Gustatory solitary tract development: a role for neuropilins.

The rostral nucleus of the solitary tract (rNST) receives orosensory information from taste bud cells in the tongue and palate via cranial nerves VII and IX. These nerves enter the brainstem, form the solitary tract (ST) and synapse with neurons in the rNST, which then relay incoming sensory information to other brain areas to process external gustatory stimuli. Factors that direct or regulate the trajectory of the developing ST are largely unknown. We used 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) to identify ST projections originating from cells in the geniculate ganglia of embryonic rats from embryonic day 14 through 18 (E14-E18). After identifying the ST fibers, immunolabeling for and protein expression analysis of the axon guidance molecules neuropilin-1 (Npn-1) and neuropilin-2 (Npn-2) and their binding partners, semaphorin-3A (Sema-3A) and semaphorin-3F (Sema-3F) were performed. The results detail the formation of ST projections into the gustatory brainstem and their relationship to developing rNST neurons. DiI-labeled ST fibers were present in the brainstem as early as E14. Npn-1 was expressed in the ST and in the trigeminal tract at E14, but levels of the protein declined through E18. The expression levels of the binding partner of Npn-1, Sema-3A, increased from E14 to E18. Npn-2 was expressed in the ST and, additionally, in radially oriented, tuft-like structures within the brainstem at E14. Expression levels of Npn-2 also declined through E18, in contrast to the expression levels of its binding partner, Sema-3F, which increased during this time period. For the first time, the time course and particular molecular components involved in development of the ST have been identified. These results indicate that the neuropilin and semaphorin families of axon guidance molecules are potential molecular participants in ST formation.

Excitatory and inhibitory synaptic function in the rostral nucleus of the solitary tract in embryonic rat.

The embryonic development of synapses in the rostral nucleus of the solitary tract (rNST) was investigated in rat to determine when synapses begin to function. Using a brain slice preparation we studied appearance of synaptic receptors on second order rNST neurons and investigated the development of postsynaptic responses elicited by afferent nerve stimulation. Prenatal excitatory and inhibitory synaptic responses were recorded as early as E14. Glutamatergic and GABAergic postsynaptic responses were detected as early as E16. Both NMDA and AMPA receptors contributed to glutamatergic postsynaptic responses. GABAergic postsynaptic responses resulted primarily from activation of GABA(A) receptors. However, functional GABA(C) receptors were also demonstrated. A glycinergic postsynaptic response was not found although functional glycine receptors were demonstrated at E16. Solitary tract (ST) stimulation-evoked EPSCs, first detected at E16, were eliminated by glutamate receptor antagonists. ST-evoked IPSPs, also detected at E16, were eliminated by GABA(A) receptor antagonist. Thus, considerable prenatal development of rNST synaptic connections occurs and this will ensure postnatal function of central taste processing circuits.

Pre- and postnatal differences in membrane, action potential, and ion channel properties of rostral nucleus of the solitary tract neurons.

There is little known about the prenatal development of the rostral nucleus of the solitary tract (rNST) neurons in rodents or the factors that influence circuit formation. With morphological and electrophysiological techniques in vitro, we investigated differences in the biophysical properties of rNST neurons in pre- and postnatal rats from embryonic day 14 (E14) through postnatal day 20. Developmental changes in passive membrane and action potential (AP) properties and the emergence and maturation of ion channels important in neuron function were characterized. Morphological maturation of rNST neurons parallels changes in passive membrane properties. Mean soma size, dendritic branch points, neurite endings, and neurite length all increase prenatally. whereas neuron resting membrane potential, input resistance, and time constant decrease. Dendritic spines, on the other hand, develop after birth. AP discharge patterns alter in pre- and postnatal stages. At E14, neurons generated a single TTX-sensitive, voltage-gated Na(+) AP when depolarized; a higher discharge rate appeared at older stages. AP amplitude, half-width, and rise and fall times all change during development. Responses to current injection revealed a number of voltage-gated conductances in embryonic rNST, including a hyperpolarization-activated inward current and a low-threshold Ca(2+) current that initiated Ca(2+) spikes. A hyperpolarization-activated, transient outward potassium current was also present in the developing neurons. Although the properties of these channels change during development, they are present before synapses form and therefore, can contribute to initial establishment of neural circuits, as well as to the changing electrophysiological properties in developing rNST neurons.

Functional development of the vagal and glossopharyngeal nerve-related nuclei in the embryonic rat brainstem: optical mapping with a voltage-sensitive dye.

We investigated functional organization of the vagus nerve (N. X)- and glossopharyngeal nerve (N. IX)-related nuclei in the embryonic rat brainstem and compared their development and spatial distribution patterns, using multiple-site optical recording with a fast voltage-sensitive dye, NK2761. Intact brainstem preparations with N. X and N. IX attached were dissected from E13-E16 rat embryos, and electrical responses evoked by N. X/N. IX stimulation were optically recorded from many loci of the stained preparations. We analyzed optical waveforms and separated fast and slow optical signals corresponding to the antidromic/orthodromic action potentials and the excitatory postsynaptic potentials (EPSPs), respectively. We constructed contour line maps of signal amplitudes and identified motor and sensory nuclei of N. X and N. IX. In the N. X-related motor nucleus (the dorsal motor nucleus of the vagus nerve: DMNV), the fast signals were distributed in multiple-peak patterns, suggesting that the neurons and/or their activity are not distributed uniformly within the motor nuclei at early developmental stages. In the sensory nucleus (the nucleus of the tractus solitarius: NTS), the EPSPs were first detected from E15 in normal physiological solution for both N. X and N. IX. The N. IX-related NTS partially overlapped with the N. X-related NTS, but the peak locations were different between these two nerves. The results obtained in this study suggest that functional organization of the N. X- and N. IX-related nuclei changes dynamically with development in the embryonic rat brainstem.

Perinatal development of inhibitory synapses in the nucleus tractus solitarii of the rat.

The nucleus tractus solitarii (NTS) plays a key role in the central control of the autonomic nervous system. In adult rats, both GABA and glycine are used as inhibitory neurotransmitter in the NTS. Using a quantitative morphological approach, we have investigated the perinatal development of inhibitory synapses in the NTS. The density of both inhibitory axon terminals and synapses increased from embryonic day 20 until the end of the second postnatal week (postnatal day 14). Before birth, only GABAergic axon terminals developed and their number increased during the first postnatal week. Mixed GABA/glycine axon terminals appeared at birth and their number increased during the first postnatal week. This suggests the development of a mixed GABA/glycine inhibition in parallel to pure GABA inhibition. However, whereas GABAergic axon terminals were distributed throughout the NTS, mixed GABA/glycine axon terminals were strictly located in the lateral part of the NTS. Established at birth, this specific topography remained in the adult rat. From birth, GABA(A) receptors, glycine receptors and gephyrin were clustered in inhibitory synapses throughout the NTS, revealing a neurotransmitter-receptor mismatch within the medial part of the NTS. Together these results suggest that NTS inhibitory networks develop and mature until postnatal day 14. Developmental changes in NTS synaptic inhibition may play an important role in shaping neural network activity during a time of maturation of autonomic functions. The first two postnatal weeks could represent a critical period where the impact of the environment influences the physiological phenotypes of adult rats.

Postnatal reorganization of primary afferent terminal fields in the rat gustatory brainstem is determined by prenatal dietary history.

Dietary manipulation has been used as an experimental strategy to gain insight into the normal development of the gustatory system. Institution of a diet low in sodium chloride (NaCl) from embryonic day 3 (E3) to E12 (E3-E12 sodium-restricted rats) yields dramatically enlarged terminal fields of the chorda tympani (CT), greater superficial petrosal (GSP), and glossopharyngeal (IX) nerves in the rostral pole of the nucleus of the solitary tract (NTS) at adulthood. To examine how this early, limited dietary manipulation affects postnatal terminal field development, we used a triple anterograde nerve label procedure at postnatal day 15 (P15), P25, P35, and > or =P40 (adults) in two groups: rats fed a commercial diet replete in sodium (controls) and E3-E12 sodium-restricted rats. Results showed an age-related decrease in terminal field volumes of all three nerves during normal development. In contrast, E3-E12 sodium-restricted rats displayed age-related increases of the CT and IX terminal fields, with the terminal field volume of the GSP remaining unchanged throughout development. NTS volume did not grow after P15; therefore, alterations in terminal field volumes are not due to parallel alterations in the size of the NTS. Our data suggest that the age-related decrease in terminal fields observed in controls may reflect activity-dependent pruning of afferent terminals, whereas terminal field increases seen in E3-E12 sodium-restricted rats may reflect cellular/molecular differences in the NTS induced predominantly by activity-independent mechanisms. These findings predict a significant difference in the development of neural coding and sensory-guided behaviors between E3-E12 sodium-restricted rats and controls.

Development of vagal afferent projections circumflex to the obex in the embryonic chick brainstem visualized with voltage-sensitive dye recording.

Using voltage-sensitive dye recording, we surveyed neural responses related to the vagus nerve in the embryonic chick brainstem. In our previous studies, we identified four vagus nerve-related response areas in the brainstem. On the stimulated side, they included (1) the nucleus of the tractus solitarius (NTS: the primary sensory nucleus) and (2) the dorsal motor nucleus of the vagus nerve (DMNV), whereas on the contralateral side, they corresponded to (3) the parabrachial nucleus (PBN: the second/higher-ordered nucleus) and (4) the medullary non-NTS region. In the present study, in addition to these areas, we identified another response area circumflex to the obex. The intensity of the optical signal in the response area was much smaller than that in the NTS/DMNV, and the spatio-temporal pattern could be discerned after signal averaging. The conduction rate to the response area was slower than that to the other four areas. Ontogenetically, the response area was distributed on the stimulated side at the 6-day embryonic stage, and it spread into the contralateral side in 7- and 8-day embryonic stages. These distribution patterns were consistent with projection patterns of vagal afferent fibers stained with a fluorescent tracer, suggesting that the response area included a primary sensory nucleus. In comparison with the functional development of the other four response areas, we traced the functional organization of vagus nerve-related nuclei in the embryonic brainstem.

The development of descending projections from the brainstem to the spinal cord in the fetal sheep.

BACKGROUND: Although the fetal sheep is a favoured model for studying the ontogeny of physiological control systems, there are no descriptions of the timing of arrival of the projections of supraspinal origin that regulate somatic and visceral function. In the early development of birds and mammals, spontaneous motor activity is generated within spinal circuits, but as development proceeds, a distinct change occurs in spontaneous motor patterns that is dependent on the presence of intact, descending inputs to the spinal cord. In the fetal sheep, this change occurs at approximately 65 days gestation (G65), so we therefore hypothesised that spinally-projecting axons from the neurons responsible for transforming fetal behaviour must arrive at the spinal cord level shortly before G65. Accordingly we aimed to identify the brainstem neurons that send projections to the spinal cord in the mature sheep fetus at G140 (term = G147) with retrograde tracing, and thus to establish whether any projections from the brainstem were absent from the spinal cord at G55, an age prior to the marked change in fetal motor activity has occurred. RESULTS: At G140, CTB labelled cells were found within and around nuclei in the reticular formation of the medulla and pons, within the vestibular nucleus, raphe complex, red nucleus, and the nucleus of the solitary tract. This pattern of labelling is similar to that previously reported in other species. The distribution of CTB labelled neurons in the G55 fetus was similar to that of the G140 fetus. CONCLUSION: The brainstem nuclei that contain neurons which project axons to the spinal cord in the fetal sheep are the same as in other mammalian species. All projections present in the mature fetus at G140 have already arrived at the spinal cord by approximately one third of the way through gestation. The demonstration that the neurons responsible for transforming fetal behaviour in early ontogeny have already reached the spinal cord by G55, an age well before the change in motor behaviour occurs, suggests that the projections do not become fully functional until well after their arrival at the spinal cord.

Extensive reorganization of primary afferent projections into the gustatory brainstem induced by feeding a sodium-restricted diet during development: less is more.

Neural development is especially vulnerable to environmental influences during periods of neurogenesis and rapid maturation. In fact, short periods of environmental manipulations confined to embryonic development lead to significant changes in morphology and function. A guiding principal emerging from studies of sensory systems is that experimentally induced effects are most dramatic in higher neural levels (e.g., cortex) and primarily involve postnatal synaptic refinements. In contrast to other sensory systems, the gustatory system is particularly susceptible to the effects of deprivation much earlier and with profound changes evident in the brainstem. Here we show that feeding pregnant rats a custom diet featuring a low-sodium content for 9 d before the tongue appears in the fetus produces extensive restructuring of the gustatory brainstem. Rats born to mothers fed the custom diet from embryonic day 3 (E3) to E12 have terminal field volumes of the greater superficial petrosal, chorda tympani, and glossopharyngeal nerves at adulthood that are expanded as much as 10 times beyond that found in rats fed a standard rat chow. The widespread alterations are not attributable to increased numbers of nerve cells, increased target size, or obvious changes in peripheral taste function. Moreover, we show that the limited period of feeding the custom diet has much larger effects than if rats were fed the diet to postweaning ages. Our results suggest that early periods of altered experience, especially during nucleus of the solitary tract neurogenesis, leads to a restructuring of the gustatory brainstem, which in turn may impact the control of sensory and homeostatic processes.

Delays in neuronal differentiation in Mash1/Ascl1 mutants.

The inactivation of a developmental transcription factor may lead to the complete absence of a specific cell type. More commonly, though, it only partially impairs its generation. The modalities of this partial effect have rarely been documented in any detail. Here, we report a novel function for the bHLH transcription factor Ascl1/Mash1 in the generation of the nucleus of the solitary tract (nTS). In Mash1(-/-) late embryos, the nTS is markedly atrophic. Tracing back the origin of this atrophy, we show that nTS precursors appear in the mutants 1 day later than in the wild type and then accumulate at a slower pace. We also show that the previously reported atrophy of the sympathetic chain in Mash1 mutants is similarly preceded by a delay of 1 to 2 days in the appearance of differentiated ganglionic cells. Finally, we provide evidence that the acceleration imposed by Mash1, regardless of the production of post-mitotic cells, affects differentiation itself, both generic and type-specific.

Glutamatergic synapses in the rat nucleus tractus solitarii develop by direct insertion of calcium-impermeable AMPA receptors and without activation of NMDA receptors.

Calcium influxes through ionotropic glutamate receptors (AMPA and NMDA receptors, AMPARs and NMDARs) are considered to be critical for the shaping and refinement of neural circuits during synaptogenesis. Using a combined morphological and electrophysiological approach, we evaluated this hypothesis at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. We confirmed that in the NTS, the first excitatory synapses appeared at embryonic day 18. We next characterized the biophysical properties of NTS AMPARs. Throughout perinatal development, both evoked and miniature EPSCs recorded in the presence of an NMDAR blocker were insensitive to polyamines and had linear current-voltage relationships. This demonstrated that AMPARs at NTS excitatory synapses were calcium-impermeable receptors composed of a majority of GluR2 subunits. We then investigated the influence of calcium influxes through NMDARs on the development of NTS synaptic transmission. We found that NMDAR expression at synaptic sites did not precede AMPAR expression. Moreover, NMDAR blockade in utero did not prevent the development of AMPAR synaptic currents and the synaptic clustering of GluR2 subunits. Thus, our data support an alternative model of synaptogenesis that does not depend on calcium influxes through either AMPARs or NMDARs. This model may be particularly relevant to the formation of neural networks devoted to basic behaviours required at birth for survival.

Development of the human nucleus of the solitary tract: a cyto- and chemoarchitectural study.

The present study investigated the prenatal development of the cyto- and chemoarchitecture of the human nucleus of the solitary tract from 9 to 35 weeks, by using Nissl staining and immunoreactivity to calbindin, calretinin, tyrosine hydroxylase and GAP-43. The nucleus began to gain heterogeneity and show different subnuclei as early as 13 weeks, and approached cytoarchitectural maturation from 21 to 25 weeks. The subnuclear division pattern observed in the fetal nucleus of the solitary tract at 25 weeks was very similar to that of the adult. Neurons immunoreactive to calbindin first appeared in the medial gastrointestinal area of the nucleus at 13 weeks, particularly within a putative gelatinosus subnucleus, while calretinin immunoreactivity during fetal life suggested the possible presence of a central subnucleus. Tyrosine hydroxylase immunoreactive neurons were seen in the medial subdivisions of the nucleus of the solitary tract as early as 13 weeks, but the population continued to increase until 25 weeks. Strong GAP-43 immunoreactivity was also present in the nucleus of the solitary tract at 13 weeks, especially in the dorsolateral and commissural subnuclei, while at 21 weeks there was a significant decline of GAP-43 expression. Results from the chemoarchitectural study showed that the human nucleus of the solitary tract expressed various neurochemical substances at an early developmental age (13 weeks), even before cellular and neuropil maturation was fully attained. Expression of these factors may play an important role in establishment and integration of viscerosensory function in the nucleus.

First localization and biochemical identification of chromogranin B- and secretoneurin-like immunoreactivity in the fetal human vagal/nucleus solitary complex.

The human vagal/nucleus solitary complex is a primary visceral relay station and an integrative brain stem area which displays a high density of chromogranin B- and secretoneurin-like immunoreactivity. In this study, we localized and biochemically identified these proteins during prenatal development. At prenatal week 11, 15, 20 and 37, we performed a chromatographic analysis to identify the molecular forms of PE-11, a peptide within the chromogranin B sequence, and secretoneurin, a peptide within secretogranin II. Their localization was studied with immunocytochemistry, and was compared to that of substance P which is well established as a functional neuropeptide in the vagal/nucleus solitary complex. At prenatal week 11, chromogranin B-, secretoneurin- and substance P-like immunoreactivities were detected consisting of varicosities, varicose fibers and single cells. At the same time, PE-11 and secretoneurin appeared as a single peak in chromatographic analysis. Prohormone convertases PC1- and PC2-like immunoreactivities were also present at week 11. In general, the density for each peptide increased during later fetal stages with the highest density at week 37. These results demonstrate that each chromogranin peptide is expressed during human fetal life in neurons of the vagal/nucleus solitary complex indicating that these peptides could be important during prenatal development.

Optical detection of convergent projections in the embryonic chick NTS.

Multiple-site optical recording of neural activity was performed in the nucleus of the tractus solitarius (NTS) of the chick embryo with stimulation of the glossopharyngeal nerve (N. IX) and vagus nerve (N. X). We measured the amplitudes of the optical signals related to glutamate-mediated excitatory postsynaptic responses, and calculated the ratio of the signal evoked by simultaneous N. IX/N. X stimulation to the signal obtained after mathematical summation of the individual N. IX and N. X responses. The ratio was significantly lower than 100% in the rostral region of the NTS, in which postsynaptic responses were elicited by both N. IX and N. X stimulations. This result means that there is a convergence of visceral inputs via the N. IX and N. X in the embryonic chick NTS. The existence of the convergence suggests that the NTS performs complex integration of information from multiple sensory inputs from the early stages of embryogenesis.

Optical mapping reveals developmental dynamics of Mg2+-/APV-sensitive components of glossopharyngeal glutamatergic EPSPs in the embryonic chick NTS.

To examine whether there are any differences in functional organization between the glossopharyngeal nerve (N. IX)- and vagus nerve (N. X)-projecting areas in the nucleus of the tractus solitarius (NTS), we performed optical recording of neural responses evoked by N. IX stimulation in 5- to 9-day-old embryonic chick brain stem preparations and compared the results with those in our previous studies concerning the N. X-related NTS. First, we investigated DL-2-amino-5-phosphonovaleric acid (APV)/Mg2+ sensitivity of the glutamatergic excitatory postsynaptic potentials (EPSPs) in the N. IX-related NTS. In 7- to 9-day-old preparations, we found regional differences in the degree of both the APV-induced reduction and Mg2+-free-induced enhancement of the EPSPs. We constructed developmental maps of spatial patterns of the APV- and Mg2+-sensitive components and showed that functional expression of the N-methyl-D-aspartate (NMDA) receptor dynamically changed during development. Second, we studied initial expression of synaptic functions in the N. IX-related NTS. In 6-day-old preparations, although action potentials alone were usually detected in normal Ringer solution, small EPSPs were elicited in a Mg2+-free solution. This result suggests that the NMDA receptor-mediated synaptic function is latently generated in the N. IX-related NTS at the 6-day-old embryonic stage and that external Mg2+ regulates the onset of synaptic functions. Developmental patterns of APV/Mg2+ sensitivity and the stage of initial expression of the glossopharyngeal EPSP were similar to those of the N. X, suggesting that the developmental sequence of the synaptic function in the NTS is the same for the N. IX- and N. X-related NTS.

Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways.

We report that the afferent relays of visceral (cardiovascular, digestive and respiratory) reflexes, differentiate under the control of the paired-like homeobox gene Phox2b: the neural crest-derived carotid body, a chemosensor organ, degenerates in homozygous mutants, as do the three epibranchial placode-derived visceral sensory ganglia (geniculate, petrosal and nodose), while their central target, the nucleus of the solitary tract, which integrates all visceral information, never forms. These data establish Phox2b as an unusual 'circuit-specific' transcription factor devoted to the formation of autonomic reflex pathways. We also show that Phox2b heterozygous mutants have an altered response to hypoxia and hypercapnia at birth and a decreased tyrosine hydroxylase expression in the petrosal chemosensory neurons, thus providing mechanistic insight into congenital central hypoventilation syndrome, which is associated with heterozygous mutations in PHOX2B.

Cardiovascular regulation and expressions of NO synthase-tyrosine hydroxylase in nucleus tractus solitarius of ovine fetus.

The purpose of this study was to examine cardiovascular responses to fourth cerebral ventricle (4V) administration of nitroglycerin (NTG) or an inhibitor of nitric oxide (NO) synthase (NOS) in the near-term ovine and to determine whether, during birth, neuronal NOS (nNOS) is induced in noradrenergic A1 neurons in the medial nucleus tractus solitarius (mNTS). In chronically instrumented fetal sheep, 4V injection of NTG (1.2 nmol), an NO donor, produced an arterial blood depressor and a moderate decrease in heart rate. Arterial blood pressure is increased by 4V administration of NG-nitro-L-arginine methyl ester (10 nmnol), an inhibitor of NOS, in fetuses. Sections of the medulla from fetuses and newborn lambs were examined by using immunolabeling with tyrosine hydroxylase (TH) antibody combined with NADPH diaphorase (NADPHd) histochemistry, a marker of nNOS activity. The NADPHd-positive cells and TH-positive cells containing NADPHd reactivity were significantly increased in the mNTS of newborns compared with the fetuses. The results suggest that during birth, there is upregulation of NADPHd/nNOS in the noradrenergic neurons of mNTS resulting in a centrally mediated reduction of fetal arterial blood pressure.

Expression of neuropeptide FF, prolactin-releasing peptide, and the receptor UHR1/GPR10 genes during embryogenesis in the rat.

Recently, several RF-amide peptides have been identified in mammals. These peptides have a similar C-terminal RF-motif and share some G-protein coupled receptors. Neuropeptide FF (NPFF) and prolactin-releasing peptide (PrRP) are expressed in the same brain areas in the adult rat and act both in prolactin release and cardiovascular regulation. Here, we characterized the embryonal expression from embryonal day 14 to postnatal day 0 of both peptide mRNAs and the mRNA distribution of UHR1/GPR10-like receptor by using in situ hybridization (ISH) and quantitative reverse transcriptase-polymerase chain reaction. NPFF mRNA was found in the spinal cord, caudal solitary tract nucleus, and surprisingly, in the medullary reticular formation. The only peripheral organs displaying NPFF mRNA expression were the lungs and the spleen. PrRP gene expression was seen in the caudal solitary tract nucleus, medullary reticular formation, pontine isthmus and liver, kidney, and testis. The receptor UHR1/GPR10 gene was expressed consistently in the medullary reticular formation and the adrenal gland but also transiently in several locations. All three genes showed weak but even ISH signal in the pituitary. These findings suggest different roles for the peptides during development and indicate that UHR1/GPR10-like receptor could also bind other ligands in addition to PrRP.

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.

Development of the cyto- and chemoarchitectural organization of the rat nucleus of the solitary tract.

The nucleus of the solitary tract (NST) is the major visceral sensory nucleus in the brainstem. The development of the rat nucleus of the solitary tract was followed during late prenatal and early postnatal life in order to determine when subnuclear organization and chemoarchitectural features develop. In Nissl-stained sections, the nucleus of the solitary tract becomes visible as a distinct cluster of cells by about E17. Between E17 and E19, a profound change in the Nissl-stained appearance of the nucleus occurred, so that by E19 all the subnuclei were discernible. Acetylcholinesterase activity in the developing NST showed an early period of rapid differentiation (E15 to E17), while by E19 the basic adult pattern of distribution of this enzyme had already been achieved. The subnuclei of the NST began to show clear differential staining for nicotinamide adenine dinucleotide phosphate diaphorase at about the same time as reactivity for that enzyme first appeared (E19). With respect to calbindin- and calretinin-immunoreactive neurons within the nucleus, many of the chemoarchitectural features associated with these two markers were obvious even by late fetal life. For example, in the central subnucleus, a strongly labelled, dense population of calbindin-immunoreactive neurons was present from E17; while in calretinin-immunoreacted material, this subnucleus was prominent because of its immunonegativity also from E17. Nevertheless, the total number of calbindin- and calretinin-immunoreactive neurons in the NST did not peak until late postnatal life. Tyrosine hydroxylase immunoreactive neurons were visible from E15, began differentiation by E17 and were distributed in a similar pattern to the adult from E19. Substance P immunoreactivity in the NST was also very similar to the adult pattern by E19. Many of these immunochemical and histochemical markers indicate a similar pattern of development, i.e. a rapid period of differentiation until E19, by which time a relatively stable adult-like pattern has been attained. The present findings indicate that many of the cyto- and chemoarchitectural features of this nucleus are present well before birth, by which time the nucleus must serve vitally important functions such as relaying information for control of respiration and the circulation.