Regeneration of taste buds in tongue grafts after reinnervation by neurons in transplanted lumbar sensory ganglia

Taste buds disappear after denervation and reappear after nerve regeneration. Sensory neurons are responsible since reinnervation by motor or autonomic fibers of peripheral nerve fail to induce bud regeneration. However, we do not know whether some neurons in all sensory ganglia can support buds or whether gustatory (i.e., taste bud inducing) neurons are localized to specific cranial ganglia. The present study was therefore pefrormed to determine whether neurons in transplanted spinal ganglia could support taste buds similarly to those in transplanted cranial ganglia. Grafts of lumbar or vagal nodose ganglia were combined with grafts of tongue's vallate papillae in the anterior chamber of rats' eyes and the papillae examined for taste buds 35 days later. Neurons were present in all transplanted ganglia, and all papillae reinnervated by them contained regenerated taste buds. Nerve fibers could be traced from the transplanted ganglia to the epithelium of the tongue grafts which bore the regenerated taste buds. Papillae transplanted without ganglia lacked buds. These findings indicate that some neurons in all sensory ganglia can induce taste bud formation. The present results could occur if gustatory neurons are intrinsically present in all sensory ganglia, but an alternative interpretation is that the tongue grafts transformed some neurons into gustatory neurons and, hence, that neuronal plasticity is involved.     

Retinal ganglion cell axons regenerate in the presence of intact sensory fibres

A novel allograft paradigm was used to test whether adult mammalian central axons regenerate within a peripheral nerve environment containing intact sensory axons. Retinal ganglion cell axon regeneration was compared following anastomosis of dorsal root ganglia grafts or conventional peripheral nerve grafts to the adult rat optic nerve. Dorsal root ganglia grafts comprised intact sensory and degenerate motor axons, whereas conventional grafts comprised both degenerating sensory and motor axons. Retinal ganglion cell axons were traced after 2 months. Dorsal root ganglia survived with their axons persisting throughout the graft. Comparable numbers of retinal ganglion cells regenerated axons into both dorsal root ganglia (1053+/-223) and conventional grafts (1323+/-881; P>0.05). The results indicate that an intact sensory environment supports central axon regeneration.     

Two neuronal populations in the head ganglia of Aplysia californiaa with egg-laying hormone-like immunoreactivity

The egg-laying hormone (ELH) is a polypeptide of known structure, which is synthesized and released by the neurosecretory bag cells in the abdominal ganglion of the mollusc, Aplysia californica. We have used immunohistochemical procedures to reveal organized groups of specific cells and fiber tracts within the pleural and cerebral ganglia in adult Aplysia which are immunopositive when reacted with antibodies generated against ELH. These antibodies are highly specific in that they only stain the bag cells within the abdominal ganglion. The ELH+ systems in the head ganglia persisted in two animals 43 days and 67 days after surgical removal of their abdominal ganglia and pleurovisceral connectives. It is therefore likely that these immunoreactive neurons and their processes are independent of the bag cell system.The pleural ganglia neurons, with ELH-like immunoreactivity, bear a striking resemblance to bag cells in somal and nuclear dimensions, neuritic morphology and association with the pleurovisceral connectives. This suggests that both populations of cells may have descended from a common precursor pool during embryogenesis. In the cerebral ganglion, a pair of bilateral clusters of 6–10 small immunopositive neurons are located on the dorsal surface in the vicinity of the C clusters, and send their processes into the neuropil. Intensely stained tracts of ELH+ fibers are prominent at all levels of section in the cerebral neuropil; stained fibers can also be traced into most of the nerves emanating from the cerebral ganglion.Although the functions of these systems, as well as the specific nature of the immunoreactive molecule(s) they contain, remain unknown, 5 ELH-like genes of Aplysia have now been cloned and 3 of these have been sequenced^26,27. Our results suggest that the immunoreactive molecules in the pleural and cerebral neurons are due to peptides controlled by one or more of these genes. ELH is known to markedly change the electrical activity of neurons in the head ganglia in vitro. This demonstration of the presence and distribution of ELH-like molecules endogenous to the head ganglia raises the possibility that ELH target neurons in the head ganglia may be activated by local sources of ELH-like neuroactive peptides in vivo.     

Innervation of single fungiform taste buds during development in rat

To determine whether the innervation of taste buds changes during postnatal development, the number of geniculate ganglion cells that innervated single fungiform taste buds were quantified in the tip- and midregions of the tongue of adult and developing rats. There was substantial variation in both the size of individual taste buds and number of geniculate ganglion cells that innervated them. Importantly, taste bud morphology and innervation were highly related. Namely, the number of labeled geniculate ganglion cells that innervated a taste bud was highly correlated with the size of the taste bud (r = 0.91, P < .0003): The larger the taste bud, the more geniculate ganglion cells that innervated it. The relationship between ganglion cell number and taste bud volume emerged during the first 40 days postnatal. Whereas there was no difference in the average number of ganglion cells that innervated individual taste buds in rats aged 10 days postnatal through adulthood, taste bud volumes increased progressively between 10 and 40 days postnatal, at which age taste bud volumes were similar to adults. The maturation of taste bud size was accompanied by the emergence of the relationship between taste bud volume and number of innervating neurons. Specifically, there was no correlation between taste bud size and number of innervating geniculate ganglion cells in 10-, 20-, or 30-day-old rats, whereas taste bud size and the number of innervating ganglion cells in 40-day-old rats were positively correlated (r = .80, P < .002). Therefore, the relationship between taste bud size and number of innervating ganglion cells develops over a prolonged postnatal period and is established when taste buds grow to their adult size. J. Comp. Neurol. 398:13–24, 1998. © 1998 Wiley-Liss, Inc.     

Regenerating afferent fibers stimulate the recovery of mauthner cell dendritic branching in the axolotl

In the medulla of the axolotl (Ambystoma mexicanum), Mauthner cells (M-cells) occur as a pair of large identifiable neurons at the level of entry of the vestibular nerve (nVIII). Each receives synapses from the ipsilateral nVIII; the terminals can be identified as club endings and are restricted to a specific set of M-cell dendritic branches. We have examined these branches for morphologic changes following long-term deafferentation in the presence and absence of nerve regeneration. Deafferentation was brought about in a group of young larvae by unilaterally severing nVIII. The nerve was allowed to regenerate in half of the larvae. In those remaining, the nVIII ganglion was damaged to preclude or limit nerve regeneration. The contralateral side served as control. After 3 months survival, the larvae were killed and the medullae prepared for microscopy. To estimate the extent of nerve regeneration, axons in the experimental nVIII tract were counted and compared with the number in the control. The mean number of axons in the nVIII tract ipsilateral to intact ganglia indicated that 69% of the fibers had regenerated. In contrast, only 31% regenerated in larvae with damaged ganglia. Electron microscopic analysis of selected sections revealed that the mean number of nVIII terminals per section through M-cells ipsilateral to destroyed ganglia was significantly less than the mean number in analogous sections through either control cells or cells ipsilateral to intact ganglia. Control and experimental M-cells were reconstructed from serial sections. Deprived M-cells had significantly reduced dendritic branching patterns in the region that normally receives nVIII input. On the other hand, the extent of branching on cells receiving regenerated afferents from intact ganglia was like that of their contralateral controls. The distribution of dendritic branches on many reinnervated M-cells, however, was broader than that on control cells. Electron microscopic examination of the displaced dendritic branches (those extending into adjacent tracts) revealed that they received vestibular synapses. Thus, in some animals, regenerated vestibular fibers were not restricted to the nVIII tract. Deafferentation of the M-cells results in a reduction of dendritic branches in the region deprived of vestibular contacts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and developmental changes in GABA-like immunoreactive neurons in the central nervous system of pond snail, Lymnaea stagnalis

We examined three-dimensionally the arrangement of gamma-aminobutyric acid (GABA)-like immunoreactive neurons in the central nervous system (CNS) of the pond snail, Lymnaea stagnalis, by a combination of immunohistochemistry and confocal laser scanning microscopy on whole-mount preparations. GABA-like immunoreactivity was detected in all ganglia of the adult CNS. The following distribution of immunoreactive cell bodies was noted in the adult snail. Buccal ganglia: one cell body and five pairs of cell bodies, cerebral ganglia: one pair of cell bodies, pedal ganglia: two single cell bodies, two pairs of cell bodies, and three pairs of cell clusters, and pleural ganglia: one pair of cell bodies. In the asymmetrical parietal ganglia, three cell bodies were located in the left parietal ganglion; three cell bodies and three cell clusters were located in the right parietal ganglion. In the single visceral ganglion, a few scattered individual cell bodies and a cell cluster were GABA-like immunoreactive. Our results showed that the occurrence of GABA is widely spread in the CNS of adult L. stagnalis. GABA-like immunoreactivity in the CNS was not detected in the embryo but was observed after hatching, although the number of stained cells was less than in the adult, with the exception of those in the cerebral ganglia where their number decreased with maturation. Our results provide detailed maps of the central GABA-like immunoreactive neurons in juveniles, immatures, and adults of L. stagnalis.     

Neurocalcin-immunoreactive neurons in the petrosal ganglion innervate the taste bud

The distribution and origin of neurocalcin-immunoreactive (NC-ir) nerve fibers in the taste bud and carotid body were examined by an immunofluorescence method. In the circumvallate papilla of the tongue, NC-ir nerve fibers made subepithelial nerve plexuses and occasionally penetrated the taste bud. However, the carotid body was devoid of ir nerve fibers. In the petrosal ganglion, 32% of neurons were immunoreactive for NC. Such neurons were mostly medium-sized to large, and scattered throughout the ganglion. In the superior cervical and intralingual ganglia, numerous ir varicose fibers surrounded postsynaptic neurons. However, NC-ir could not be detected in cell bodies of these neurons. The retrograde tracing method indicated that NC-ir petrosal neurons innervated taste buds in the circumvallate papilla. NC-ir neurons may have a gustatory function in the petrosal ganglion.     

Neuron/target plasticity in the peripheral gustatory system

Taste bud volume on the anterior tongue in adult rats is matched by an appropriate number of innervating geniculate ganglion cells. The larger the taste bud, the more geniculate ganglion cells that innervate it. To determine if such a match is perturbed in the regenerated gustatory system under different dietary conditions, taste bud volumes and numbers of innervating neurons were quantified in adult rats after unilateral axotomy of the chorda tympani nerve and/or maintenance on a sodium-restricted diet. The relationship between taste bud size and innervation was eliminated in rats merely fed a sodium-restricted diet; individual taste bud volumes were smaller than predicted by the corresponding number of innervating neurons. Surprisingly, the relationship was disrupted in a similar way on the intact side of the tongue in unilaterally sectioned rats, with no diet-related differences. The mismatch in these groups was due to a decrease in average taste bud volumes and not to a change in numbers of innervating ganglion cells. In contrast, individual taste bud volumes were larger than predicted by the corresponding number of innervating neurons on the regenerated side of the tongue; again, with no diet-related differences. However, the primary variable responsible for disrupting the function on the regenerated side was an approximate 20% decrease in geniculate ganglion cells available to innervate taste buds. Therefore, the neuron/target match in the peripheral gustatory system is susceptible to surgical and/or dietary manipulations that act through multiple mechanisms. This system is ideally suited to model sensory plasticity in adults.     

The structure of the fourth abdominal ganglion of the crayfish, Procambarus clarki (Girard). I. Tracts in the ganglionic core

The organization of the fourth abdominal ganglion of the crayfish, Procambarus clarki, was studied with the light microscope in serial sections stained with osmium ethyl gallate. This ganglion is composed of a ventral rind of somata and a core of alternating layers of through-tracts and commissures. The longitudinal tracts of the ganglion are named according to the system in use for the orthopteran insects, because the basic plans of the crustacean and insect ventral ganglia exhibit striking anatomical parallels. The dorsal tracts are the largest and the most regular in their path through the ganglion. In the ventral posterior quadrant of the ganglion the tracts diverge from the basic plan to pass around the major synaptic neuropil and the bases of the peripheral nerves. This paper reports the three-dimensional anatomy of the major longitudinal through-tracts, internal tracts and commissures, and bases of peripheral nerves. Landmark features of the ganglion--including the tracts, the major artery of the vascular system, the shape of the ganglionic core in section, and prominent single cells, all of which make it possible to recognize specific regions of the ganglion--are described.     

Morphological and morphometrical changes in dorsal root ganglion neurons innervating the regenerated lizard tail

The variations occurring in neurons from dorsal root ganglia that provide innervation to the regenerated tail of the lizard (vicarious ganglia) are analysed. Vicarious ganglion neurons, when compared to control ganglion neurons (i.e. ganglia from the same animal that were not involved in the reinnervation process), show a size increase of the soma (cell hypertrophy) which applies to all cell types and sub-types. No statistically significant differences in the relative percentage of neurofilament-poor (type D) and neurofilament-rich (type L) neurons were found between vicarious dorsal root ganglia compared to controls in all animals. On the contrary, within L neuron sub-types, a statistically significant increase in sub-type L2 (very rich in neurofilaments), and the appearance of sub-type L3 neuron which is not detectable in controls, were demonstrated in vicarious dorsal root ganglia. In spite of these variations in size and percentage distribution, no structural and ultrastructural differences of the various cell types and sub-types are detectable, except for the appearance of the sub-type L3 neurons. However, this neuron sub-type might not be considered specific of hypertrophy since the same morphological features have been observed, in normal conditions, in lizard dorsal root ganglia from cervical and lumbar spinal levels that provide innervation to limb plexuses.     

The neural induction of taste buds in the salivary ducts of the lingual gland of von Ebner

Taste buds appear in the vallate papilla when tongue grafts are combined with sensory ganglia grafts in the anterior chamber of rats' eyes. After 50 days, many of the tongue grafts (but not the ganglion grafts) developed cysts, the papilla atrophied, and fewer taste buds were found in them than at earlier postoperative times. A study was therefore undertaken to determine whether exteriorizing the tongue graft (i.e., placing it in the confines of a corneal biopsy opening with the epithelial surface of the papilla facing outward) would better preserve the morphology of long-term grafts. In isogenic adult Lewis rats, tongue grafts were exteriorized alone or with nodose ganglia and examined after 90 to 100 days. The tongue grafts were better preserved irrespective of whether they were transplanted with or without a nodose ganglion. In addition, taste buds were now found in the epithelium of the ducts of von Ebner's lingual salivary gland. These glands lie just below the vallate papilla and because their ducts empty into the base of the papilla, portions of the ducts are unavoidably contained in tongue grafts. It appeared that intrinsic nerve fibers of the eye, just like nerve fibers from ganglia, induced the formation of ductal buds since ductal buds appeared in ten of 12 tongue grafts transplanted without added ganglia. Of significance was the observation that intense cholinesterase activity was seen in intragemmal nerve fibers of ganglionic neurons whereas no such activity was seen in intragemmal fibers when they were derived from intrinsic nerve fibers of the eye. These results indicate that exteriorization of tongue grafts better preserves their structure, that epithelium in the ducts of von Ebner's gland can be transformed into taste bud cells, that intrinsic nerve fibers of the eye can induce taste bud formation, and that there are two types of sensory neurons (based on intragemmal cholinesterase activity of their nerve fibers) which can cause taste bud development.     

Progressive morphological abnormalities observed in rat spinal motor neurons at extended intervals after axonal regeneration.

It is generally accepted that mammalian spinal motor neurons return to normal after axotomy if their regenerated axons successfully reinnervate appropriate peripheral targets. However, morphological abnormalities, recently observed in spinal motor neurons examined 1 year after nerve crush injury, raise the possibility that delayed perikaryal changes occur after regeneration is complete. In order to distinguish between chronic and progressive alterations in neurons with long-term regenerated axons, rat spinal motor neurons and dorsal root ganglion cells were examined at 5 and 10 months following unilateral sciatic nerve crush. Neurons with regenerated axons were identified by retrograde labelling with horseradish peroxidase. The structural properties of neurons ipsilateral to nerve injury were compared to those of neurons from the spinal cord and dorsal root ganglia on the contralateral side and from age-matched control rats. At 5 months postcrush, the morphology of motor and sensory neurons ipsilateral to injury was comparable to that of control cells. However, several features of the motor neurons with regenerated axons distinguished them from control motor neurons at 10 months postcrush. Mean perikaryal area of ipsilateral spinal motor neurons was larger than the means for control motor neurons (p less than .001). Ipsilateral spinal motor neurons also appeared clustered within the spinal cord and had thicker dendrites. Dorsal root ganglion cells with regenerated axons were slightly larger than control cells at 10 months postcrush but they exhibited no other morphological changes. The present findings indicate that spinal motor neurons are progressively altered after their regenerated axons have reestablished functional synapses with their peripheral targets.     

The ability of axons to regenerate their growth cones depends on axonal type and age, and is regulated by calcium, cAMP and ERK

The processes activated at the time of axotomy and leading to the formation of a new growth cone are the first step in regeneration, but are still poorly characterized. We investigated this event in an in vitro model of axotomy performed on dorsal root ganglia and retinal explants. We observed that the dorsal root ganglion axons and retinal ganglion cell axons, which had grown out on a poly d-lysine/laminin substrate at the time of culture preparation greatly differed in their regenerative response after a subsequent in vitro lesion made far from the cell body. The majority of axons of adult dorsal root ganglia but only a small percentage of axons of adult retinal ganglion cells regenerated new growth cones within four hours after in vitro axotomy, though both kinds of axons were growing before the lesion. The depletion of extracellular calcium and the inhibition of extracellular-signal regulated kinase 1,2 (ERK) and protein kinase A (PKA) at the time of injury significantly impaired the capacity of dorsal root ganglia axons to re-initiate growth cones without affecting growth cone motility. Pharmacological treatments directed at increasing the level of cAMP promoted growth cone regeneration in adult retinal ganglion cell axons in spite of the low regenerative potential exhibited in normal conditions. Understanding the cellular mechanisms activated at the time of lesion and leading to the formation of a new growth cone is necessary for devising treatments aimed at enhancing the regenerative response of injured axons.     

Serotoninergic reinnervation of regenerating tentacular sensory organs in a pulmonate snail, Cryptomphalus aspersa.

Several ontogenetic studies performed in different species suggest a developmental role for 5-HT neurons. The 5-HT system interconnecting the CNS and the tentacular sensory organs in pulmonates is a suitable model for studying the postulated developmental role of 5-HT neurons. In this paper we describe the behavior of the 5-HT fibers during the early stages of blastema reinnervation, primordium formation and differentiation of regenerating tentacular sensory organs in the pulmonate snail Cryptomphalus aspersa. Our results show that the regeneration process allows the development of a normal pattern of 5-HT innervation of the regenerated sensory organs and suggest that 5-HT could be involved in reciprocal developmental interactions with regenerating tentacular tissues.     

Neuronal localization of pancreatic polypeptide (PP) and vasoactive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris)

Nerve fibres and cell bodies displaying vasoactive intestinal polypeptide (VIP) or pancreatic polypeptide (PP) immunoreactivity were demonstrated in ganglia of the earthworm (Lumbricus terrestris). VIP cell bodies were found in the most anterior ganglion of the ventral nerve cord, the subpharyngeal ganglion. Immunoreactive nerves were seen running in the center of the cord until about the 10th segment. PP cell bodies were found in the cerebral ganglion where VIP was lacking, in the subpharyngeal ganglion and in more posteriorly located ganglia of the ventral nerve cord. PP nerve fibres could be followed below the 10th segment of the cord.     

Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs.

Sensory ganglia that innervate taste buds and gustatory papillae (geniculate and petrosal) are reduced in volume by about 40% in mice with a targeted deletion of the gene for brain-derived neurotrophic factor (BDNF). In contrast, the trigeminal ganglion, which innervates papillae but not taste buds on the anterior tongue, is reduced by only about 18%. These specific alterations in ganglia that innervate taste organs make possible a test for roles of lingual innervation in the development of appropriate number, morphology, and spatial pattern of fungiform and circumvallate papillae and associated taste buds. We studied tongues of BDNF null mutant and wild-type littermates and made quantitative analyses of all fungiform papillae on the anterior tongue, the single circumvallate papilla on the posterior tongue, and all taste buds in both papilla types. Fungiform papillae and taste buds were reduced in number by about 60% and were substantially smaller in diameter in mutant mice 15-25 days postnatal. Remaining fungiform papillae were selectively concentrated in the tongue tip region. The circumvallate papilla was reduced in diameter and length by about 40%, and papilla morphology was disrupted. Taste bud number in the circumvallate was reduced by about 70% in mutant tongues, and the remaining taste buds were smaller than those on wild-type tongues. Our results demonstrate a selective dependence of taste organs on a full complement of appropriate innervation for normal growth and morphogenesis. Effects on papillae are not random but are more pronounced in specific lingual regions. Although the geniculate and petrosal ganglia sustain at least half of their normal complement of cell number in BDNF -/- mice, remaining ganglion cells do not substitute for lost neurons to rescue taste organs at control numbers. Whereas gustatory ganglia and the taste papillae initially form independently, our results suggest interdependence in later development because ganglia derive BDNF support from target organs and papillae require sensory innervation for morphogenesis.     

Topographic organization of serotonergic and dopaminergic neurons in the cerebral ganglia and their peripheral projection patterns in the head areas of the snail Helix pomatia.

The distribution of monoaminergic neurons within the cerebral ganglia was investigated in the pulmonate snail Helix pomatia. Simultaneous serotonin and tyrosine hydroxylase double immunostaining revealed that the immunoreactive cell groups are concentrated in a putative monoaminergic center on the ventral surface of the cerebral ganglia. Simultaneous cobalt (Co)- and nickel (Ni)-lysine backfills of cerebral nerves were combined with 5, 6-dihydroxytryptamine pigment-labelling of serotonergic neurons, or with fluorescence immunocytochemistry of dopaminergic neurons. This showed that the serotonergic and dopaminergic cell groups can be divided into smaller subgroups on the basis of their axonal projections into different cerebral nerves. These subgroups show a topographic organization within the serotonergic and dopaminergic neuronal clusters. In the serotonergic system, the different regions of the head are represented in a rostrocaudal direction, whereas a caudorostral organization is characteristic for the dopaminergic system. No serotonin- or dopamine-immunoreative cell bodies but numerous fibers were observed in the head areas, indicating that these are innervated by cerebral monoaminergic neurons and show different innervation patterns. Serotonin-immunoreactive fibers mostly innervate muscle fibers, whereas dopamine-immunoreactive processes do not innervate effector cells, but terminate within the nerve branches of the head areas. On the basis of their innervation pattern, we suggest that dopaminergic neurons may take part in en route modulation of sensory afferent and efferent processes in an as yet unknown manner. The serotonergic neurons, on the other hand, may play a direct role in the modulation of muscle function.     

Regeneration of axons and synaptic connections by touch sensory neurons in the leech central nervous system

In studies of axonal regeneration, it has been difficult to determine (a) whether growth along the normal pathway is important for restoration of connections with previous targets and (b) whether the new synapses resemble the old in strength and location. To address these problems at the level of individual nerve cells, we have studied touch (T) sensory neurons in the leech after their axons have been severed and we have confirmed that their axons regenerate electrical connections with some of their usual synaptic targets in the central nervous system. Injections of horseradish peroxidase and Lucifer Yellow dye into separate T cells in unoperated animals showed that T cell axons typically run close to one another within single ganglia or from ganglion to ganglion. Knowledge of one T cell's arborizations thus revealed the groundplan of others in the same ganglia and the sites of apparent contact with its synaptic targets. For regenerating axons, those sprouts that encountered the normal pathway (as marked by homologous axons) grew preferentially along it. Despite the striking coincidence of old and new pathways, regenerated branching patterns within the ganglionic neuropils were usually incomplete and sometimes had atypical branches. Synaptic connections with normal targets (other T cells as well as S and C cells) were abnormally weak physiologically. The numbers of apparent contacts seen with the light microscope were also lower than normal. In addition, the strength of the synaptic potentials, normalized to the number of contacts (calculated as microvolts per contact), was generally smaller in the regenerated connections than in the controls, and smallest at earliest times, during the first 6 weeks following injury. It thus appears to be characteristic of T cell regeneration that axon regrowth is aided by the recognition of specific pathways and that successful regeneration, as assayed anatomically and physiologically, occurs frequently but usually incompletely.     

An identified pleural ganglion interneuron inhibits patterned motor activity in the buccal ganglia of the snail, Helisoma

Neuron, Pl1, an interneuron that inhibits patterned motor output underlying feeding in the snail, Helisoma, is identified. The soma of neuron Pl1 is in the pleural ganglion and its axon projects through the pedal and cerebral ganglia to the buccal ganglia. A train of action potentials in neuron Pl1 suppresses rhythmic activity in the buccal pattern generator even in the presence of strong pharmacological stimulation with serotonin.