Double retrograde neuronal labeling through divergent axon collaterals,using two fluorescent tracers with the same excitation wavelength which label different features of the cell

Summary Recent studies show that several fluorescent substances are transported retrogradely through axons to their parent cell bodies and label in different colors different features of the cell at the same 360 nm excitation wavelength. Thus, Bisbenzimide (Bb) and Nuclear Yellow (NY; Hoechst S 769121) produce green and golden-yellow retrograde labeling of the neuronal nucleus. True Blue (TB) and Fast Blue (FB) produce blue retrograde labeling of the neuronal cytoplasm. In the present study the possibility of retrograde double labeling of neurons by way of divergent axon collaterals using combinations of Bb or NY with TB or FB has been explored in rat and cat. The findings show that in these animals these tracer combinations are transported retrogradely through two axon collaterals to one and the same cell. Neurons which are retrogradely double-labeled with these tracer combinations display a blue fluorescent cytoplasm and a white or golden-yellow fluorescent nucleus at the same 360 nm excitation wavelength. Therefore, these tracer combinations can be successfully used to demonstrate the existence of divergent axon collaterals in the brain.     

Retrograde labeling of striato-nigral neurons in the rat by three different tracers

The three tracers horseradish peroxidase (HRP), 4',6-diamidino-2-phenylindol-2HCl (DAPI) and Fast Blue (FB) differ in retrograde labeling of striato-nigral neurons. After a 24 h survival, injection of DAPI into the ventral tegmentum labeled numerous cells throughout the neostriatum, whereas an identical amount of HRP only labeled cells in circumscribed areas of the neostriatum. Injections of FB labeled a substantial number of neostriatal neurons after a survival time of 4 days, but not after 24 h. In addition, differences between retrograde staining of striato-nigral neurons and layer V pyramidal cells of the ipsilateral neocortex, also labeled in these experiments, were found.     

Significant differences in the retrograde labeling of spinothalamic tract cells by horseradish peroxidase and the fluorescent tracers fast blue and diamidino yellow

Summary The laminar distributions of spinothalamic tract cells retrogradely labeled by the fluorescent tracers Fast Blue and Diamidino Yellow and by free or lectin-coupled horseradish peroxidase have been found to be significantly different. The total numbers of cells labeled by each method are similar, but nearly twice as many lamina I cells are labeled by the fluorescent tracers and more lamina V cells are labeled by peroxidase. Injection site spread and spurious labeling due to leakage or fibers of passage do not account for these differences. These results indicate that both horseradish peroxidase and fluorescent tracers may be selectively transported and, thus, that the cautious use of both methods should be recommended for analyses of afferent populations.     

Efferent neurons to the labyrinth of Salamandra salamandra as revealed by retrograde transport of horseradish peroxidase

Application of horseradish peroxidase to the severed VIIIth nerve of Salamandra salamandra resulted in heavy bilateral labeling of neurons of the medullary reticular formation. These neurons closely resemble the Mauthner neuron in their widespread dendritic ramification. In most preparations axon collaterals are seen to leave the medulla via the contralateral VIIIth nerve. It is suggested that these neurons are labyrinthine efferents.     

Axonal branching in the projections from the paramedian reticular nucleus to the cerebellar cortex

Injections of fluorescent tracers into cat cerebellar cortex gave evidence of collateral axonal branching of neurons situated in the paramedian reticular nucleus. These branched reticulocerebellar projections were distributed to opposing sides of the cerebellum, in particular the anterior lobe and the ansiform lobule. No topographical organization was observed in the PRN. Less than 30% of ipsilaterally projecting reticulocerebellar fibers had contralaterally directed collateral branches. These results are in keeping with a bilateral fastigial projection to the PRN forming a feedback loop circuit through which orthostatic reflexes may be mediated.     

Cerebellar afferents from the paramedian reticular nucleus studied with retrograde transport of horseradish peroxidase

Summary Details in the cerebellar projections from the paramedian reticular nucleus (PRN) were studied in cats and monkeys by means of retrograde axonal transport of horseradish peroxidase (HRP). In the cat the majority of the fibres projects to the anterior lobe and to the vermis of the posterior lobe (with the exception of lobules VIIB and VIIIA). A less conspicuous projection was found to the lobulus simplex, the crura and the flocculus. The cerebellar nuclei, the paramedian lobule and the paraflocculus appear to be weakly connected with the PRN. A similar distribution of the cerebellar afferent fibres was found in the monkey material. The three subgroups of the PRN in the cat are not equal in their projection. The dorsal group appears to be connected with the greater part of the cerebellar cortex and with all nuclei. The ventral group lacks a connection with lobulus IX, the flocculus and the paraflocculus, and the accessory group appears to have its strongest connection with lobulus I (lingula), the flocculus and the vermal lobules VII–X. The findings are discussed in relation to other studies on the efferent and afferent connections of the nucleus.On leave from the Laboratory of Neurobiology and Department of Anatomy, Faculty of Science, Mahidol University, Bankok, Thailand, under the Fellowship Program of the Norwegian Agency for International Development (NORAD)     

A double-labelling study of reticular collaterals to the spinal cord and cerebellum of the North American opossum

Injections of horseradish peroxidase into either the spinal cord or cerebellum label neurons in the gigantocellular and lateral reticular nuclei of the North American opossum. In order to determine if neurons which project to the spinal cord and cerebellum are intermingled in these two nuclei and if single neurons provide collaterals to both areas, we have employed fluorescent markers in double-labelling experiments. Our results show that reticular neurons innervating either the spinal cord or cerebellum are often close together and that a few provide collaterals to both areas. Neurons providing such collaterals are rare, however, comprising 2% or less of those innervating either target alone.     

Distribution of cochlear efferents and olivo-collicular neurons in the brainstem of rat and guinea pig

Summary In rat and guinea pig, cochlear efferents to the two ears were labeled simultaneously with different fluorescent tracers. It was found that in both species only few (1–3%) olivo-cochlear neurons were double-labeled and project to both cochleae. In most periolivary regions large olivocochlear neurons (OCN) projecting to the ipsilateral and contralateral side are intermingled and form a continuous cell column between the facial nucleus and lateral lemniscus. In a second series of experiments in rat, cochlear efferents and ascending olivo-collicular neurons were labeled. Olivo-cochlear and olivo-collicular neurons are intermingled in the lateral superior olive (LSO) and in the ventromedial periolivary region. No double-labeled neurons were found that project to the cochlea and the inferior colliculus.     

Cerebellar afferents from neurons in the extraocular motor nuclei: A fluorescent retrograde double‐labeling study in the sheep

The fluorescent retrograde double labeling technique has been used to identify within the extraocular motor nuclei of the sheep the neurons projecting to the cerebellum and to provide evidence whether they are motor neurons sending collaterals to the cerebellum or a separate population of neurons. The study was performed on eight sheep. The fluorescent tracers used were Fast Blue and the diamidino yellow dihydrochloride. In one and the same animal a fluorescent tracer was injected into the extraocular muscles (EOMs) and the other into bilateral points of the vermal folia II‐V and paramedian lobule, or into the vermal folia VI,VIIA and VIIB, or into the underlying fastigial nuclei. Within the oculomotor, trochlear, and abducens nuclei, almost all of the motor neurons were labeled by the tracer injected into the EOMs and only a few cells were fluorescent for the tracer infiltrated into the cerebellum. These latter labelings were present bilaterally, and their number and distribution did not show apparent differences after injecting the paramedian lobule and the vermal folia or the fastigial nucleus. Along the rostrocaudal extent of the oculomotor and trochlear nuclei, the neurons projecting to the cerebellum were intermingled with the motor neurons located in the nuclear area facing the medial longitudinal fasciculus. In the abducens nucleus they were restricted to the caudal pole of the nucleus, which is located ventrolaterally to the genu of the facial nerve. Double‐labeled neurons were never found. The absence of double‐labeled cells, in spite of the efficiency of the tracer infiltration into the EOMs and into the cerebellum, demonstrates that the cerebellar projections from the extraocular motor nuclei are not collaterals of the motor neurons, but axons of a separate population of neurons. Anat Rec 254:490–495, 1999. © 1999 Wiley‐Liss, Inc.     

Putative cholinergic afferents of the chick hyperstriatum ventrale: a combined acetylcholinesterase and retrograde fluorescence labelling study

The retrograde fluorescent tracers Fast Blue and Nuclear Yellow were injected into the intermediate part of the medial hyperstriatum ventrale (IMHV) of the chick telencephalon. After the appropriate survival times, the brains were processed for acetylcholinesterase (AChE) histochemistry using the thiocholine technique. In some telencephalic regions, fluorescent retrograde labelling and brown AChE reaction product were observed simultaneously in the same neurones by using a combination of ultraviolet and bright-field illumination. These AChE-positive afferents of IMHV arose from the hippocampus, septum, septal nuclei, hyperstriatum ventrale and neostriatum.     

Distribution of corticospinal neurons with collaterals to lower brain stem reticular formation in cat

Summary The fluorescent retrograde double-labeling technique has been used to determine whether corticospinal neurons in the cat sensorimotor cortex distribute collaterals to the lower brain stem reticular formation. In this study the fluorescent tracers Nuclear Yellow and Diamidino Yellow 2HCl were used in combination with Fast Blue. One tracer was injected unilaterally in the spinal cord and the other was injected ipsilaterally in the bulbar medial reticular formation. The distribution of the retrogradely labeled neurons was studied in the contralateral hemisphere. In the sensorimotor cortex a large population of neurons was found which were labeled from the spinal cord and were double-labeled from the brain stem. These branching neurons were concentrated in the rostromedial part of area 4 and the adjoining lateral part of area 6. In this region the percentages of corticospinal neurons which were double-labeled from the brain stem ranged from 5% laterally to 30% medially. In two cats it was demonstrated by means of the anterograde transport of HRP that the corticobulbar fibers from this region which must include the corticospinal collaterals are distributed to the reticular formation of the lower brain stem. In view of the fact that the double-labeled neurons are concentrated in the anterior part of the motor cortex, those branching neurons are in all likelihood involved in the control of neck, back and shoulder movements. This control is probably exerted by way of two routes i.e. by way of the direct corticospinal connections to spinal interneurons, and by way of the indirect cortico-reticulospinal connections established by the cortical fibers to the bulbar reticular formation. The present findings suggest that this dual control may be exerted by one and the same cell.Supported in part by Grant 13-46-91 of FUNGO/ZWO (Dutch Organization for Fundamental Research in Medicine)     

Responses of medullary reticulospinal and other reticular neurons to somatosensory and brainstem stimulation in anesthetized or freely-moving ovariectomized rats with or without estrogen treatment

Summary The medial medullary reticular formation (mMRF) is probably involved in controlling lordosis, a feminine mating reflex which requires both estrogen priming and appropriate somatosensory input(s). We have recorded single-unit activity of antidromically identified reticulospinal (RS) and unidentified (UI) neurons in mMRF of ovariectomized rats with or without estrogen treatment to investigate neurohormonal mechanisms regulating lordosis. The units were recorded in both acute and chronic preparations, the latter involving implanted floating wire electrodes to allow the influence of estrogen on a particular unit to be followed for several days.A substantial number of RS and UI units in both acute and chronic preparations were either excited or inhibited by a lordosis-eliciting somatosensory stimulation, indicating that the lordosis-eliciting sensory inputs did reach mMRF. The majority of these units responded promptly to the stimulation, and could participate in triggering the short-latency lordosis reflex. Electrical stimulation of several brainstem locations revealed that there was an extensive and specific convergence on mMRF neurons between inputs from the lordosis-eliciting stimulation and mesencephalic central gray, which has been shown to relay lordosis-inducing estrogen influence from hypothalamus to lower brainstem. Therefore, mMRF neurons can receive both the estrogen influence and the lordosis-eliciting inputs and integrate them. Although no apparent estrogen influence was detected in chronic preparations, statistical comparisons of results from acute preparations with or without estrogen treatment suggest that estrogen can increase the proportion of the neurons excited by the lordosis-eliciting stimulation and facilitate neuronal excitability. Both effects are consistent with the prevailing notion that the net lordosis-inducing influence of estrogen is facilitatory, and they may be mechanisms for making lordosis elicitable.Supported by NIH grant HD 05751     

Distribution of corticospinal neurons with collaterals to the lower brain stem reticular formation in monkey (Macaca fascicularis)

Summary An earlier retrograde double-labeling study in cat showed that up to 30% of the corticospinal neurons in the medial and anterior parts of the precruciate motor area represent branching neurons which project to both the spinal cord and the reticular formation of the lower brain stem. These neurons were found to be concentrated in the rostral portion of the motor cortex, from where axial and proximal limb movements can be elicited. In the present study the findings in the macaque monkey are reported. The fluorescent retrograde tracer DY was injected unilaterally in the spinal cord at C2 and the fluorescent tracer FB was injected ipsilaterally in the medial tegmentum of the medulla oblongata. In the contralateral hemisphere large numbers of single DY-labeled corticospinal neurons and single FBlabeled corticobulbar neurons were present. A substantial number of DY-FB double-labeled corticospinal neurons were also found, which must represent branching neurons projecting to both the spinal cord and the bulbar reticular formation. These neurons were present in: 1. The anterior portion of the cingulate corticospinal area in the lower bank of the cingulate sulcus; 2. The supplementary motor area (SMA); 3. The rostral part of precentral corticospinal area; 4. The upper portion of the precentral face representation area; 5. The caudal bank of the inferior limb of the arcuate sulcus; 6. The posterior part of the insula. In these areas 10% to 30% of the labeled neurons were double-labeled. The functional implications of the presence of branching corticospinal neurons in these areas is discussed.Abbreviations A nucleus ambiguus - AS arcuate sulcus - C cuneate nucleus - Cing. S. cingulate sulcus - corp. call. corpus callosum - CS central sulcus - Cx external cuneate nucleus - DCN dorsal column nuclei - dl dorsolateral intermediate zone - IO inferior olive - IP intraparietal sulcus - Lat. Fis. lateral fissure - LR lateral reticular nucleus - LS lunate sulcus - ML medial lemniscus - MLF medial longitudinal fascicle - mn motoneuronal pool - MRF medial reticular formation - Occ. occipital pole - P pyramid - PG pontine grey - PS principle sulcus - RB restiforme body - RF reticular formation - S solitary nucleus - SPV spinal trigeminal complex - STS superior temporal sulcus - Sup. Col. superior colliculus - TB trapezoid body - VC vestibular complex - vm ventromedial intermediate zone - III nucleus oculomotorius - VI nucleus abducens - VII nucleus, n. facialis - X motor nucleus n. vagus - XII nucleus hypoglossus Supported in part by grant 13-46-96 of FUNGO/ZWO (Dutch organisation for fundamental research in medicine)     

刺激延髓背角诱发的小鼠脑干网状结构内向Vmo投射的GABA能神经元突触后反应的电生理学研究

本实验利用谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠制备离体脑片,结合TMR逆行束路追踪技术,在荧光镜和红外镜头下,利用膜片钳全细胞记录的方法对电刺激延髓背角(即三叉神经脊束核尾侧亚核,Vc)诱发的小鼠小细胞网状结构(PCRt)内GABA能和TMR逆标的运动前神经元的突触后反应及其反应类型、药理学特征进行系统的研究。结果显示:(1)高频刺激(20 Hz)Vc,在PCRt内GFP和TMR双标的运动前神经元上可记录到兴奋性的突触后电流(EP-SCs),波形显示为单突触反应;(2)电压钳记录模式下,α-氨基羟甲基恶唑丙酸受体(AMPA受体)拮抗剂氨基-2-硝基喹啉-2,3-二酮(CNQX)及N-甲基-D-天冬氨酸受体(NMDA受体)阻断剂D,L-2-氨基-5-磷酸基戊酸(AP-5)均可显著降低刺激Vc所诱发的小鼠PCRt内GFP和TMR双标神经元的EPCSs的幅值;(3)电流钳模式下,刺激Vc,在PCRt内GFP和TMR双标的神经元上记录到兴奋性突触后电位,其幅度与刺激强度在一定范围内呈正相关。以上结果提示,小鼠PCRt内向三叉神经运动核(Vmo)发出投射的GABA能运动前神经元可通过其细胞膜上AMPA受体或NMDA受体的介导,对口面部伤害性信息发挥整合和调控作用,以实现对口面部伤害性反射活动的精确调节。     

Diamidino yellow dihydrochloride (DY·2HCl); a new fluorescent retrograde neuronal tracer,which migrates only very slowly out of the cell

Summary Earlier studies showed that Nuclear Yellow (NY), True Blue (TB) and Fast Blue (FB) are transported retrogradely through axons to their parent cell bodies. NY produces a yellow fluorescent labeling of the neuronal nucleus at 360 nm excitation wavelength, while TB and FB produce a blue fluorescence of the cytoplasm at this same wavelength. Therefore, NY may be combined with TB or FB in double-labeling experiments demonstrating the existence of axon collaterals. However, retrograde neuronal labeling with TB or FB requires a relatively long survival time, while NY requires a short survival time since NY migrates rapidly out of the retrogradely labeled neurons. This complicates double-labeling experiments since TB and FB must be injected first and NY later, a short time before the animal is sacrificed. We report a new yellow fluorescent tracer which labels mainly the nucleus and migrates much more slowly out of the retrogradely labeled neurons than NY. This new tracer can be used instead of NY in combination with TB or FB in double-labeling experiments and unlike NY can be injected at the same time as TB or FB. The new tracer is a diamidino compound (no. 28826) which is commercially available. It will be referred to as Diamidino Yellow Dihydrochloride (DY·2HCl). According to the present study DY·2HCl is transported over long distances in rat and cat, and produces a yellow fluorescence of the neuronal nucleus at 360 nm excitation wavelength, resembling that obtained with NY. When combined with TB or FB, DY·2HCl is as effective as NY in double labeling of neurons by way of divergent axon collaterals.Supported in part by Grant 13-46-91 of FUNGO/ZWO Dutch Organization for Fundamental Research in Medicine

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不同神经示踪剂组合对大鼠脊髓运动神经元的逆行标记效率比较

目的:探讨神经示踪剂荧光金(FG)、真蓝(TB)和荧光红(FR)两两组合对脊髓运动神经元的标记效率差异,为再生神经重支配准确性研究奠定基础.方法;采用大鼠胫神经示踪模型,采取神经内注射与神经横断后近侧断端浸泡(20 min)2种方式,分别对FG、TB和FR的两两组合进行示踪试验.示踪术后5d,取脊髓腰膨大段冷冻纵切,共聚焦显微镜进行显微成像和计数.结果:FG联合TB示踪标记的运动神经元数量最多,其次为FG联合FR,而FR联合TB组标记细胞数最少,双标比例也最小.神经断端浸泡方式使用示踪剂时标记效率仅为神经内注射的2/3左右.结论:FG联合TB以及FG联合FR示踪对脊髓运动神经元的标记效果较好,且神经内注射使用示踪剂效果优于持续20 min的神经断端浸泡.     

Axonal branching of medullary swallowing neurons projecting on the trigeminal and hypoglossal motor nuclei: demonstration by electrophysiological and fluorescent double labeling techniques

Summary The projections of ventral medullary reticular neurons on both trigeminal (Vth) and hypoglossal (XIIth) motor nucleus were studied in sheep anesthetized with halothane. In a first series of experiments, extracellular microelectrodes were used to record the activity of medullary swallowing interneurons (SINs) located in the ventral region (around the nucleus ambiguus) of the swallowing center. Antidromic activation after electrical stimulation of the Vth and XIIth nuclei was tested in 83 SINs. For 38 SINs a clear antidromic activation was observed and for 8 of them the response was triggered by stimulation of either nucleus. As confirmed by the reciprocal collision test, these 8 SINs had branched axons sending information to both nuclei tested. Average latencies for antidromic activation of branched SINs after stimulation of the XIIth and the Vth motor nucleus were 2.2±0.6 ms and 2.7±0.8 ms respectively. The axonal conduction velocity of these neurons was 4.4±1.3 m/s for the collateral to the Vth motor nucleus and 2.7±0.7 m/s for axons projecting to the XIIth motor nucleus. In a second series of experiments the double retrograde labeling technique was used to confirm the existence of neurons with branched axons in the medullary regions corresponding to the swallowing center. Small and well localized injections of Fast Blue (FB) and Diamidino Yellow (DY) fluorescent tracers were made in the Vth and in the XIIth motor nucleus respectively. A relatively large number of double-labeled cells was found in the ventral region of swallowing center (reticular formation around the nucleus ambiguus, 2–4 mm in front of obex). Such neurons (supplying both the XIIth and the Vth motor nucleus) appeared mixed with those innervating only either the XIIth or the Vth motor nucleus. Each type of neuron, i.e. single or double labeled, was shown to have bilateral distribution with an ipsilateral predominance.     

Identification and input-output properties of bulbar reticular neurons involved in the cerebral cortical control of trigeminal motoneurons in cats

Summary Neurons found in the medial bulbar reticular formation were activated by stimulation of the orbital gyms and responded with antidromic spike potentials to selective stimulation of either the masseter or anterior digastric motoneuron pool in the trigeminal motor nucleus in cats anesthetized with -chloralose. These two kinds of reticular neurons were assumed to be inhibitory neurons projecting to masseter motoneurons (IM neurons) and excitatory neurons projecting to anterior digastric motoneurons (ED neurons), involved in the effects of stimulation of the orbital gyrus on trigeminal motoneurons: inhibition of masseter motoneurons and excitation of anterior digastric motoneurons.Input-output properties of IM and ED neurons were studied intracellularly with the following results: (1) stimulation of the orbital gyrus evoked EPSPs in im and ED neurons with mono- and polysynaptic latencies; and (2) stimulation of the lingual nerve evoked a spike potential in a few IM and ED neurons after a rather long latency, indicating that the pathways involved in the cortical control of trigeminal motoneurons via IM and ED neurons were basically separate from those responsible for the reflex control by the peripheral inputsIntracellular injection of horseradish peroxidase revealed that both IM and ED neurons were small or medium in size and the former were smaller than the latter, while none of the large reticular neurons directly projected to the trigeminal motor nucleus. This suggests a possible functional differentiation among bulbar reticular neurons according to cell size.Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Arrangement of neurons in the medullary reticular formation and raphe nuclei projecting to thoracic, lumbar and sacral segments of the spinal cord in the cat

Summary The distribution of neurons in the medullary reticular formation and raphe nuclei projecting to thoracic, lumbar and sacral spinal segments was studied, using the technique of retrograde transport of horseradish peroxidase (HRP), alone or in combination with nuclear yellow (NY). Retrogradely labeled cells were observed in the lateral tegmental field (FTL), paramedian reticular nucleus, magnocellular reticular nucleus (Mc), in the gigantocellular nucleus (Gc), lateral reticular nucleus (LR), lateral paragigantocellular nucleus (PGL), rostral ventrolateral medullary reticular formation (RVR), as well as in the medullary raphe nuclei following the injection of the tracer substance(s) into various levels of the spinal cord. The FTL, the ventral portion of the paramedian reticular nucleus (PRv), Mc, LR, PGL and the raphe nuclei were found to project to thoracic, lumbar and sacral spinal segments. This projection was bilateral; the contralaterally projecting fibers crossed the midline at or near their termination site. The dorsal portion of the paramedian reticular nucleus (PRd), Gc and the RVR projected mainly to thoracic segments. This projection was unilateral. Experiments in which the HRP-injection was combined with lesion of the spinal cord showed that some descending raphe-spinal axons coursed presumably alongside the central canal. Experiments with two tracer substances suggested that some reticulo and raphe-spinal neurons had axon collaterals terminating both in thoracic and sacral spinal segments.Abbreviations CC Central Canal - FTL Lateral Tegmental Field - Gc Gigantocellular Nucleus - IO Inferior Olive - LR Lateral Reticular Nucleus - Mc Magnocellular Reticular Nucleus - Nc Cunetae Nucleus - Ng Gracile Nucleus - P Pyramidal Tract - PGL Lateral Paragigantocellular Nucleus - PRd Paramedian Reticular Nucleus,dorsal portion - PRv Paramedian Reticular Nucleus, ventral portion - RB Restiform Body - Ro Nucleus Raphe Obscurus - Rm Nucleus Raphe Magnus - Rpa Nucleus Raphe Pallidus - RVR Rostral Ventrolateral Medullary Reticular Formation - TSp5 Tractus Spinalis Nervi Trigemini - V4 Fourth Ventricle - 12N Hypoglossal Nerve - A B C D E and F correspond to levels Fr 16.0 Fr 14.7 Fr 12.7 Fr 11.6 Fr 10.0 and Fr 9.2 posterior to the frontal zero