Injections Of Fluoro-Gold Research Articles

Systemic administration of fluorogold for anatomical pre‐labeling of autonomic and motor neurons in the rat spinal cord compromises urodynamic recordings in acute but not long‐term studies

The use of anatomical tracer injections into peripheral tissues for retrograde labeling of spinal cord neurons may compromise physiological experiments in combined functional and morphological studies. We investigated whether a systemic injection of a retrogradely transported tracer, fluorogold (FG), may provide an alternative to direct injections into end organs for combined anatomical and physiological studies of the lower urinary tract. Urodynamic studies including cystometrogram recordings and external urethral sphincter electromyography were used as functional outcome measures. Pre-labeling of spinal cord neurons by intraperitoneal (i.p.) administration of FG resulted in a transient decrease in voiding efficiency, increase in resting pressure as well as increase in bladder size and weight at 5-7 days after the tracer administration. In contrast, there were no urodynamic or end-organ effects detected at 6-8 weeks after the i.p. injection of FG. We suggest that pre-labeling of spinal autonomic and motor neurons using i.p. administration of FG may be a useful tool when combining anatomical and functional outcome measures in long-term but not acute studies.

Spinal projections from the presumptive midbrain locomotor region in the mouse

The mesencephalic locomotor region (MLR) plays an important role in the control of locomotion, but there is ongoing debate about the anatomy of its connections with the spinal cord. In this study, we have examined the spinal projections of the mouse precuneiform nucleus (PrCnF), which lies within the boundaries of the presumptive MLR. We used both retrograde and anterograde labeling techniques. Small clusters of labeled neurons were seen in the medial portion of the PrCnF following fluoro-gold injections in the upper cervical spinal cord. Fewer labeled neurons were seen in the PrCnF after upper thoracic injections. Following the injection of anterograde tracer (biotinylated dextran amine) into the PrCnF, labeled fibers were clearly observed in the spinal cord. These fibers traveled in the ventral and lateral funiculi, and terminated mainly in the medial portions of laminae 7, 8, and 9, as well as area 10, with an ipsilateral predominance. Our observations indicate that projections from the PrCnF to the spinal cord may provide an anatomical substrate for the role of the MLR in locomotion.

Novel Model to Assess Laryngeal Function, Innervation, and Reinnervation

Laryngeal paralysis remains an unsolved problem, and the behavior of the laryngeal muscles following injury to the native innervation appears to be a function of denervation and reinnervation. The aim of the present study was to develop a reliable, accurate, and multifaceted animal model for study of laryngeal function, innervation, and reinnervation. A spontaneous-breathing anesthesia technique, suspension laryngoscopy, endoscopic evaluation of the rat larynx, and transoral injection of a retrograde neuronal tracer, hydroxystilbamidine (FluoroGold), were developed. We submitted 14 rats to the developed technique to map the brain stem projections of the superior laryngeal nerve and the recurrent laryngeal nerve and to determine the feasibility and accuracy of the endoscopic injection technique. This endoscopic technique provided full evaluation of the rat larynx. We performed transoral endoscopic injection of FluoroGold and transcervical application of the tracer to transected superior laryngeal and recurrent laryngeal nerves in 14 different rats and successfully created a neural projection map for the superior laryngeal nerve, the recurrent laryngeal nerve, and the cervical ganglia. A reliable, accurate model for the characterization of laryngeal function, routes of innervation, and sources of spontaneous reinnervation following recurrent laryngeal nerve resection has been developed. This stable and reproducible model can serve as a dependable tool in future investigations of laryngeal nerve injury and recovery.

Chondroitinase ABC promotes recovery of adaptive limb movements and enhances axonal growth caudal to a spinal hemisection.

A number of studies have shown that chondroitinase ABC (Ch'ase ABC) digestion of inhibitory chondroitin sulfate glycosaminoglycans significantly enhances axonal growth and recovery in rodents following spinal cord injury (SCI). Further, our group has shown improved recovery following SCI in the larger cat model. The purpose of the current study was to determine whether intraspinal delivery of Ch'ase ABC, following T10 hemisections in adult cats, enhances adaptive movement features during a skilled locomotor task and/or promotes plasticity of spinal and supraspinal circuitry. Here, we show that Ch'ase ABC enhanced crossing of a peg walkway post-SCI and significantly improved ipsilateral hindlimb trajectories and integration into a functional forelimb-hindlimb coordination pattern. Recovery of these complex movements was associated with significant increases in neurofilament immunoreactivity immediately below the SCI in the ipsilateral white (p = 0.033) and contralateral gray matter (p = 0.003). Further, the rubrospinal tract is critical in the normal cat during skilled movements that require accurate paw placements and trajectories like those seen during peg walkway crossing. Rubrospinal connections were assessed following Fluoro-Gold injections, caudal to the hemisection. Significantly more retrogradely labeled right (axotomized) red nucleus (RN) neurons were seen in Ch'ase ABC-treated (23%) compared with control-treated cats (8%; p = 0.032) indicating that a larger number of RN neurons in Ch'ase ABC-treated cats had axons below the lesion level. Thus, following SCI, Ch'ase ABC may facilitate axonal growth at the spinal level, enhance adaptive features of locomotion, and affect plasticity of rubrospinal circuitry known to support adaptive behaviors in the normal cat.

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions.

The hypothalamic paraventricular nucleus (PVN) regulates numerous homeostatic systems and functions largely under the influence of forebrain inputs. Glutamate is a major neurotransmitter in forebrain, and glutamate neurosignaling in the PVN is known to mediate many of its functions. Previous work showed that vesicular glutamate transporters (VGluTs; specific markers for glutamatergic neurons) are expressed in forebrain sites that project to the PVN; however, the extent of this presumed glutamatergic innervation to the PVN is not clear. In the present study retrograde FluoroGold (FG) labeling of PVN-projecting neurons was combined with in situ hybridization for VGluT1 and VGluT2 mRNAs to identify forebrain regions that provide glutamatergic innervation to the PVN and its immediate surround in rats, with special consideration for the sources to the anterior versus posterior PVN. VGluT1 mRNA colocalization with retrogradely labeled FG neurons was sparse. VGluT2 mRNA colocalization with FG neurons was most abundant in the ventromedial hypothalamus after anterior PVN FG injections, and in the lateral, posterior, dorsomedial, and ventromedial hypothalamic nuclei after posterior PVN injections. Anterograde tract tracing combined with VGluT2 immunolabeling showed that 1) ventromedial nucleus-derived glutamatergic inputs occur in both the anterior and posterior PVN; 2) posterior nucleus-derived glutamatergic inputs occur predominantly in the posterior PVN; and 3) medial preoptic nucleus-derived inputs to the PVN are not glutamatergic, thereby corroborating the innervation pattern seen with retrograde tracing. The results suggest that PVN subregions are influenced by varying amounts and sources of forebrain glutamatergic regulation, consistent with functional differentiation of glutamate projections.

Amygdaloid projections to the ventral striatum in mice: direct and indirect chemosensory inputs to the brain reward system

Rodents constitute good models for studying the neural basis of sociosexual behavior. Recent findings in mice have revealed the molecular identity of the some pheromonal molecules triggering intersexual attraction. However, the neural pathways mediating this basic sociosexual behavior remain elusive. Since previous work indicates that the dopaminergic tegmento-striatal pathway is not involved in pheromone reward, the present report explores alternative pathways linking the vomeronasal system with the tegmento-striatal system (the limbic basal ganglia) by means of tract-tracing experiments studying direct and indirect projections from the chemosensory amygdala to the ventral striato-pallidum. Amygdaloid projections to the nucleus accumbens, olfactory tubercle, and adjoining structures are studied by analyzing the retrograde transport in the amygdala from dextran amine and fluorogold injections in the ventral striatum, as well as the anterograde labeling found in the ventral striato-pallidum after dextran amine injections in the amygdala. This combination of anterograde and retrograde tracing experiments reveals direct projections from the vomeronasal cortex to the ventral striato-pallidum, as well as indirect projections through different nuclei of the basolateral amygdala. Direct projections innervate mainly the olfactory tubercle and the islands of Calleja, whereas indirect projections are more widespread and reach the same structures and the shell and core of nucleus accumbens. These pathways are likely to mediate innate responses to pheromones (direct projections) and conditioned responses to associated chemosensory and non-chemosensory stimuli (indirect projections). Comparative studies indicate that similar connections are present in all the studied amniote vertebrates and might constitute the basic circuitry for emotional responses to conspecifics in most vertebrates, including humans.

<b>The study of migraine model rat with photophobia </b><b>in the upper cervical spinal cord </b>

Background and purposes: Photophobia is characteristic and important sign of the migraine headache. Recently, we found that the response of dura-sensitive neuron to photic stimulation was enhanced after dura inflammation. Some previous papers also have reported that neurons located in the dorsal horn of the C2 spinal cord (C2 neurons) receive photic inputs suggesting that C2 neurons somehow involved in photophobia. However, how the C2 neurons are involved in photophobia associated with migraine is unknown. Therefore, the change in neuronal excitability of C2 nociceptive neurons was analyzed in rat model with dural inflammation. Methods: The Sprague-Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and the small hole was made in the frontal bone, and 10% mustard oil (MO) was applied to the dura surface around the transverse sinus (migraine rat). Photic stimulation was applied to the eye in migraine and vehicle-treated rats, and c-Fos like immunoreactive (LI) cells and pERK-immunoreactive (IR) cells in C2 were precisely analyzed. To identify trigeminal ganglion (TG) neurons innervating the retina, the fluorogold (FG) solution (4% in distilled water, 10 µl) was injected to the retina using a 31-gauge needle. Substance P (SP), calcitonin generelated peptide (CGRP), TRPV1 immunoreactivities were also detected in the ratina. Results: Following MO application to the dura, a large number of c-Fos-LI cells was expressed in the transition zone between subnuclei interpolaris and caudalis (Vi/Vc) zone bilaterally and was also in the ipsilateral C2. Photic stimulation to the eye in migraine rat caused an expression of pERK-IR cells in C2, and many of them also showed c-Fos immunoreactivity. FG-labeled trigeminal ganglion cells were observed in the first branch-innervated area of the TG following FG injection into the retina. Many SP, CGRP and TRPV1 immunoreactivities were detected in nerve fibers in the retina. The present findings suggested that C2 neurons are involved in photophobia associated with migraine, and also suggested that the unmyelinated trigeminal afferents innervating the retina might be one of the sources of photic information in photophobia.

Identification of NO/sGC signalling in the bulbospinal respiratory pathway after C2–C3 hemisection of the spinal cord in rats

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylyl cyclase (β1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2–C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around β1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2–C3 hemisection by a loss of β1sGC positivity. The results confirm, that β1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.

Projections of calcitonin gene-related peptide immunoreactive neurons in the vagal ganglia of the rat

We have studied the connections of calcitonin gene-related peptide immunoreactive (CGRP-ir) sensory neurons in the ganglia of the vagus nerve. Many CGRP-ir neurons were identified in the jugular ganglion located in the cranial cavity, while fewer CGRP-ir neurons were found in the nodose ganglion located at the level of the jugular foramen. Application of Fluorogold to the cut end of the cervical vagus nerve resulted in many Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Application of Fluorogold to the cut end of the subdiaphragmatic vagus nerve resulted in Fluorogold-labeled neurons mostly in the nodose ganglion with only a few labeled neurons in the jugular ganglion. Injection of Fluorogold into the heart resulted in Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Double labeling combining CGRP immunohistochemistry and Fluorogold retrograde tracing showed that in cases of both the application of Fluorogold to the cut end of the cervical vagus nerve and the injection of Fluorogold into the heart, about 40% of the Fluorogold-labeled neurons in the jugular ganglion expressed CGRP-like immunoreactivity. These results indicate that many CGRP-ir neurons in the jugular ganglion innervate the cervical and thoracic visceral organs, including the heart, but only a few CGRP-ir neurons project to the abdominal visceral organs.

Neuroanatomical Study of the A11 Diencephalospinal Pathway in the Non-Human Primate

BackgroundThe A11 diencephalospinal pathway is crucial for sensorimotor integration and pain control at the spinal cord level. When disrupted, it is thought to be involved in numerous painful conditions such as restless legs syndrome and migraine. Its anatomical organization, however, remains largely unknown in the non-human primate (NHP). We therefore characterized the anatomy of this pathway in the NHP.Methods and FindingsIn situ hybridization of spinal dopamine receptors showed that D1 receptor mRNA is absent while D2 and D5 receptor mRNAs are mainly expressed in the dorsal horn and D3 receptor mRNA in both the dorsal and ventral horns. Unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the cervical spinal enlargement labeled A11 hypothalamic neurons quasi-exclusively among dopamine areas. Detailed immunohistochemical analysis suggested that these FG-labeled A11 neurons are tyrosine hydroxylase-positive but dopa-decarboxylase and dopamine transporter-negative, suggestive of a L-DOPAergic nucleus. Stereological cell count of A11 neurons revealed that this group is composed by 4002±501 neurons per side. A 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication with subsequent development of a parkinsonian syndrome produced a 50% neuronal cell loss in the A11 group.ConclusionThe diencephalic A11 area could be the major source of L-DOPA in the NHP spinal cord, where it may play a role in the modulation of sensorimotor integration through D2 and D3 receptors either directly or indirectly via dopamine formation in spinal dopa-decarboxylase-positives cells.

Tuberoinfundibular peptide of 39 residues modulates the mouse hypothalamic-pituitary-adrenal axis via paraventricular glutamatergic neurons.

Neurons in the subparafascicular area at the caudal border of the thalamus that contain the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39) densely innervate several hypothalamic areas, including the paraventricular nucleus (PVN). These areas contain a matching distribution of TIP39's receptor, the parathyroid hormone receptor 2 (PTH2R). Frequent PTH2R coexpression with a vesicular glutamate transporter (VGlut2) suggests that TIP39 could presynaptically regulate glutamate release. By using immunohistochemistry we found CRH-ir neurons surrounded by PTH2R-ir fibers and TIP39-ir axonal projections in the PVN area of the mouse brain. Labeling hypothalamic neuroendocrine neurons by peripheral injection of fluorogold in PTH2R-lacZ knock-in mice showed that most PTH2Rs are on PVN and peri-PVN interneurons and not on neuroendocrine cells. Double fluorescent in situ hybridization revealed a high level of coexpression between PTH2R and VGlut2 mRNA by cells located in the PVN and nearby brain areas. Local TIP39 infusion (100 pmol) robustly increased pCREB-ir in the PVN and adjacent perinuclear zone. It also increased plasma corticosterone and decreased plasma prolactin. These effects of TIP39 on pCREB-ir, corticosterone, and prolactin were abolished by coinfusion of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonopentanoic acid (AP-5; 30 pmol each) and were absent in PTH2R knockout mice. Basal plasma corticosterone was slightly decreased in TIP39 knockout mice just before onset of their active phase. The present data indicate that the TIP39 ligand/PTH2 receptor system provides facilitatory regulation of the hypothalamic-pituitary-adrenal axis via hypothalamic glutamatergic neurons and that it may regulate other neuroendocrine systems by a similar mechanism.

Deficits in bladder function following spinal cord injury vary depending on the level of the injury

Loss of bladder function is an important consequence of a spinal cord injury (SCI) but is rarely assessed in animal studies of SCI. Here, we use a simple outcome measure (volume of retained urine) to assess bladder dysfunction over time following moderate contusion injuries at 3 different thoracic levels (T1, T4, or T9) and complete crush injuries (T1 vs. T9). The volume of urine retained in the bladder was measured daily for fourteen days post injury by anesthetizing the animals with isoflurane, expressing the bladder, and weighing the urine. To compare bladder deficits with the degree of impairment of hindlimb motor function, locomotion was assessed using the BBB open field rating scale. Rats with contusions at T4 and T9 exhibited bladder impairments reflected by increased urine retention from 1 to 12 days post injury. In contrast, rats with contusions at T1 exhibited minimal deficits (smaller volumes of retained urine). Lesion size and overall functional impairment were comparable between groups based on quantitative assessments of lesion area at the epicenter and BBB locomotor scores. Moreover, a sector analysis of sparing of different portions of the white matter revealed no differences in sparing of different funiculi between the groups. Injections of Fluorogold into lumbar segments led to retrograde labeling of a larger number of neurons in the pontine micturition center (PMC) following T1 injury when compared to T4 or T9. Thus, moderate contusion lesions at T1 spare a critical descending pathway able to mediate at least reflex voiding in rats.

Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality

PurposeThe number of retinal ganglion cells (RGC) is often used as an outcome measure in neuroprotection. The gold standard for staining RGC is retrograde labeling, e.g. with fluorogold (FG). However, this method alone does not permit to differentiate between viable and dead cells, because dying cells only avoid being counted once they have undergone complete microglial-phagocytosis. To differentiate between viable and dead but still existent RGC, we additionally stained FG-labeled RGC with calcein-acetoxymethylester (CAM). MethodsThe left optic nerves of rats were crushed 6 days after stereotactical injection of FG into both superior colliculi. The right eyes served as controls. Retinal whole mounts were prepared 2, 5, 8 or 11 days after optic nerve crush (ONC), and incubated for 30min in culture media containing 0.01% CAM. RGC densities were determined in defined areas at different eccentricities under a fluorescence microscope using the appropriate filters. Twice-positive RGC were counted after merging both filters. ResultsThe loss of RGC induced by ONC is identified earlier when these cells are detected by FG+CAM rather than by FG-labeling alone. The percentages of FG-positive RGC stained with CAM were 83% in controls, 68% on day 2, 48% on day 5, 26% on day 8, and 9% on day 11 after ONC. The decay rate of FG-prelabeled RGC appears accelerated and becomes more linear when only viable RGC positive for CAM are counted. ConclusionsThe staining of FG-prelabeled RGC with CAM permits the discrimination between dead and viable RGC in retinal whole mounts, which enables to quantify RGC degeneration earlier after injury than by using microglial-phagocytosis-dependant retrograde labeling alone.

Loss of propriospinal neurons after spinal contusion injury as assessed by retrograde labeling

We studied the number, location and size of long descending propriospinal tract neurons (LDPT), located in the cervical enlargement (C3–C6 spinal levels), and short thoracic propriospinal neurons (TPS), located in mid-thoracic spinal cord (T5–T7 spinal levels), 2, 6 and 16 weeks following a moderate low thoracic (T9) spinal cord contusion injury (SCI; 25 mm weight drop) and subsequent injections of fluorogold into the upper lumbosacral enlargement (L2–L4 spinal levels). Retrograde labeling showed that ∼23% of LDPT and 10% of TPS neurons were labeled 2 weeks after SCI, relative to uninjured animals. No additional significant decrease in number of labeled LDPT and TPS cells was found at the later time points examined, indicating that the maximal loss of propriospinal neurons in these two subpopulations occurs within the first 2 weeks post-SCI. The distribution of labeled cells post-moderate SCI was similar to normal in terms of their location within the gray matter. However, there was a significant change in the size (cross sectional area) of labeled neurons following injury, relative to uninjured controls, indicating a loss in the number of the largest class of propriospinal neurons. Interestingly, the number of labeled LDPT and TPS neurons was not significantly different following different injury severities. Although the rostro–caudal extent of the lesion site expanded between 2 and 16 weeks following injury, there was no significant difference in the number of propriospinal neurons that could be retrogradely labeled at these time points. Possible reasons for these findings are discussed.

Urocortin 2 increases c-Fos expression in serotonergic neurons projecting to the ventricular/periventricular system

Serotonin plays an important role in the regulation of anxiety states and physiological responses to aversive stimuli. Intracerebroventricular (i.c.v.) injection of the stress- and anxiety-related neuropeptide urocortin 2 (Ucn 2) increases c-Fos expression in serotonergic neurons in the dorsal (DRD) and caudal (DRC) parts of the dorsal raphe nucleus. These regions contain a subset of serotonergic neurons that projects via the dorsal raphe periventricular tract to periventricular structures, including the subfornical organ and ependymal layer, and to the ventricular system. To determine if Ucn 2 activates ventricle/periventricular-projecting serotonergic neurons in the midbrain raphe complex, we made i.c.v. injections of the retrograde tracer Fluoro-Gold into the lateral ventricle, followed 7 days later by i.c.v. injection of Ucn 2. The DRD at − 8.18 mm and the DRC at −8.54 mm and −9.16 mm bregma were analyzed using a combined bright field and immunofluorescence technique. Approximately 40% of the ventricle/periventricular-projecting neurons in the subdivisions sampled were serotonergic. Urocortin 2 increased c-Fos expression in ventricle/periventricular-projecting serotonergic neurons in the DRC and in non-ventricle/periventricular-projecting serotonergic neurons in the DRD and DRC. Of the total population of ventricle/periventricular-projecting serotonergic neurons in the DRC at −8.54 and −9.16 mm bregma, 35% expressed c-Fos following Ucn 2 injections. These data are consistent with previous studies showing that i.c.v. injection of Ucn 2 activates subpopulations of serotonergic neurons restricted to the mid-rostrocaudal DRD and DRC and further demonstrate that these include both subsets of serotonergic neurons that do and do not project to the ventricle/periventricular system.

Spinally‐projecting C1 neurons are not responsible for increased arterial pressure after nasal stimulation

Nasal stimulation (NS) can produce a sympathetically‐mediated increase in arterial blood pressure (BPa). Our objective was to determine if the increased BPa seen after NS occurs through activation of spinally‐projecting C1 RVLM neurons. Using Fos (a marker for activated neurons) and PNMT (a marker for C1 neurons), we found that NS activates both C1 and non‐C1 RVLM neurons. Only 7–24% of activated RVLM neurons were C1 neurons. Next we used Fos and PNMT immunohistochemistry with Fluorogold injections into the T3 IML to identify spinally‐projecting neurons activated by NS. Of all spinally‐projecting RVLM neurons, 10% were activated non‐C1 neurons, while 4% were activated C1 neurons. Of all activated RVLM neurons, 12% were non‐C1 spinally‐projecting neurons, while 4% were C1 spinally‐projecting neurons. Of all C1 neurons, 9% were activated and spinally‐projecting. Only 2% of all RVLM neurons were spinally‐projecting C1 neurons activated by NS. Finally, anti‐DβH‐saporin was injected into the T3 IML to selectively kill spinally‐projecting catecholaminergic neurons. In 3 of 12 rats, 77% of the spinally‐projecting C1 neurons within the RVLM were selectively killed. We found that successful ablation of spinally‐projecting C1 neurons did not eliminate the cardiovascular responses to NS. We therefore conclude that spinally‐projecting C1 neurons are not responsible for the increased BPa seen after NS.

Alteration of Primary Afferent Activity following Inferior Alveolar Nerve Transection in Rats

BackgroundIn order to evaluate the neural mechanisms underlying the abnormal facial pain that may develop following regeneration of the injured inferior alveolar nerve (IAN), the properties of the IAN innervated in the mental region were analyzed.ResultsFluorogold (FG) injection into the mental region 14 days after IAN transection showed massive labeling of trigeminal ganglion (TG). The escape threshold to mechanical stimulation of the mental skin was significantly lower (i.e. mechanical allodynia) at 11-14 days after IAN transection than before surgery. The background activity, mechanically evoked responses and afterdischarges of IAN Aδ-fibers were significantly higher in IAN-transected rats than naive. The small/medium diameter TG neurons showed an increase in both tetrodotoxin (TTX)-resistant (TTX-R) and -sensitive (TTX-S) sodium currents (INa) and decrease in total potassium current, transient current (IA) and sustained current (IK) in IAN-transected rats. The amplitude, overshoot amplitude and number of action potentials evoked by the depolarizing pulses after 1 μM TTX administration in TG neurons were significantly higher, whereas the threshold current to elicit spikes was smaller in IAN-transected rats than naive. Resting membrane potential was significantly smaller in IAN-transected rats than that of naive.ConclusionsThese data suggest that the increase in both TTX-S INa and TTX-R INa and the decrease in IA and Ik in small/medium TG neurons in IAN-transected rats are involved in the activation of spike generation, resulting in hyperexcitability of Aδ-IAN fibers innervating the mental region after IAN transection.

Origins of glutamatergic terminals in the inferior colliculus identified by retrograde transport and expression of VGLUT1 and VGLUT2 genes

Terminals containing vesicular glutamate transporter (VGLUT) 2 make dense axosomatic synapses on tectothalamic GABAergic neurons. These are one of the three types of glutamatergic synapses in the inferior colliculus (IC) identified by one of three combinations of transporter protein: VGLUT1 only, VGLUT2 only, or both VGLUT1 and 2. To identify the source(s) of these three classes of glutamatergic terminals, we employed the injection of Fluorogold (FG) into the IC and retrograde transport in combination with in situ hybridization for VGLUT1 and VGLUT2 mRNA. The distribution of FG-positive soma was consistent with previous reports. In the auditory cortex, all FG-positive cells expressed only VGLUT1. In the IC, the majority of FG-positive cells expressed only VGLUT2. In the intermediate nucleus of the lateral lemniscus, most FG-positive cells expressed VGLUT2, and a few FG-positive cells expressed both VGLUT1 and 2. In the superior olivary complex, the majority of FG-positive cells expressing VGLUT2 were in the lateral superior olive, medial superior olive, and some periolivary nuclei. Fewer FG-positive cells expressed VGLUT1&2. In the ventral cochlear nucleus, almost all FG-positive cells expressed VGLUT1&2. On the other hand in the dorsal cochlear nucleus, the vast majority of FG-positive cells expressed only VGLUT2.Our data suggest that (1) the most likely sources of VGLUT2 terminals in the IC are the intermediate nucleus of the lateral lemniscus, the dorsal cochlear nucleus, the medial and lateral superior olive, and the IC itself, (2) VGLUT1 terminals in the IC originate only in the ipsilateral auditory cortex, and (3) VGLUT1&2 terminals in IC originate mainly from the VCN with minor contributions from the SOC and the lateral lemniscal nuclei.

Origins of Glutamatergic Terminals in the Inferior Colliculus Identified by Retrograde Transport and Expression of VGLUT1 and VGLUT2 Genes

Terminals containing vesicular glutamate transporter (VGLUT) 2 make dense axosomatic synapses on tectothalamic GABAergic neurons. These are one of the three types of glutamatergic synapses in the inferior colliculus (IC) identified by one of three combinations of transporter protein: VGLUT1 only, VGLUT2 only, or both VGLUT1 and 2. To identify the source(s) of these three classes of glutamatergic terminals, we employed the injection of Fluorogold (FG) into the IC and retrograde transport in combination with in situ hybridization for VGLUT1 and VGLUT2 mRNA. The distribution of FG-positive soma was consistent with previous reports. In the auditory cortex, all FG-positive cells expressed only VGLUT1. In the IC, the majority of FG-positive cells expressed only VGLUT2. In the intermediate nucleus of the lateral lemniscus, most FG-positive cells expressed VGLUT2, and a few FG-positive cells expressed both VGLUT1 and 2. In the superior olivary complex (SOC), the majority of FG-positive cells expressing VGLUT2 were in the lateral superior olive, medial superior olive, and some periolivary nuclei. Fewer FG-positive cells expressed VGLUT1&2. In the ventral cochlear nucleus, almost all FG-positive cells expressed VGLUT1&2. On the other hand in the dorsal cochlear nucleus, the vast majority of FG-positive cells expressed only VGLUT2. Our data suggest that (1) the most likely sources of VGLUT2 terminals in the IC are the intermediate nucleus of the lateral lemniscus, the dorsal cochlear nucleus, the medial and lateral superior olive, and the IC itself, (2) VGLUT1 terminals in the IC originate only in the ipsilateral auditory cortex, and (3) VGLUT1&2 terminals in IC originate mainly from the VCN with minor contributions from the SOC and the lateral lemniscal nuclei.

Calcitonin gene-related peptide immunoreactive neurons innervating the soft palate, the root of tongue, and the pharynx in the superior glossopharyngeal ganglion of the rat

We have examined whether calcitonin gene-related peptide immunoreactive (CGRP-ir) neurons in the glossopharyngeal ganglia innervate the soft palate, the root of tongue, and the pharynx of the rat. Immunohistochemical observations revealed that numerous CGRP-ir neurons are located in the superior glossopharyngeal ganglion located ventrolateral to the medulla oblongata in the cranial cavity, and that CGRP-ir neurons are also located in the inferior glossopharyngeal ganglion at the jugular foramen. When Fluorogold was injected into the soft palate, the root of tongue, or the pharyngeal constrictor muscles, many retrogradely Fluorogold-labeled neurons were found in the superior glossopharyngeal ganglion and the nodose ganglion, and several Fluorogold-labeled neurons were found in the inferior glossopharyngeal ganglion. Double labeling with immunohistochemistry for CGRP and Fluorogold showed that in every case of injections of Fluorogold into the soft palate, the root of tongue, or the pharynx, about 30% of the Fluorogold-labeled neurons in the superior glossopharyngeal ganglion expressed CGRP-like immunoreactivity, while no double-labeled neurons were found in the inferior glossopharyngeal ganglion or the nodose ganglion. These results indicate that nociceptive sensory information from the soft palate, the root of tongue, and the pharynx might be conveyed by the neurons in the superior glossopharyngeal ganglion to the nucleus tractus solitarii.