Injection Of Cholera Toxin B Subunit Research Articles

Differential innervation of tissues located at traditional acupuncture points in the rat forehead and face

ObjectivesTo compare the neural pathways associated with the tissues located at different traditional acupuncture points in the rat forehead and face using the cholera toxin B subunit (CTB) neural tracing...

Perineural capsaicin induces the uptake and transganglionic transport of choleratoxin b subunit by nociceptive c-fiber primary afferent neurons

The distribution of spinal primary afferent terminals labeled transganglionically with the choleratoxin B subunit (CTB) or its conjugates changes profoundly after perineural treatment with capsaicin. Injection of CTB conjugated with horseradish peroxidase (HRP) into an intact nerve labels somatotopically related areas in the ipsilateral dorsal horn with the exceptions of the marginal zone and the substantia gelatinosa, whereas injection of this tracer into a capsaicin-pretreated nerve also results in massive labeling of these most superficial layers of the dorsal horn. The present study was initiated to clarify the role of C-fiber primary afferent neurons in this phenomenon. In L5 dorsal root ganglia, analysis of the size frequency distribution of neurons labeled after injection of CTB-HRP into the ipsilateral sciatic nerve treated previously with capsaicin or resiniferatoxin revealed a significant increase in the proportion of small neurons. In the spinal dorsal horn, capsaicin or resiniferatoxin pretreatment resulted in intense CTB-HRP labeling of the marginal zone and the substantia gelatinosa. Electron microscopic histochemistry disclosed a dramatic, ∼10-fold increase in the proportion of CTB-HRP-labeled unmyelinated dorsal root axons following perineural capsaicin or resiniferatoxin. The present results indicate that CTB-HRP labeling of C-fiber dorsal root ganglion neurons and their central terminals after perineural treatment with vanilloid compounds may be explained by their phenotypic switch rather than a sprouting response of thick myelinated spinal afferents which, in an intact nerve, can be labeled selectively with CTB-HRP. The findings also suggest a role for GM1 ganglioside in the modulation of nociceptor function and pain.

Differential content of vesicular glutamate transporters in subsets of vagal afferents projecting to the nucleus tractus solitarii in the rat

The vagus nerve contains primary visceral afferents that convey sensory information from cardiovascular, pulmonary, and gastrointestinal tissues to the nucleus tractus solitarii (NTS). The heterogeneity of vagal afferents and their central terminals within the NTS is a common obstacle for evaluating functional groups of afferents. To determine whether different anterograde tracers can be used to identify distinct subpopulations of vagal afferents within NTS, we injected cholera toxin B subunit (CTb) and isolectin B4 (IB4) into the vagus nerve. Confocal analyses of medial NTS following injections of both CTb and IB4 into the same vagus nerve resulted in labeling of two exclusive populations of fibers. The ultrastructural patterns were also distinct. CTb was found in both myelinated and unmyelinated vagal axons and terminals in medial NTS, whereas IB4 was found only in unmyelinated afferents. Both tracers were observed in terminals with asymmetric synapses, suggesting excitatory transmission. Because glutamate is thought to be the neurotransmitter at this first primary afferent synapse in NTS, we determined whether vesicular glutamate transporters (VGLUTs) were differentially distributed among the two distinct populations of vagal afferents. Anterograde tracing from the vagus with CTb or IB4 was combined with immunohistochemistry for VGLUT1 or VGLUT2 in medial NTS and evaluated with confocal microscopy. CTb-labeled afferents contained primarily VGLUT2 (83%), whereas IB4-labeled afferents had low levels of vesicular transporters, VGLUT1 (5%) or VGLUT2 (21%). These findings suggest the possibility that glutamate release from unmyelinated vagal afferents may be regulated by a distinct, non-VGLUT, mechanism.

Targeted ablation of mesenteric projecting sympathetic neurons reduces the hemodynamic response to pain in conscious, spinal cord-transected rats.

Individuals with spinal cord injuries above thoracic level 6 (T(6)) experience episodic bouts of life-threatening hypertension as part of a condition termed autonomic dysreflexia. The paroxysmal hypertension can be caused by a painful stimulus below the level of the injury. Targeted ablation of mesenteric projecting sympathetic neurons may reduce the severity of autonomic dysreflexia by reducing sympathetic activity. Therefore, cholera toxin B subunit (CTB) conjugated to saporin (SAP; a ribosomal inactivating protein that binds to and inactivates ribosomes) was injected into the celiac ganglion to test the hypothesis that targeted ablation of mesenteric projecting sympathetic neurons reduces the pressor response to pain in conscious, spinal cord-transected rats. Nine Sprague-Dawley male rats underwent a spinal cord transection between thoracic vertebrae 4 and 5. Following recovery (5 wk), all rats were instrumented with a radio telemetry device for recording arterial pressure and bilateral catheters in the gluteus maximus muscles for the infusion of hypertonic saline (hNa(+)Cl(-)). Subsequently, the hemodynamic responses to intramuscular injection of hNa(+)Cl(-) (100 microl and 250 microl, in random order) were determined. Following the experiments in the no celiac ganglia injected condition (NGI), rats received injections of CTB-SAP (n = 5) or CTB (n = 3) into the celiac ganglia. CTB-SAP rats, compared with NGI and CTB rats, had reduced pressor responses to hNa(+)Cl(-). Furthermore, the number of stained neurons in the celiac ganglia and spinal cord (segments T(6)-T(12)), was reduced in CTB-SAP rats. Thus, CTB-SAP retrogradely transported from the celiac ganglia is effective at ablating mesenteric projecting sympathetic neurons and reducing the pressor response to pain in spinal cord-transected rats.

Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.

Exposure to an open-field arena increases c-Fos expression in a subpopulation of neurons in the dorsal raphe nucleus, including neurons projecting to the basolateral amygdaloid complex

Serotonergic systems in the dorsal raphe nucleus are thought to play an important role in the regulation of anxiety states. To investigate responses of neurons in the dorsal raphe nucleus to a mild anxiety-related stimulus, we exposed rats to an open-field, under low-light or high-light conditions. Treatment effects on c-Fos expression in serotonergic and non-serotonergic cells in the midbrain raphe nuclei were determined 2 h following open-field exposure or home cage control (CO) conditions. Rats tested under both light conditions responded with increases in c-Fos expression in serotonergic neurons within subdivisions of the midbrain raphe nuclei compared with CO rats. However, the total numbers of serotonergic neurons involved were small suggesting that exposure to the open-field may affect a subpopulation of serotonergic neurons. To determine if exposure to the open-field activates a subset of neurons in the midbrain raphe complex that projects to forebrain circuits regulating anxiety states, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons projecting to the basolateral amygdaloid complex (BL) in combination with c-Fos immunostaining to identify cells that responded to open-field exposure. Rats received a unilateral injection of CTb into the BL. Seven to 11 days following CTb injection rats were either, 1) exposed to an open-field in low-light conditions, 2) briefly handled or 3) left undisturbed in home cages. Dual immunostaining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunoreactive BL-projecting neurons in open-field-exposed rats compared with handled and control rats. Dual immunostaining for tryptophan hydroxylase and CTb revealed that a majority (65%) of BL-projecting neurons were serotonergic, leaving open the possibility that activated neurons were serotonergic, non-serotonergic, or both. These data are consistent with the hypothesis that exposure to anxiogenic stimuli activates a subset of neurons in the midbrain raphe complex projecting to amygdala anxiety circuits.

Exposure to an open-field arena increases c-Fos expression in a distributed anxiety-related system projecting to the basolateral amygdaloid complex

Anxiety states and anxiety-related behaviors appear to be regulated by a distributed and highly interconnected system of brain structures including the basolateral amygdala. Our previous studies demonstrate that exposure of rats to an open-field in high- and low-light conditions results in a marked increase in c-Fos expression in the anterior part of the basolateral amygdaloid nucleus (BLA) compared with controls. The neural mechanisms underlying the anatomically specific effects of open-field exposure on c-Fos expression in the BLA are not clear, however, it is likely that this reflects activation of specific afferent input to this region of the amygdala. In order to identify candidate brain regions mediating anxiety-induced activation of the basolateral amygdaloid complex in rats, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons with direct afferent projections to this region in combination with c-Fos immunostaining to identify cells responding to exposure to an open-field arena in low-light (8–13 lux) conditions (an anxiogenic stimulus in rats). Adult male Wistar rats received a unilateral microinjection of 4% CTb in phosphate-buffered saline into the basolateral amygdaloid complex. Rats were housed individually for 11 days after CTb injections and handled (HA) for 2 min each day. On the test day rats were either, 1) exposed to an open-field in low-light conditions (8–13 lux) for 15 min (OF); 2) briefly HA or 3) left undisturbed (control). We report that dual immunohistochemical staining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunopositive basolateral amygdaloid complex-projecting neurons in open-field-exposed rats compared with HA and control rats in the ipsilateral CA1 region of the ventral hippocampus, subiculum and lateral entorhinal cortex. These data are consistent with the hypothesis that exposure to the open-field arena activates an anxiety-related neuronal system with convergent input to the basolateral amygdaloid complex.

CVL‐projecting NTS neurons receive input from a distinct subset of vagal afferents

The vagus nerve contains primary visceral afferents that convey information from cardiovascular, pulmonary, and gastrointestinal tissues to the nucleus tractus solitarii (NTS). We previously showed that two tract tracers, cholera toxin B subunit (CTb) and isolectin B4 (IB4), label distinct populations of vagal afferents. In this study, we tested whether these fibers contact different populations of NTS neurons, including NTS neurons that project to the caudal ventrolateral medulla (CVL). To label CVL‐projecting NTS neurons, the retrograde tracer Fluorogold (2% in 0.9% saline; 50 nl, Fluorochrome Inc.) was injected into CVL; vagal afferents were labeled with injections of CTb and IB4 (2% solution, 1–4 ul) into the vagus nerve. Male Sprague‐Dawley rats (350–425 g) were used and surgeries were performed under isoflurane anesthesia. Seven days later, rats were perfused and vibratome sections through NTS were examined using dual immunocytochemistry for Fluorogold (rabbit anti‐FG, Chemicon) and either CTb (goat anti‐CTb, List Biological Laboratories) or IB4 (goat anti‐IB4, Vector Laboratories). Primary antibodies were visualized using fluorescent secondary antibodies. By confocal microscopy, we found that CVL‐projecting NTS neurons were more than twice as likely to receive somatic input from CTb‐labeled afferents (79%) than IB4‐labeled afferents (34%). In somatic nerves, CTb and IB4 label myelinated and unmyelinated afferents, respectively. Therefore, our findings suggest that CVL‐projecting NTS neurons receive input mainly from myelinated vagal afferents that contain a distinct population of transporters, neurotransmitters, and receptors.Support: NIH HL56301

Facial-Hypoglossal Nerve Anastomosis Using Laser Nerve Welding

The aim of this study is to compare laser nerve welding to microsurgical suturing of hypoglossal-facial nerve anastomosis (HFA), and a result of immediate to delayed repair, and to evaluate the effect of laser nerve welding on HFA for reanimation of facial palsy. The first group of five rats underwent immediate HFA by microsurgical suturing and the second group of five rats by CO2 laser welding. The third group of five rats underwent delayed HFA by microsurgical suturing, and the fourth group of five rats by laser nerve welding. The fifth group of five rats served as controls, with intact hypoglossal and facial nerve. In all rats of the four different treatment groups, cholera toxin B subunit (CTb) was injected in the epineurium distal to the anastomosis site on the postoperative 6th week and in the normal hypoglossal nerve in the five rats of the control group. Neurons labeled CTb of hypoglossal nuclei were positive immunohistochemically, and the numbers were counted. In the immediate HFA groups, CTb-positive neurons were 751 +/- 247 in the laser welding group (n = 5) and 888 +/- 60 in the microsurgical suturing group (n = 5). There was no significant difference (P = 0.117). In the delayed HFA groups, CTb-positive neurons were 749 +/- 54 in the laser welding group (n = 5) and 590 +/- 169 in the microsurgical suturing group (n = 5). The difference was not significant (P = 0.116). There was no significant difference between immediate and delayed anastomosis in the laser welding group (P = 0.600), but there was significance between immediate and delayed anastomosis in the microsurgical suturing group (P = 0.009). Injected CTb in intact hypoglossal neurons (n = 5) were labeled 1,003 +/- 52. No dehiscence in the laser welding site of nerve anastomosis was seen at the time of re-exploration for injection of CTb in all 10 rats. This study shows that the regeneration of anastomosed hypoglossal-facial nerve was affected similarly by laser welding and microsurgical suturing, and more effective, especially in delayed repair.

Cholera toxin B subunit and isolectin B4 label distinct populations of vagal afferents projecting to the nucleus tractus solitarii

The vagus nerve contains primary visceral afferents that convey information from cardiovascular, pulmonary, and gastrointestinal tissues to nucleus tractus solitarii (NTS). We tested the hypothesis that different anterograde tracers will label distinct populations of primary afferents. In somatic nerves, cholera toxin B subunit (CTb) and isolectin B4 (IB4) have been shown to label myelinated and unmyelinated fibers, respectively. We also examined the vesicular glutamate transporter 2 (VGlut2) content of these afferents. Male Sprague-Dawley rats (275–350 g) received injections of either CTb or IB4 (2% solution, 1–4 μl) into the vagus nerve under isoflurane anesthesia. Seven days later, rats were perfused and vibratome sections through NTS were examined with dual immunocytochemistry for either CTb (goat anti-CTb, List) or IB4 (goat anti-IB4, Vector) and VGlut2 (guinea pig anti-VGlut2, Chemicon), visualized using fluorescent secondary antibodies. CTb and IB4 produced distinct patterns of afferent labeling in NTS following vagal nerve injections. CTb produced both anterograde labeling in NTS and retrograde labeling of vagal motor neurons, while IB4 labeled only afferents. By confocal microscopy, we found VGlut2 in a subset of CTb-labeled vagal afferents, but VGlut2 was rarely found in IB4-labeled afferents. By electron microscopy we found that CTb afferents were both myelinated and unmyelinated, but IB4 afferents were only unmyelinated. These findings suggest that these tracers label largely distinct, but partially overlapping, populations of vagal afferents to NTS with distinct phenotypes. Support: NIH HL56301

Chicken suprachiasmatic nuclei: I. Efferent and afferent connections

The avian circadian system is composed of multiple inputs, oscillators, and outputs. Among its oscillators are the pineal gland, retinae, and a hypothalamic structure assumed to be homologous to the mammalian suprachiasmatic nucleus (SCN). Two structures have been suggested as this homolog -- the medial SCN (mSCN) and the visual SCN (vSCN). The present study employed biotin dextran amine (BDA) and cholera toxin B subunit (CTB) as anterograde and retrograde tracers to investigate the connectivity of the mSCN and vSCN in order to address this issue. Intravitreal injections of CTB were used to determine whether one or both of these structures receives afferent input from retinal ganglion cells. Both the vSCN and mSCN receive terminal retinal input, with the strongest input terminating in the vSCN. Precise iontophoretic injections of BDA and CTB in the mSCN and vSCN were used to identify efferents and afferents. The avian mSCN and vSCN collectively express more efferents and afferents than does the mammalian SCN. A subset of these connections matches the connections that have been established in rodent species. Individually, both the mSCN and vSCN are similar to the mammalian SCN in terms of their connections. Based on these data and other studies, we present a working model of the avian SCN that includes both the mSCN and vSCN as hypothalamic oscillators. We contend that both structures are involved in a suprachiasmatic complex that, as a functional group, may be homologous to the mammalian SCN.

Direct pyrogenic input from prostaglandin EP3 receptor‐expressing preoptic neurons to the dorsomedial hypothalamus

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.

Laser Welding of Rat's Facial Nerve

The aim of this study is to compare regeneration of the severed nerves that were repaired by laser welding with those repaired by microsurgical suturing and evaluate the value in use of laser nerve welding in the head and neck area. In 12 rats the buccal branches of the facial nerves on the both sides were transected, and CO2 laser welding of the epineurium was performed on the right side and microsurgical suture technique was applied on the left side. In six rats Cholera Toxin B Subunit (CTb) was injected in the epineurium distal to the nerve anastomosis site at postoperative week 4. Another six rats were treated exactly in the same way in postoperative week 8. Six normal rats were used as controls. Intact facial nerve was observed after injection of CTb as well. Neurons of facial nuclei labeled positively by CTb were detected immunohistochemically, and the numbers were counted. CTb-positive neurons in the control group were 1311 +/- 258 (n = 6). CTb-positive neurons in the group (n = 6) with laser nerve welding were 1174 +/- 122 in postoperative week 4 and 1562 +/- 565 in postoperative week 8. CTb-positive neurons in the group (n = 6) with microsurgical suture were 1066 +/- 89 in postoperative week 4 and 1443 +/- 531 in postoperative week 8. CTb-positive neurons were seen significantly more in the group with laser welding than in the group with microsurgical suture in postoperative week (P = 0.028), but there was not much difference in postoperative week 8 (P = 0.463). None of 12 rats showed dehiscence at the nerve anastomosis done by laser welding. This study shows that nerve regeneration is more apparent in the nerve repaired by laser welding than in that repaired by microsurgical suture.

Ultrastructural evidence for a direct excitatory pathway from the nucleus retroambiguus to lateral longissimus and quadratus lumborum motoneurons in the female golden hamster

During mating, the female golden hamster displays a stereotyped specific receptive posture, characterized by lordosis of the back, elevation of the tail, and extension of the legs. Muscles involved in this posture are thought to be iliopsoas, cutaneus trunci, lateral longissimus (LL), and quadratus lumborum (QL). Lesion studies in rats suggest that mating behavior is controlled by the mesencephalic periaqueductal gray (PAG). The PAG does not project directly to the motoneurons innervating the muscles involved in mating, but is thought to make use of the nucleus retroambiguus (NRA) as relay. The NRA is located ventrolaterally in the most caudal medulla, and projects directly to iliopsoas and cutaneus trunci motoneuronal cell groups. The question is whether this is also true for LL and QL muscles. Retrograde HRP tracing experiments revealed that LL and QL motoneurons are located medially in the ventral horn of the T12-L6 and T13-L4 segments, respectively. A subsequent ultrastructural study combined wheatgerm agglutinin-conjugated horseradish peroxidase injections in the NRA with cholera-toxin B-subunit injections in LL and QL muscles. The results revealed monosynaptic contacts between anterogradely labeled NRA-fiber terminals with retrogradely labeled dendrites of both LL and QL motoneurons. Almost all these terminals had asymmetrical synapses and contained spherical vesicles, suggesting an excitatory function of this NRA-motoneuronal pathway. These results correspond with the hypothesis that in hamster the PAG-NRA-motoneuronal projection not only involves motoneurons of iliopsoas and cutaneus trunci but also of LL and QL.

Intergeniculate leaflet and ventral lateral geniculate nucleus afferent connections: An anatomical substrate for functional input from the vestibulo‐visuomotor system

The intergeniculate leaflet (IGL) has widespread projections to the basal forebrain and visual midbrain, including the suprachiasmatic nucleus (SCN). Here we describe IGL-afferent connections with cells in the ventral midbrain and hindbrain. Cholera toxin B subunit (CTB) injected into the IGL retrogradely labels neurons in a set of brain nuclei most of which are known to influence visuomotor function. These include the retinorecipient medial, lateral and dorsal terminal nuclei, the nucleus of Darkschewitsch, the oculomotor central gray, the cuneiform, and the lateral dorsal, pedunculopontine, and subpeduncular pontine tegmental nuclei. Intraocular CTB labeled a retinal terminal field in the medial terminal nucleus that extends dorsally into the pararubral nucleus, a location also containing cells projecting to the IGL. Distinct clusters of IGL-afferent neurons are also located in the medial vestibular nucleus. Vestibular projections to the IGL were confirmed by using anterograde tracer injection into the medial vestibular nucleus. Other IGL-afferent neurons are evident in Barrington's nucleus, the dorsal raphe, locus coeruleus, and retrorubral nucleus. Injection of a retrograde, trans-synaptic, viral tracer into the SCN demonstrated transport to cells as far caudal as the vestibular system and, when combined with IGL injection of CTB, confirmed that some in the medial vestibular nucleus polysynaptically project to the SCN and monosynaptically to the IGL, as do cells in other brain regions. The results suggest that the IGL may be part of the circuitry governing visuomotor activity and further indicate that circadian rhythmicity might be influenced by head motion or visual stimuli that affect the vestibular system.

Collateralization of climbing and mossy fibers projecting to the nodulus and flocculus of the rat cerebellum

Collateralization of mossy and climbing fibers was investigated using cortical injections of cholera toxin b-subunit in the rat vestibulocerebellum. Injections were characterized by their retrograde labeling within the inferior olive. Collateral labeling was plotted using color-coded density profiles of the whole cerebellar cortex. Injections in the medial part of the nodulus resulted in olivary labeling that was restricted to the rostral part of the dorsal cap. Climbing fiber collaterals were found in medial and lateral nodular zones as well as in the ventral paraflocculus and adjacent flocculus. Injections in the intermediate part of the nodulus resulted in olivary labeling of the beta-subnucleus but could also involve the ventrolateral outgrowth. In the latter case, climbing fiber collaterals were found in the two floccular zones and in a small region in the lateral-most part of crus I. All nodular injections showed a bilaterally symmetric distribution of collateral mossy fiber rosettes that was mostly confined to the vestibulocerebellum and originated predominantly from the vestibular nuclei. Injections in the flocculus labeled the caudal part of the dorsal cap and/or the ventrolateral outgrowth. Mossy fiber rosettes were observed throughout the vestibulocerebellum but also included other regions of the cerebellar cortex in a bilaterally symmetric pattern corresponding with a more widespread precerebellar origin. Climbing fibers originating in the rostral dorsal cap, labeled from an injection in the ventral paraflocculus, collateralize to a medial and lateral zone in the nodulus. Climbing fiber collaterals were usually accompanied by subjacent labeling of mossy fiber rosettes. These results demonstrate that some nodular and floccular zones are related and, at least partially, share a common input.

Immunohistochemical characterization of cardiac vagal preganglionic neurons in the rat

Cardiac vagal preganglionic neurons (CVN) control cardiac activity by negative chronotropic, dromotropic and inotropic effects. We attempted to characterize the distribution and neuronal properties of the CVN by using double labeling with the retrograde tracer cholera toxin B subunit (CTb) and immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP) or nitric oxide synthase (NOS). Injection of CTb into the sinoatrial ganglia resulted in many retrogradely labeled of neurons in the dorsal motor nucleus of the vagus (DMV), the compact (AmC), semicompact (AmS), loose (AmL), external (AmE) formations of the nucleus ambiguus, and the intermediate zone (IZ) between DMV and the nucleus ambiguus. Almost all CTb-labeled neurons showed ChAT immunoreactivity in the DMV, AmC, AmS, AmL and IZ, but most of the CTb-labeled neurons showed no ChAT immunoreactivity in the AmE. Most of the CTb-labeled neurons were double-labeled with CGRP immunoreactivity in the AmC, AmS and AmL, but a few double-labeled neurons were found in the DMV, IZ and AmE. A few CTb-labeled neurons were double-labeled with NOS immunoreactivity only in the DMV. No TH-immunoreactive neurons were found among the CVN. These results indicate that there are four kinds of neurons among the CVN: non-cholinergic CVN in the AmE, cholinergic and CGRP-containing CVN in the AmC, AmS and AmL, and cholinergic or cholinergic and NOS-containing CVN in the DMV.

Activation of adrenal preganglionic neurons during autonomic dysreflexia in the chronic spinal cord-injured rat

Autonomic dysreflexia (AD) occurs in a majority of high paraplegic and quadriplegic patients and is particularly characterized by a paroxysmal hypertension elicited by somatic or visceral stimuli. We have previously shown that plasma adrenaline and noradrenaline levels were significantly increased during episodes of AD in the 30-day spinal cord-injured (SCI) rats, suggesting the participation of adrenal catecholamines in the cardiovascular changes associated to AD. Thus, adrenal sympathetic preganglionic neurons (SPN) could be activated by visceral afferences leading to AD. The aim of this study was then to demonstrate whether visceral stimulation that induces AD activates adrenal SPN in chronic SCI rats. To this end, a retrograde tracer, the cholera toxin B subunit (CTB), was combined with the immunocytochemical detection of Fos protein after visceral stimulation. Chronic SCI rats received a CTB injection into the adrenal gland and, 3 days later, were stimulated by repetitive distension of the colon. Results showed that this stimulation elicited typical hypertensive episodes of AD and a significant increase in the number of double-labeled neurons (CTB/Fos immunoreactive neurons) in the thoracic spinal cord below the level of injury (T4 segment) when compared to the stimulated non-SCI rats. In conclusion, visceral stimulations in the chronic SCI rats activate adrenal SPN, which could induce release of catecholamines by the adrenal medulla. The present study brings new data on the spinal mechanisms of AD cardiovascular dysfunctions.

Subnuclear distribution of afferents from the oral, pharyngeal and laryngeal regions in the nucleus tractus solitarii of the rat: a study using transganglionic transport of cholera toxin.

The central distributions of afferents from the oral cavity, the pharynx, the larynx and the esophagus to the nucleus tractus solitarii (NTS) were examined by using transganglionic anterograde transport of the cholera toxin B subunit (CT-b). Injections of CT-b into the body of the tongue and the hard palate resulted in heavy labeling of the lateral subnucleus (l-NTS) of the NTS rostral to the area postrema. Injection into the root of the tongue resulted in heavy labeling of the l-NTS, the dorsal half of the medial (m-NTS), the intermediate (im-NTS) and the interstitial (is-NTS) subnuclei rostral to the area postrema. Injections into the soft palate and the pharynx resulted in a similar labeling pattern in the is-NTS, im-NTS and m-NTS to that in the case of the root of the tongue, but this labeling extended rostrocaudally. Heavy labeling of the medial aspect of the l-NTS was found in the case of the soft palate, but the labeling was sparse in the case of the pharynx. Moderate labeling was also found in the commissural subnucleus (co-NTS). Injection into the larynx resulted in labeling of the is-NTS throughout the NTS, and of the rostral half of im-NTS. Injection into the esophagus resulted in heavy labeling of the central subnucleus, and moderate labeling of the co-NTS and the caudal half of im-NTS. A few but consistent anterogradely labeled terminals were found to appose retrogradely labeled small neurons in the rostral tip of the dorsal motor nucleus of vagus in the cases of injections into the root of the tongue, the soft palate, the pharynx, and the larynx. These results have characterized the viscerotopic representation of afferent projections from the oral and the cervical visceral organs to the subnuclei of the NTS.