV-thalamic lemniscal pathway is sparse and develops late. Excess cell death occurs in the embryonic Lmx1b(-)(/)(-) PrV, but not in Lmx1b/Bax double null mutants. Expression of Drg11, a downstream transcription factor essential for PrV development and pattern formation, is abolished in PrV, but not in the V ganglion. Consequently, whisker patterns fail to develop in PrV by birth. Rescued PrV cells in Lmx1b/Bax double (-)(/)(-)s failed to rescue whisker-related PrV pattern formation. Thus, Lmx1b and Drg11 may act in the same genetic signaling pathway that is essential for PrV pattern formation.

Thalamic afferent and efferent connectivity to cerebral cortical areas with direct projections to identified subgroups of trigeminal premotoneurons in the rat.

The roles of supramedullary brain mechanisms involved in the control of jaw movements are not fully understood. To address this issue, a series of retrograde (Fluorogold, FG) and anterograde (biotinylated dextran amine, BDA) tract-tracing studies were done in rats. At first, we identified projection patterns from defined sensorimotor cortical areas to subgroups of trigeminal premotoneurons that are located in defined brainstem areas. Focal injections of FG into these brainstem areas revealed that the rostralmost part of lateral agranular cortex (rmost-Agl), the rostralmost part of medial agranular cortex (rmost-Agm), and the rostralmost part of primary somatosensory cortex (rmost-S1) preferentially project to brainstem areas containing jaw-closing premotoneurons, jaw-opening premotoneurons and a mixture of both types of premotoneurons, respectively. The thalamic reciprocal connectivities to rmost-Agl, rmost-Agm, and rmost-S1 were then investigated following cortical injections of FG or BDA. We found many retrogradely FG-labeled neurons and large numbers of axons and terminals labeled anterogradely with BDA in the dorsal thalamus mainly on the side ipsilateral to the injection sites. The rmost-Agl had strong connections with the ventral lateral nucleus (VL), ventromedial nucleus (VM), parafascicular nucleus, and posterior nucleus (Po); the rmost-Agm with the ventral anterior nucleus, VL, VM, central lateral nucleus, paracentral nucleus, central medial nucleus, mediodorsal nucleus and Po; and the rmost-S1 with the ventral posteromedial nucleus and Po. The present results suggest that the descending multiple pathways from the cerebral cortex to jaw-closing and jaw-opening premotoneurons have unique functional roles in jaw movement motor control.

Effects of CO2 laser irradiation of the gingiva during tooth movement.

Patients often feel pain or discomfort in response to orthodontic force. It was hypothesized that CO(2) laser irradiation may reduce the early responses to nociceptive stimuli during tooth movement. The distribution of Fos-immunoreactive (Fos-IR) neurons in the medullary dorsal horn of rats was evaluated. Two hrs after tooth movement, Fos-IR neurons in the ipsilateral part of the medullary dorsal horn increased significantly. CO(2) laser irradiation to the gingiva just after tooth movement caused a significant decrease of Fos-IR neurons. PGP 9.5- and CGRP-positive nerve fibers were observed in the PDL of all study groups. The maximum temperature below the mucosa during CO(2) laser irradiation was less than 40 degrees C. It was suggested that CO(2) laser irradiation reduced the early responses to nociceptive stimuli during tooth movement and might not have adverse effects on periodontal tissue.

Trigeminothalamic barrelette neurons: natural structural side asymmetries and sensory input-dependent plasticity in adult rats.

In the rodent trigeminal principal nucleus (Pr5) the barrelette thalamic-projecting neurons relay information from individual whiskers to corresponding contralateral thalamic barreloids. Here we investigated the presence of lateral asymmetries in the dendritic trees of these neurons, and the morphometric changes resulting from input-dependent plasticity in young adult rats. After retrograde labeling with dextran amines from the thalamus, neurons were digitally reconstructed with Neurolucida, and metrically and topologically analyzed with NeuroExplorer. The most unexpected and remarkable result was the observation of side-to-side asymmetries in the barrelette neurons of control rats. These asymmetries more significantly involved the number of low-grade trees and the total dendritic length, which were greater on the left side. Chronic global input loss resulting from infraorbital nerve (IoN) transection, or loss of active touch resulting from whisker clipping in the right neutralized, or even reversed, the observed lateral differences. While results after IoN transection have to be interpreted in the context of partial neuron death in this model, profound bilateral changes were found after haptic loss, which is achieved without inflicting any nerve damage. After whisker trimming, neurons on the left side closely resembled neurons on the right in controls, the natural dendritic length asymmetry being reversed mainly by a shortening of the left trees and a more moderate elongation of the right trees. These results demonstrate that dendritic morphometry is both side- and input-dependent, and that unilateral manipulation of the sensory periphery leads to bilateral morphometric changes in second order neurons of the whisker-barrel system. The presence of anatomical asymmetries in neural structures involved in early stages of somatosensory processing could help explain the expression of sensory input-dependent behavioral asymmetries.

Influence of subcortical inhibition on barrel cortex receptive fields.

Influence of subcortical inhibition on barrel cortex receptive fields. By the time neural responses driven by vibrissa stimuli reach the barrel cortex, they have undergone significant spatial and temporal transformations within subcortical relays. A major regulator of these transformations is thought to be subcortical GABA-mediated inhibition, but the actual degree of this influence is unknown. We used disinhibition produced by GABA receptor antagonists to unmask the excitatory sensory responses that are normally suppressed by inhibition in the main subcortical sensory relays to barrel cortex; principal trigeminal (Pr5) and primary thalamic (VPM) nuclei. We found that, within subcortical relays, inhibition only slightly suppresses short-latency receptive field responses, but robustly suppresses long-latency center and surround receptive field responses. However, the long-latency subcortical effects of inhibition are mostly not reflected in the barrel cortex. The most robust effect of subcortical inhibition on barrel cortex responses is to transiently suppress the receptive field responses of high-frequency sensory stimuli. This transient adaptation caused by subcortical inhibition recovers within a few stimuli and gives way to a steady-state adaptation that is independent of subcortical inhibition.

Posterior lateral hypothalamic axon terminals are in contact with trigeminal premotor neurons in the parvicellular reticular formation of the rat medulla oblongata.

This study was performed to understand the anatomical substrates of hypothalamic modulation of jaw movements. After cholera toxin B subunit (CTb) injection into the parvicellular reticular formation (RFp) of the rat medulla oblongata, where many trigeminal premotor neurons have been known to exist, numerous CTb-labeled neurons were found in the posterior lateral hypothalamus (PLH) bilaterally with a clear-cut ipsilateral dominance. After ipsilateral injections of biotinylated dextran amine (BDA) into the PLH and CTb into the motor trigeminal nucleus (Vm), the prominent distribution of BDA-labeled axon terminals around CTb-labeled neurons was found in the RFp region just ventral to the nucleus of the solitary tract and medial to the spinal trigeminal nucleus ipsilateral to the injection sites. Within the neuropil of the RFp, BDA-labeled axon terminals made an asymmetrical synaptic contact predominantly with dendrites and additionally with somata of the RFp neurons, some of which were labeled with CTb. It was further revealed that these BDA-labeled axon terminals were immunoreactive for vesicular glutamate transporter 2. The present data suggest that the PLH plays an important role in the control of jaw movements by exerting its glutamatergic excitatory action upon RFp neurons presynaptic to trigeminal motoneurons.

Instructive role of a peripheral pattern for the central patterning of the trigeminal projection at the brainstem and thalamus revealed by an artificially altered whisker pattern.

The central patterning mechanism of neuronal circuits is an important issue in developmental neuroscience. We report here the role of a peripheral whisker pattern for the patterning of the trigeminal projection at the brainstem and thalamus in the mouse somatosensory system. The whisker pattern was manipulated by infecting the embryonic epidermis with adenovirus harboring Shh. The ectopic expression of Shh led to the induction of extra whiskers and displacement of whiskers, where these whiskers were histologically normal. The altered whisker pattern was isomorphically represented in the brainstem (barrelette: subnuclei principalis and subnuclei interpolaris), thalamus (barreloid) and cortex (barrel) as revealed by cytochrome oxidase staining. The barrelette-like pattern of the parvalbumin became discernible by immunostaining at P7 in subnuclei principalis and at P4 in subnuclei interpolaris in normal mice. These are the barrelette neurons projecting to the thalamus and the local circuit within the barrelette. The barrelette-like parvalbumin pattern also exhibits the altered whisker pattern induced by the adenovirus harboring Shh. These results highlight the role the peripheral whisker pattern for the central patterning of the brainstem, thalamus, and cortex in the mouse somatosensory system.

Occipital afferent activation of second order neurons in the trigeminocervical complex in rat.

Stimulation of the greater occipital nerve produces excitation of second order neurons in the trigeminocervical complex. Given that neck pain is very common in primary headache disorders, this convergent excitation may play a role in pain referral from cervical structures. While previous studies have demonstrated a physiological model for this convergence, this study sought an anatomical approach to examine the distribution of second order neurons in the trigeminocervical complex receiving greater occipital nerve input. In addition, the role of glutamatergic NMDA receptor activation within the trigeminocervical complex in response to cervical afferents was studied. Noxious stimulation of the occipital muscle in rat using mustard oil and mineral oil produced significantly altered Fos expression in the trigeminocervical complex compared with the surgical control (H(4)=31.3, P<0.001, Kruskal-Wallis). Baseline expression was 11 (median, range 4, 17) fos positive cells in the trigeminocervical complex, occipital muscle treated with mustard oil produced 23 (17, 33) and mineral oil a smaller effect of 19 (15, 25) fos positive cells, respectively (P=0.046). The effects of both mustard and mineral oil were reversed by the NMDA-receptor antagonist MK801. This study introduces a model for examining trigeminocervical complex activity after occipital afferent stimulation in the rat that has good anatomical resolution and demonstrates involvement of glutamatergic NMDA receptors at this important synapse.

Synaptic Ras GTPase activating protein regulates pattern formation in the trigeminal system of mice.

The development of ordered connections or "maps" within the nervous system is a common feature of sensory systems and is crucial for their normal function. NMDA receptors are known to play a key role in the formation of these maps; however, the intracellular signaling pathways that mediate the effects of glutamate are poorly understood. Here, we demonstrate that SynGAP, a synaptic Ras GTPase activating protein, is essential for the anatomical development of whisker-related patterns in the developing somatosensory pathways in rodent forebrain. Mice lacking SynGAP show only partial segregation of barreloids in the thalamus, and thalamocortical axons segregate into rows but do not form whisker-related patches. In cortex, layer 4 cells do not aggregate to form barrels. In Syngap(+/-) animals, barreloids develop normally, and thalamocortical afferents segregate in layer 4, but cell segregation is retarded. SynGAP is not necessary for the development of whisker-related patterns in the brainstem. Immunoelectron microscopy for SynGAP from layer 4 revealed a postsynaptic localization with labeling in developing postsynaptic densities (PSDs). Biochemically, SynGAP associates with the PSD in a PSD-95-independent manner, and Psd-95(-/-) animals develop normal barrels. These data demonstrate an essential role for SynGAP signaling in the activity-dependent development of whisker-related maps selectively in forebrain structures indicating that the intracellular pathways by which NMDA receptor activation mediates map formation differ between brain regions and developmental stage.

Formation of whisker-related principal sensory nucleus-based lemniscal pathway requires a paired homeodomain transcription factor, Drg11.

Little is known about the molecular mechanisms underlying the formation of the principal sensory nucleus (PrV) of the trigeminal nerve, a major relay station for somatotopic pattern formation in the trigeminal system. Here, we show that mice lacking Drg11, a homeodomain transcription factor, exhibit defects within the PrV, which include an aberrant distribution of Drg11-/- cells, altered expression of a molecular marker, unusual projections of primary afferents from trigeminal ganglion cells, and, subsequently, increased cell death. In addition, surviving PrV cells exhibit delayed and more spatially restricted ascending projections to the ventral posterior medial nucleus of the thalamus (VPm). These early embryonic abnormalities in the PrV lead to the failure to develop whisker-related patterns in the PrV, VPm, and somatosensory cortex. By contrast, somatotopic patterns exist in the spinal trigeminal subnuclei interpolaris (SpVi) and subnuclei caudalis (SpVc) and the dorsal column nucleus-based lemniscal and cortical pathway. Thus, the deficits in the trigeminal system of Drg11-/- mice are specific to the PrV. Our results demonstrate that Drg11 is essential for proper cellular differentiation and, subsequently, for the formation of the whisker-related lemniscal and cortical structures.

Coeruleotrigeminal suppression of nociceptive sensorimotor function during inflammation in the craniofacial region of the rat.

Descending action from the locus coeruleus (LC) on the trigeminal sensorimotor function was evaluated in a rat model of oral-facial inflammation. For the induction of oral-facial inflammation, mustard oil (20% solution in 20microl mineral oil) was injected into the region of the temporomandibular joint (TMJ). One week before testing, rats received bilateral lesions of the LC using a cathodal current. The electromyogram (EMG) threshold, which is the threshold intensity for the onset of EMG activity of the masseter muscle evoked by pressure on the TMJ region, was used in the present study as an indicator of the trigeminal sensorimotor function. Following mustard oil injection, in the LC-lesioned rats, EMG thresholds significantly decreased at 30min, which lasted up to 240min. In contrast, EMG thresholds in the LC-intact rats returned to the level before injection after 180min. Systemic naloxone (1.3mg/kg, i.v.) produced a further decrease of EMG thresholds in both the LC-intact and LC-lesioned rats. Under the existence of naloxone, EMG thresholds in the LC-lesioned rats were significantly lower than those of the LC-intact rats. These results suggest that oral-facial inflammation activates the coeruleotrigeminal modulating system and that an action of this system is independent of the opioid depressive mechanism.

Differences between SHR and WKY following the airpuff startle stimulus in the number of Fos expressing, RVLM projecting neurons.

The neurocircuitry responsible for excessive stress-induced cardiovascular responses in genetic hypertensive rats remains elusive. Prior studies detailed a differential cardiovascular response profile to airpuff startle stimuli between Spontaneously Hypertensive (SHR) and Wistar Kyoto (WKY) rats. We recently identified strain differential Fos expression in the rostroventrolateral medulla (RVLM) and several RVLM projecting sites following airpuff startle. The current study sought to define RVLM projecting neurons that also express Fos following placement in the test chamber and administration of the airpuff startle stimulus. Unilateral iontophoretic micro-injections of fluorogold were made into the RVLM of 9-10 week old SHR and WKY rats. Two to three weeks later, animals were subjected to a series of 60 airpuff startle stimuli. Brains were double labeled for Fos and fluorogold. Single fluorogold and single Fos cells, and double labeled cells were found in the nucleus tractus solitarius (NTS), caudal ventral lateral medulla (CVLM), Kölliker fuse (KF), ventral lateral, lateral, and dorsal central gray, lateral hypothalamus (LH), and paraventricular nucleus of the hypothalamus (PVN). These data are consistent with the notion that the RVLM receives differential excitatory and/or inhibitory input from higher brain centers, perhaps contributing to differential Fos expression in the RVLM, differential autonomic responding, or both.

Contributions of the vestibular nucleus and vestibulospinal tract to the startle reflex.

The startle reflex is elicited by strong and sudden acoustic, vestibular or trigeminal stimuli. The caudal pontine reticular nucleus, which mediates acoustic startle via the reticulospinal tract, receives further anatomical connections from vestibular and trigeminal nuclei, and can be activated by vestibular and tactile stimuli, suggesting that this pontine reticular structure could mediate vestibular and trigeminal startle. The vestibular nucleus, however, also projects to the spinal cord directly via the vestibulospinal tracts, and therefore may mediate vestibular startle via additional faster routes without a synaptic relay in the hindbrain. In the present study, the timing properties of the vestibular efferent pathways mediating startle-like responses were examined in rats using electrical stimulation techniques. Transient single- or twin-pulse electrical stimulation of the vestibular nucleus evoked bilateral, startle-like responses with short refractory periods. In chloral hydrate-anesthetized rats, hindlimb electromyogram latencies recorded from the anterior biceps femoris muscle were shorter than those for stimulation of the trigeminal nucleus, and similar to those for stimulation of the caudal pontine reticular nucleus or ventromedial medulla. In awake rats, combining vestibular nucleus stimulation with either acoustic stimulation or trigeminal nucleus stimulation enhanced the whole-body startle-like responses and led to strong cross-modal summation without collision effects. In both chloral hydrate-anesthetized and awake rats, combining vestibular nucleus stimulation with ventromedial medulla stimulation produced a symmetrical collision effect, i.e. a loss of summation at the same positive and negative stimulus intervals, indicating a continuous connection between the vestibular nucleus and ventromedial medulla in mediating vestibular startle. By contrast, combining trigeminal nucleus stimulation with ventromedial medulla stimulation resulted in an asymmetric collision effect when the trigeminal nucleus stimulation preceded ventromedial medulla stimulation by 0.5 ms, suggesting that a monosynaptic connection between the trigeminal nucleus and ventromedial medulla mediates trigeminal startle. We propose that the vestibulospinal tracts participate strongly in mediating startle produced by activation of the vestibular nucleus. The convergence of the vestibulospinal tracts with the reticulospinal tract within the spinal cord therefore provides the neural basis of cross-modal summation of startling stimuli.

Projections from subnucleus oralis of the spinal trigeminal nucleus to contralateral thalamus via the relay of juxtatrigeminal nucleus and dorsomedial part of the principal sensory trigeminal nucleus in the rat.

Following injection of HRP into contralateral thalamus, retrogradely labeled cells were observed in principal sensory trigeminal nucleus (Vp) and an area of juxtatrigeminal nucleus (JX) formerly described by John and Tracey (1987). When PHA-L was delivered to dorsomedial part of the subnucleus oralis (Vodm), PHA-L labeled terminals were seen in dorsomedial part of the Vp (Vpdm) and in the JX region. Comparing the distribution of PHA-L labeled terminal field with that of HRP labeled JX neurons showed that the labeled terminals and neurons were overlapped closely in the JX. The distribution patterns of the labeled terminals and JX neurons were also the same: viewed on the coronal planes caudal-rostrally, both of the labelings began to appear at the levels where the facial nerve root was just broken. Rostrally, at middle levels of the motor trigeminal nucleus (Vmo), the labelings showed their typical view covering dorsal and ventral JX (dJX, vJX). The labelings disappeared at rostral poles of the Vmo and Vp. When injections of PHA-L into the Vodm and HRP into the contralateral thalamus was made in one rat, the contacts between Vodm projecting terminals labeled with PHA-L and HRP labeled trigemino-thalamic neurons were seen in the JX and also in the Vpdm. Then, electron microscopic (EM) study was done, injections of kainic acid into the Vodm and HRP into the contralateral thalamus was performed simultaneously. After EM embedding, the JX and Vpdm regions were selected, ultrathin sections were cut and observed with EM. In both areas, axo-somatic and axo-dendritic synapses were seen between degenerated boutons and HRP labeled somata or dendrites. Namely, the Vodm projecting terminals synapsed on trigemino-thalamic neurons in the JX and Vpdm. Anyway, axo-dendritic synapses was the main type of observed synapses. Thus, the present work demonstrated 1. the JX containing a group of trigemno-thalamic neurons was a target of special projections froin the Vodm; 2. The Vodm neurons projected to the contralateral thalamus through the relay of JX and Vpdm neurons.