Development of the rat phrenic nucleus and its connections with brainstem respiratory nuclei

The development of phrenic motoneurons and descending bulbospinal projections to the cervical spinal cord have been examined in prenatal and early postnatal rats with the aid of the carbocyanine dyes DiI and DiA. Phrenic motoneurons could be identified by retrograde labelling as early as E13, while aggregation of phrenic motoneurons into a column and the formation of dendritic bundles became apparent from E16. The initial phrenic motoneuron dendritic bundles were oriented in the dorsolateral and ventromedial directions, while ventrolaterally directed bundles entering the marginal zone appeared by E16, and rostrocaudal bundles were clearly visible by E21. The column of phrenic motoneurons extended rostrocaudally from C2 to C6 at E13 and E14, but this became confined to the C3-5 segments by E21. Two-way tracing of connections between putative brainstem respiratory centres and cervical spinal cord with the carbocyanine dyes, DiI and DiA, indicated that brainstem bulbospinal neurons in the position of the adult ventral respiratory group (VRG) and medial parabrachial (MPB) nuclei appeared to project to the cervical cord white matter as early as E15 and may contribute axons to the grey matter of the cervical cord as early as E17 These findings are consistent with electrophysiological studies of respiratory function development in the fetal rat, which found relatively regular rhythmic phrenic discharge by E20 to 21. In summary, our findings indicate that the structural differentiation of phrenic motoneurons is well-advanced prior to birth and that the descending pathways involved in the control of respiratory function are in place several days before birth.     

The ultrastructure and synaptic architecture of phrenic motor neurons in the spinal cord of the adult rat

Summary Although light microscopic studies have analysed phrenic motor neurons in several different species, there has never been an ultrastructural investigation of identified phrenic motor neurons. In addition, electrophysiological studies have raised questions relating to the function of phrenic motor neurons which may be answered only by direct electron microscopic investigation. Thus, the present study was carried out to provide a detailed ultrastructural analysis of identified phrenic motor neurons. Phrenic motor neurons in the spinal cord of the rat were labelled by retrogradely transported horseradish peroxidase (HRP) after transecting the phrenic nerve in the neck and applying the enzyme directly to the central stump of the transected nerve. The results showed that the general ultrastructural characteristics of phrenic motor neurons were similar to those previously reported for other spinal motor neurons. However, phrenic primary dendrites appeared to be isolated from all other dendritic profiles in the neuropil. Primary dendrites were not fasciculated. Fasciculation occurred only among the more distal secondary and tertiary phrenic dendritic branches. Direct dendrodendritic or dendrosomatic apposition was rarely seen; gap junctions between directly apposing phrenic neuronal membranes were not observed. The membranes of adjacent phrenic neuronal profiles were most frequently separated by intervening sheaths of astroglial processes. Myelinated phrenic axons and a phrenic axon collateral were identified. The initial portion of the phrenic axon collateral was cone-shaped, lacked myelin, and thus resembled a miniature axon hillock. In one instance, a large accumulation of polyribosomes was observed within the hillock-like structure of a phrenic axon collateral. Eight morphological types of synaptic boutons, M, P, NF_s, S, NF_f, F, G and C were classified according to criteria used by previous investigators. Most of these endings (M, NF_s, NF_f, S and F) made synaptic contact with profiles of labelled phrenic somata and dendrites. F, NF_f, and S boutons also terminated on phrenic axon hillocks. C and G boutons contacted exclusively phrenic somata and small calibre dendrites, respectively. P boutons established axo-axonic synaptic contacts with the M and NF_s bouton. The morphological findings of the present study provide new data that may be related to phrenic synchronized output and presynaptic inhibition of primary afferents terminating on phrenic motor neurons.     

Monoaminergic and GABAergic terminations in phrenic nucleus of rat identified by immunohistochemical labeling

The termination patterns of axons in the phrenic nucleus immunoreactive to synthetic enzymes for catecholamines and for serotonin and GABA were studied in rats. Spinal cord tissue in which phrenic motoneurons were retrogradely labeled with horseradish peroxidase was incubated with antisera against dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, 5-hydroxytryptamine, and GABA to identify presumptive terminations of monoaminergic and GABAergic neurons onto identified phrenic motoneurons. In the C3 to C5 spinal cord, 5-hydroxytryptamine-, dopamine beta-hydroxylase- and GABA-like positive terminals with varicosities formed a dense network, with presumptive synaptic contacts on dendrites and somas of phrenic motoneurons. A similar pattern of terminations was also observed in adjacent (non-respiratory muscle) motoneuron pools. There were fewer phenylethanolamine-N-methyltransferase-positive terminal arborizations in the cervical spinal cord compared to thoracic spinal cord; phenylethanolamine-N-methyltransferase terminals were not seen in the vicinity of phrenic motoneurons. These results suggest that phrenic motoneuronal activity is influenced by multiple supraspinal inputs utilizing different neurotransmitters. These transmitters also mediate inputs to other (nearby) spinal motoneurons and thus are not unique for signal transmission to phrenic motoneurons.     

Transganglionic transport of the lectin soybean agglutinin (Glycine max) following injection into the sciatic nerve of the adult rat

The lectin soybean agglutinin (SBA) from Glycine max binds to small-sized dorsal root ganglion cells and their central terminals in the superficial dorsal horn of the spinal cord. Here we investigated the ability of SBA and SBA conjugated to horseradish peroxidase(SBA-HRP) to trace thin calibre afferents into the spinal cord from a peripheral nerve. Following injection into the sciatic nerve, labelled cells in the dorsal root ganglion were predominantly small-sized but some medium-sized cells were also labelled. Colocalisation studies of transported SBA with various neuronal markers showed that all neurons that transported SBA-HRP showed SBA binding, indicating high uptake specificity for the conjugate. 15% were immunoreactive for RT97 indicating that some axons were myelinated, and 54% also expressed binding sites for isolectin B4 from Griffonia simplicifolia, a selective marker for a subpopulation of unmyelinated afferents. With regard to neurotransmitter content, 43% of the SBA cells contained calcitonin gene-related peptide, 33% contained substance P and 2.5% somatostatin. In addition, 3% contained carbonic anhydrase. Centrally, injection of SBA in the sciatic nerve resulted in labelled terminals in somatotopically appropriate regions of laminae I–II of the dorsal horn, and in the gracile nucleus. A few neurons in the dorsal horn were labelled indicating that some transneuronal transport of SBA had occurred. The results show that SBA can be used as a transganglionic tracer to label fine calibre primary afferents that project to laminae I–II of the spinal cord and the gracile nucleus. It appears to label more afferents than isolectin B4, including also a subpopulation of myelinated afferents.     

Identification of the neural pathway underlying spontaneous crossed phrenic activity in neonatal rats

Cervical spinal cord hemisection at C2 leads to paralysis of the ipsilateral hemidiaphragm in rats. Respiratory function of the paralyzed hemidiaphragm can be restored by activating a latent respiratory motor pathway in adult rats. This pathway is called the crossed phrenic pathway and the restored activity in the paralyzed hemidiaphragm is referred to as crossed phrenic activity. The latent neural pathway is not latent in neonatal rats as shown by the spontaneous expression of crossed phrenic activity. However, the anatomy of the pathway in neonatal rats is still unknown. In the present study, we hypothesized that the crossed phrenic pathway may be different anatomically in neonatal and adult rats. To delineate this neural pathway in neonates, we injected wheat germ agglutinin conjugated to horseradish peroxidase (WGA–HRP), a retrograde transsynaptic tracer, into the phrenic nerve ipsilateral to hemisection. We also injected cholera toxin subunit B–horseradish peroxidase (BHRP) into the ipsilateral hemidiaphragm following hemisection in other animals to determine if there are midline-crossing phrenic dendrites involved in the crossed phrenic pathway in neonatal rats. The WGA–HRP labeling was observed only in the ipsilateral phrenic nucleus and ipsilateral rostral ventral respiratory group (rVRG) in the postnatal day (P) 2, P7, and P28 hemisected rats. Bilateral labeling of rVRG neurons was shown in P35 rats. The BHRP study showed that many phrenic dendrites cross the midline in P2 neonatal rats at both rostral and caudal parts of the phrenic nucleus. There was a marked reduction of crossing dendrites observed in P7 and P28 animals and no crossing dendrites observed in P35 rats. The present results suggest that the crossed phrenic pathway in neonatal rats involves the parent axons from ipsilateral rVRG premotor neurons that cross at the level of obex as well as decussating axon collaterals that cross over the spinal cord midline to innervate ipsilateral phrenic motoneurons following C2 hemisection. In addition, midline-crossing dendrites of the ipsilateral phrenic motoneurons may also contribute to the crossed phrenic pathway in neonates.     

The accessory phrenic nerve in the rat

Summary Reinnervation studies of the diaphragm led us to reinvestigate the normal anatomy of the phrenic nerve of the rat. The phrenic nerve originates from the cervical nerve roots C₄ and C₅. In 16 out of 19 normal rats an accessory phrenic nerve was observed receiving its segmental fibres from C₆. The number of myelinated axons of the accessory phrenic nerve varied from 41 to 101 (mean: 64.3, i.e. about 15% of the average number of axons in the common phrenic nerve). The accessory phrenic nerve innervates the dorsal part of the costal and the lateral part of the crural region, whereas the remaining parts of the hemidiaphragm are supplied by the segments C₄ and C₅. There is no evidence for any additional contribution to the motor innervation of the diaphragm from intercostal nerves.Direktor: Prof. Dr. W. ZenkerThis study was supported by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich.     

Bötzinger-complex expiratory neurons monosynaptically inhibit phrenic motoneurons in the decerebrate rat

We examined respiratory neurons in the Bötzinger complex of the medulla oblongata in 18 vagotomized, paralyzed, ventilated, and decerebrated rats and tested the hypothesis that bulbospinal expiratory neurons in this region monosynaptically inhibit phrenic motoneurons. First, we surveyed the types of respiratory neurons found in the Bötzinger complex; only 11 of the 98 (～11%) examined were bulbospinal, and all discharged only during late expiration (E2), usually with an augmenting discharge frequency (AUG). Then, we examined the spinal projections of 34 E2-AUG neurons using antidromic activation and found that all projected as far as the C4 or C5 segments of the spinal cord but no further caudally. Most (30, ～88%) had only unilateral projections, the majority (25, ～83%) ipsilateral, but 4 neurons (～12%) had bilateral projections. Their axons could be antidromically activated at low currents (less than 10 μA) in the dorsal-lateral part of the spinal cord at the C2–3 border; 0.5–1.2 mm (mean±SD 0.84±0.23 mm) below the dorsal surface and 0.7–1.5 mm (1.19±0.25 mm) lateral from the midline. We sought evidence for connections from bulbospinal E2-AUG neurons to 118 phrenic motoneurons by computing spike-triggered averages (STAs) of their intracellular potentials triggered by the action potentials of 38 unilaterally-projecting E2-AUG neurons. Resting phrenic motoneuron membrane potentials ranged from –40 to –75 mV (–56±8 mV) and fluctuations with the respiratory cycle from 7 to 20 mV (14±4 mV). Of the 118 STAs computed, hyperpolarizations were evident in 18 (～15%) STAs, evoked by 11 of 38 (～29%) E2-AUG neurons. Their amplitudes varied from 35 to 550 μV (105±113 μV), 10–90% fall times from 0.4 to 0.9 ms (0.63±0.17 ms), and half-amplitude widths from 1.3 to 3.2 ms (2.0±0.52 ms). Most (16/95, ～17%) of the STAs that displayed hyperpolarizations were associated with ipsilateral trigger neurons but some (2/23, ～9%) resulted from contralateral trigger neurons. We conclude that Bötzinger-complex, expiratory neurons project to the C4 and/or C5 segments of the cervical spinal cord but no further caudal. Their axons are located dorsolaterally in the upper cervical segments of the spinal cord, and they monosynaptically inhibit phrenic motoneurons during the late part of expiration.     

Structural and functional characteristics of individual phrenic motoneurons

Summary Intracellular recording and staining techniques were applied to the study of cat phrenic motoneurons. Spontaneously driven phrenic cells possessed individualistic depolarization and spiking patterns that were a function of the conduction velocity in the different motor axons. Staining of phrenic motoncurons with Procion yellow indicated that fast conducting cells with small slow-wave depolarizations were large in size while slow conducting cells with large depolarizations were small in size. This implicated differences in membrane input resistance between large and small cells, although an unequal distribution of inputs to the individual components could not be discounted.On the average, phrenic motoneurons had a smaller dendritic surface area and smaller dendritic dominance than lumbosacral motoneurons. These factors help to explain the higher membrane resistances and longer time constants of phrenic cells.Phrenic dendrites were found to project in all directions away from the cell body and form ellipsoidal receptive fields that overlapped with other phrenic fields. It is speculated that the close approximation of phrenic dendrites with one another could, in part, be responsible for the high degree of synchronization among the different phrenic units.     

Morphology and synaptic relationships of physiologically identified low-threshold dorsal root axons stained with intra-axonal horseradish peroxidase in the cat and monkey

The arborizations and synaptic relationships of intra-axonally stained horseradish peroxidase- (HRP) labeled primary afferent fibers to the dorsal horn of the cat and monkey spinal cord have been studied by light and electron microscopic methods. The light microscopic arborizations of the afferent fiber types (hair follicle afferents, pacinian corpuscle afferents, type I and type II slowly adapting afferents) are similar to those described by Brown and his colleagues (1) in the cat. The synaptic profiles formed by labeled afferents contain rounded synaptic vesicles. In serial thin sections, it was found that single dorsal root axons may make hundreds or thousands of synapses with neuronal structures of the dorsal horn. The vast majority of synaptic contacts are on the dendritic trees of dorsal horn neurons. The synapses made by these low-threshold afferent axons are almost all in the deeper laminae (III-VI) of the dorsal horn. The hair follicle afferent axons and the pacinian corpuscle afferents have numerous vesicle-containing structures that synapse on them to form either axoaxonal synapses or dendroaxonal synapses. The slowly adapting afferent axons are less often found to be postsynaptic to axons or dendrites. It is concluded that different physiological classes of primary afferent axons have different morphological characteristics, both at the light and electron microscopic level.     

Excitatory interactions between phrenic motoneurons in the cat

Summary 1. Interactions between phrenic motoneurons have been analysed in anaesthetized, paralyzed cats after C3 to C7 deafferentation. Effects of electrical stimulation of the C5 phrenic axons have been studied on thin filaments dissected from the stimulated nerve. Repetitive stimulation could elicit, after the primary direct response of the stimulated axons, a secondary response named Recurrent Response, RR. 2. RRs have been obtained in 117/186 phrenic axons. They appear sporadically (mean occurrence: 3.75 RRs elicited by 100 shocks of stimulation) at a constant latency. They originate from a spinal mechanism since they persist after C2 transection and disappear after section of the ventral roots. 3. The mechanism responsible for RR shows spatial and temporal facilitation. The RR probability increases with the number of antidromically invaded motoneurons as revealed by changes either of stimulation intensity or of central respiratory drive. However, RR could be evoked in a motoneuron without an antidromic volley in its axon. 4. Systemic injections of nicotinic blocking drugs such as dihydro--erytroidin or mecamylamine decrease or suppress the occurrence of RR; therefore, cholinergic synapses are involved in the RR generating process. 5. RR are assumed to be due to direct excitatory interactions between homonymous motoneurons. Recurrent axon collaterals impinging directly on neighbouring motoneurons would link together the different motoneurons of the phrenic pool. The functional significance of this phenomenon is discussed.     

Is there electrical coupling between phrenic motoneurons in cats?

The possibility of electrical coupling between phrenic motoneurons was studied in anesthetized cats. In animals with C4-C7 dorsal roots cut (spinal cord intact), no sign of short-latency increase in the firing probability was observed in one phrenic rootlet following stimulation of the other phrenic rootlet. Intracellular recording from 21 phrenic motoneurons, in cats spinalized at the C1 segment, revealed no graded, short-latency antidromic depolarizations, even when the collision technique was used. Conditioning depolarizations of the impaled motoneurons never resulted in an increase of cell firing following test antidromic activation of adjacent motoneurons. It is concluded that the short-term discharge synchronization, observed within the phrenic nucleus by other authors, must be due to the action of chemical synapses.     

初级传入纤维在发育阶段小鼠腰髓背角内生长特点的研究(英文)

本研究通过将亲脂性羰花青染料 Di I置入胚胎 12 d(E12 )到生后 3 d(P3 )小鼠背根神经节内进行追踪的方法 ,观察了初级传入纤维在小鼠腰段脊髓背角内生长过程和终止的形态特点。结果显示 ,初级传入纤维在 E13时生长至脊髓后索 ,在此停留 2d,到 E15时开始进入脊髓灰质。 E16和 E17时 ,生长至脊髓灰质内的初级传入纤维的数量增加并向腹角延伸。有相当数量的初级传入纤维进入脊髓背角深层。至 E18,背角深层的传入纤维的密度进一步增加 ,分支的状态变得更为复杂。一些分支延伸到背角浅层。在出生后 ,初级传入纤维在脊髓背角的分布特点无明显变化 ,但是在背角前层的纤维数量进一步增多。另外 ,还观察到初级传入纤维发出侧支向对侧脊髓背角的投射。这种向对侧生长的纤维侧支开始于 E16,来源于同侧背角内侧部的传入纤维。至E18和出生时 ,向对侧背角生长的侧支的来源部位增加到两个。本研究的结果表明 ,小鼠脊髓背角内初级传入纤维的板层分布模式形成于胚胎发育的晚期和出生后的早期 ,此后初级传入纤维在背角内进行进一步的精细调整 ,达到与成年时一致的状态。     

Bilateral reflex effects on phrenic nerve activity in response to single-shock vagal stimulation

The bilateral reflex actions of vagus nerve afferent signals on phrenic efferent activity have been tested by unilateral graded single shock electrical stimulation. An early excitation (latency 3–5 msec) was more prominent in the phrenic nerve contralateral to the stimulated vagus. Spinal cord hemisection at C₃ eliminated both contralateral and ipsilateral responses: thus, both were mediated via descending tracts in the contralateral cord. A bilaterally symmetrical early inhibition (latency 8–12 msec) followed the early excitation. The electrical thresholds for evoking the early responses and the temperature for blocking these responses during graded vagal cooling were closely similar to the threshold and blocking temperature for pulmonary stretch receptor afferents. Higher stimulus strengths evoked a strong, bilaterally similar, late excitation (latency 12–20 msec) followed by a late inhibition. At very high stimulus strengths a third excitation (latency 25–30 msec) could appear. Sometimes these responses were followed by lowered phrenic activity for the remainder of inspiration. Single shock stimulation of the intact vagus nerve or of the peripheral end of the cut recurrent laryngeal nerve provoked. by the contraction of laryngeal muscles, a strong, short latency (12 msec) inhibition of phrenic activity mediated by superior laryngeal nerve afferents. The implications of these results with respect to the reflex pathways of the different responses and their possible integration in the central respiratory control mechanisms are discussed.     

An electron microscopic analysis of the left phrenic nerve in the rat

In this electron microscopic study, the axonal categories in the left phrenic nerve at its entrance to the diaphragm have been determined. At a level 3 mm rostral to the diaphragm, the left phrenic nerve contains approximately 700 axons: 57% are myelinated and 43% are unmyelinated. The dorsal root ganglion cells give rise to 31% of the myelinated axons and the ventral root contributes 69%. Of the unmyelinated axons, the dorsal root ganglion cell contributes 59%, the cervical sympathetic chain 24%, and 17% course through the ventral roots. These ventral root unmyelinated axons are presumably preganglionic efferents since the proximal stump of the ventral root showed no decrease in unmyelinated axons after ventral rhizotomy.     

Membrane potential changes of phrenic motoneurons during fictive vomiting, coughing, and swallowing in the decerebrate cat.

1. The patterns of membrane potential changes of phrenic motoneurons were compared during fictive vomiting, fictive coughing, and fictive swallowing in decerebrate, paralyzed cats. These fictive behaviors were identified by motor nerve discharge patterns similar to those recorded from the muscles of nonparalyzed animals. Phrenic motoneurons (n = 54) were identified by antidromic activation from the thoracic phrenic nerve. Intracellular recordings were obtained from 27 motoneurons during fictive vomiting, 40 during fictive coughing, and 27 during fictive swallowing. Sixteen motoneurons were recorded during both fictive coughing and fictive swallowing, eight during both fictive coughing and fictive vomiting, and two during both fictive vomiting and fictive swallowing. Seven motoneurons were studied during all three behaviors. 2. Fictive vomiting, typically evoked by electrical stimulation of abdominal vagal afferents, was characterized by a series of bursts of coactivation of phrenic and abdominal motor nerves, culminating in an expulsion phase in which abdominal discharge was prolonged both with respect to phrenic discharge and to abdominal discharge during the preceding retching phase. During fictive vomiting, phrenic motoneurons depolarized abruptly, and the amplitude of depolarization was significantly greater than during control inspirations. They then repolarized slowly throughout the phrenic burst, rapidly repolarizing at the end of each phrenic burst during retching and reaching a level similar to that observed during expiration. During the expulsion phase, the pattern was initially the same. However, after the cessation of phrenic discharge, the membrane potential repolarized slowly until the end of the abdominal burst, exhibiting greater synaptic noise than during expiration. One phrenic motoneuron, presumably innervating the periesophageal region of the diaphragm, received a strong hyperpolarization just before the onset of the emetic episode and fired for shorter periods during fictive vomiting than did other phrenic motoneurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anatomy of primary afferents and projection neurones in the rat spinal dorsal horn with particular emphasis on substance P and the neurokinin 1 receptor

The dorsal horn of the spinal cord plays an important role in transmitting information from nociceptive primary afferent neurones to the brain; however, our knowledge of its neuronal and synaptic organisation is still limited. Nociceptive afferents terminate mainly in laminae I and II and some of these contain substance P. Many projection neurones are located in lamina I and these send axons to various parts of the brain, including the caudal ventrolateral medulla (CVLM), parabrachial area, periaqueductal grey matter and thalamus. The neurokinin 1 (NK1) receptor on which substance P acts is expressed by certain neurones in the dorsal horn, including approximately 80 % of lamina I projection neurones. There is also a population of large NK1 receptor-immunoreactive neurones with cell bodies in laminae III and IV which project to the CVLM and parabrachial area. It has been shown that the lamina III/IV NK1 receptor-immunoreactive projection neurones are densely and selectively innervated by substance P-containing primary afferent neurones, and there is evidence that these afferents also target lamina I projection neurones with the receptor. Both types of neurone are innervated by descending serotoninergic axons from the medullary raphe nuclei. The lamina III/IV neurones also receive numerous synapses from axons of local inhibitory interneurones which contain GABA and neuropeptide Y, and again this input shows some specificity since post-synaptic dorsal column neurones which also have cell bodies in laminae III and IV receive few contacts from neuropeptide Y-containing axons. These observations indicate that there are specific patterns of synaptic connectivity within the spinal dorsal horn.     

Distribution of five peptides,three general neuroendocrine markers,and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: An immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I–II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I–III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I–II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

The development of cranial nerve and visceral afferents to the nucleus of the solitary tract in the rat

We have used carbocyanine dye tracing techniques to examine the distribution of afferents from the facial, trigeminal and vagal nerves to the nucleus of the solitary tract (NST) in the developing rat (E13 to P13). Crystals of DiI (1, 1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) were placed (unilaterally) into the facial or trigeminal ganglia, or into the cervical vagus nerve, and the sections examined with a laser scanning confocal microscope. Inputs from some peripheral structures (tongue, aortic arch, right atrium and lung) to the NST were also analyzed to provide information on the distribution of organ-specific afferents. No afferents were labeled following DiI placement in the above sites at E13. At E14, a few axons from the geniculate ganglion of the facial nerve were present in the NST anlage, but these were restricted to the area adjacent to the solitary tract. These axons began to invade the medial NST at E15. By E17, facial afferent axons had become widespread throughout rostral NST and from E19 the distribution of DiI labeling displayed a morphologically mature pattern. DiI-labeled afferent axons from the trigeminal nerve first emerged into the NST anlage at E14, initially coursing medially to penetrate the ventricular zone. Between E15 and E17, axonal density increased markedly but after E17 became progressively confined to the lateral NST. Axons from the vagus nerve first appeared in the caudal NST as early as E14 and coursed directly into the proliferative zone of the alar plate at all rostrocaudal levels by E15. From E19 through postnatal life, the distribution of vagal afferent axons was essentially stable with particularly dense label in the caudal NST. Cranial nerve afferents to the NST appear to be distributed to appropriate sites from the beginning of ingrowth, with the exception of trigeminal afferents, where some small initial exuberance was found. The terminal fields derived from selected peripheral organs such as lung, right atrium, aortic arch and tongue were also predominantly distributed to appropriate subnuclei from the beginning of ingrowth into the NST, although organ-specific afferent fields appeared to develop dense arbors somewhat later than did individual cranial nerves. Electron microscopy was used to examine regional synapse development in the rat NST. There was some delay between the ingrowth of afferents to the NST (E15) and the first appearance of synaptic thickenings. The earliest synapses were simple (usually) symmetrical membrane thickenings (from E17) and vesicles did not appear until E19. High synaptic density within the C subnucleus appeared during early postnatal life. Synaptic glomeruli, which are a characteristic feature of afferent input to the adult NST, had not developed by birth, indicating that the pre- and perinatal function of the NST must be mediated through simpler, single, axodendritic inputs to NST neurons. Accepted: 22 March 2001     

痛温觉和本体觉传入纤维在小鼠脊髓内的不同发育特点(英文)

本研究通过采用钙基因相关肽(CGRP)和小牛白蛋白(PV)分别标记胚胎15d(E15)到生后3d(P3)小鼠脊髓的痛温觉和本体觉两种初级传入纤维,观察了这两种纤维在小鼠脊髓内投射和终止的发育变化。结果显示:CGRP样免疫阳性(LI)纤维最早于E16出现在脊髓背角浅层,并在E17和E18时逐渐向背角外侧和深层终止。在出生后,CGRPLI纤维在背角的分布特点无明显变化,但是在背角浅层的纤维数量进一步增加,分支状态更为复杂。另外,还在E16时开始出现向对侧脊髓背角发出侧支投射的CGRP-LI纤维,至生后早期,向对侧投射的纤维数量增多。PVLI纤维最早于E15进入脊髓灰质。E16时,已有较多的PVLI纤维到达中间带灰质和腹角。随着发育阶段的增长,脊髓腹角内PVLI本体觉纤维和终末的数量和密度逐渐增加,并于生后早期(P0P3)时达到最高水平。本体觉传入纤维的终末在E17时开始与脊髓腹角内的运动神经元形成密切的接触。本实验结果表明痛温觉和本体觉传入纤维在脊髓内的终止形成于小鼠胚胎晚期和生后早期,并具有时空特异性。这为深入理解感觉信息在脊髓传递和调节的形成过程提供了依据。     

Rexed's laminae and the acid phosphatase (FRAP)-band in the superficial dorsal horn of the neonatal rat spinal cord

The pattern of Rexed's lamination and the situation of the FRAP-reactive band in the superficial dorsal horn were studied in transverse sections of the cervical cord of the rat from the day of birth (P0) to postnatal day P20. Laminae I, II and III increased moderately in thickness during this period, but the outer (IIo) and inner (IIi) zones of lamina II underwent opposite evolutions. Lamina IIi nearly doubled in thickness while lamina IIo decreased markedly. The FRAP-band was large on days P2 and P5 virtually covering lamina IIi, then shrank to a thinner strip in its dorsal part. It is suggested that lamina IIi growth is due to the massive arrival of FRAP-reactive thin primary axons followed by large afferents from deep layers occurring during the postnatal period, and the subsequent establishment of synaptic connections with the developing dendritic trees of dorsal horn cells.