The role of feedback from the vocal organ. I. Maintenance of stereotypical vocalizations by adult zebra finches

The stereotypical vocal patterns of adult male zebra finches (Poephila guttata) were examined before and after elimination of auditory feedback and/or feedback from the vocal organ (the syrinx). Elimination of auditory feedback was accomplished via bilateral removal of the cochleae, whereas feedback from the syrinx was eliminated by cutting hypoglossal afferent fibers while leaving hypoglossal efferents intact. Very little or no disruption of song was observed in birds which underwent deafening as well as unilateral deafferentation of the syrinx. Control experiments showed that the minor deficits observed were not attributable to lesion of pulmonary fibers in the descending branch of the vagus. There was also little deficit in song behavior of birds that were deafened and subjected to bilateral deafferentation of the syrinx. These results are consistent with the hypothesis that stable song patterns in adult passerine birds are not dependent on peripheral sources of feedback, but may be governed by a learned central control program.     

Afferents from Vocal Motor and Respiratory Effectors Are Recruited during Vocal Production in Juvenile Songbirds

Learned behaviors require coordination of diverse sensory inputs with motivational and motor systems. Although mechanisms underlying vocal learning in songbirds have focused primarily on auditory inputs, it is likely that sensory inputs from vocal effectors also provide essential feedback. We investigated the role of somatosensory and respiratory inputs from vocal effectors of juvenile zebra finches (Taeniopygia guttata) during the stage of sensorimotor integration when they are learning to imitate a previously memorized tutor song. We report that song production induced expression of the immediate early gene product Fos in trigeminal regions that receive hypoglossal afferents from the tongue and syrinx (the main vocal organ). Furthermore, unilateral lesion of hypoglossal afferents greatly diminished singing-induced Fos expression on the side ipsilateral to the lesion, but not on the intact control side. In addition, unilateral lesion of the vagus reduced Fos expression in the ipsilateral nucleus of the solitary tract in singing birds. Lesion of the hypoglossal nerve to the syrinx greatly disrupted vocal behavior, whereas lesion of the hypoglossal nerve to the tongue exerted no obvious disruption and lesions of the vagus caused some alterations to song behavior. These results provide the first functional evidence that somatosensory and respiratory feedback from peripheral effectors is activated during vocal production and conveyed to brainstem regions. Such feedback is likely to play an important role in vocal learning during sensorimotor integration in juvenile birds and in maintaining stereotyped vocal behavior in adults.     

Neural pathways for bilateral vocal control in songbirds

Ipsilateral and contralateral projections of nucleus robustus archistriatalis (RA), a telencephalic vocal premotor nucleus, to respiratory-vocal nuclei in the brainstem were defined in adult male Wasserschlager canaries, grey catbirds, and zebra finches, three songbird species that appear to differ in the degree of lateralized syringeal dominance. In all three species, ipsilateral projections of RA to the medulla included the tracheosyringeal part of the hypoglossal nucleus (XIIts), that innervates the syrinx, the bird's vocal organ, the suprahypoglossal area (SH), and two respiratory-related nuclei, retroambigualis (RAm) and parambigualis (PAm; Reinke and Wild [1998] J Comp Neurol 391:147-163). Projections of RA to the contralateral XIIts, SH and RAm, were substantial in canaries, which use the left side of the syrinx predominantly during singing; less pronounced in catbirds, which have no lateral dominance for song control; and least pronounced in zebra finches, in which there is a right-sided dominance for song control. There were no obvious differences in the number of crossed projections in birds injected in the left or right RA. Local sources of inputs to XIIts and RAm were defined anatomically in zebra finches and canaries. RAm, including neurons in close proximity to XIIts, was found to project to XIIts and the suprahypoglossal area bilaterally but predominantly ipsilaterally. RAm also had reciprocal connections with its contralateral homologue. These results suggest a pattern of connections between premotor and motor respiratory-vocal nuclei that may be involved in bilateral control of vocal output at medullary levels, a control that involves a high degree of coordination between vocal and respiratory structures on both sides of the body.     

Innervation of the syrinx of the zebra finch (Taeniopygia guttata)

In songbirds, the learning and maintenance of song is dependent on auditory feedback, but little is known about the presence or role of other forms of sensory feedback. Here, we studied the innervation of the avian vocal organ, the syrinx, in the zebra finch. Using a combination of immunohistochemistry, immunofluorescence and neural tracing with subunit B of cholera toxin (CTB), we analysed the peripheral and central endings of the branch of the hypoglossal nerve that supplies the syrinx, the tracheosyringeal nerve. In the syringeal muscles, we show the presence of numerous choline acetyl transferase‐like immunoreactive en plaque motor endplates and substance P‐like immunoreactive, thin and varicose free nerve endings. Substance P‐like immunoreactive free nerve endings were also present in the luminal syringeal tissues, especially in the luminal epithelium of the trachea and pessulus. Also, by a combination of immunofluorescence and transganglionic tracing following injections of CTB in the tracheosyringeal nerve, we identified as central targets of the syringeal receptors the caudolateral part of the interpolaris subnucleus of the descending trigeminal tract, a caudolateral region of the nucleus tractus solitarius, and a lateral band of the principal sensory trigeminal nucleus. Further studies are required to determine the sensory modalities of these receptors and the connections of their specific synaptic targets.     

Androgen affects cholinergic enzymes in syringeal motor neurons and muscle

We examined the role of testosterone (T) in regulating the weight of the songbird syrinx and the activity of two cholinergic enzymes, choline acetyltransferase (CAT) and acetylcholinesterase (AChE). Castration of adult male zebra finches or neonatal canaries results in a lowering of syringeal weight and CAT and AChE activity. Administration of T for 1–4 weeks restores syringeal weight and AChE to intact levels in male zebra finches. Activity of CAT in muscle is not fully restored. Ovariectomy of female zebra finches and canaries does not affect these syringeal parameters, but T administration to ovariectomized females for 1 month increased syringeal weight and AChE activity. In the zebra finch tracheosyringealis nerve, activity of CAT and AChE is decreased on moonth after castration. T administration to castrates maintains nerve AChE activity but not CAT. In contrast to changes in the syrinx, tongue muscles do not change in weight or enzyme activity when circulating T levels are altered. Effects of muscle use and disuse were found on syringeal weight and AChE activity, but an androgenic effect also operates in addition. Results suggest that one mechanism for T regulation of singing in passerine birds is through induction of specific enzymatic proteins in androgen target neurons and muscles.     

Bilateral interactions within the vocal control pathway of Birds: Two evolutionary alternatives

Bilateral interactions between the right and left sides of the vocal control pathway of three avian species, the budgerigar (Melopsittacus undulatus), the zebra finch (Poephila guttata), and the canary (Serinus can-arius), were compared by using microstimulation. Stimulation of the neo-striatal nucleus, hyperstriatum ventrale pars caudale (HVc), caused a constant-latency increase in neural activity of the tracheosyringeal (ts) nerve. This nerve supplies the avian vocal organ, the syrinx. Unilateral stimulation of HVc in budgerigars caused equal responses in both right and left ts nerves, whereas in canaries and zebra finches, much larger responses with lower thresholds and shorter latencies were evoked in the ipsilateral ts nerve than in the contralateral ts nerve. Furthermore, contralateral responses in the oscines were seen only at high rates of stimulation, whereas ipsilateral responses could be evoked by a single stimulus pulse. Simultaneous bilateral stimulation of HVc in budgerigars evoked a greater than linear summation of responses on each ts nerve, evidence for convergence of ipsi- and contralateral inputs onto the same set of motor neurons. No such summation was detected in zebra finches or canaries. The bilateral distribution of excitation within the song control pathway of budgerigars should lead to coactivation of bilaterally paired muscles on each side of the syrinx. This type of interaction should ensure that the two sides of the parrot syrinx operate as parts of a unitary, or single-voiced, syrinx. In contrast, the two sides of the song control pathway in canaries and zebra finches are almost completely isolated from each other, and are thus free to act independently. This type of organization lends itself to the separate control of simultaneously produced, harmonically unrelated sound elements by the two independent sound sources present in the songbird's two-voiced syrinx.     

Lateralization and effects of adult androgen in a sexually dimorphic neuromuscular system controlling song in zebra finches

Unlike many other sexually dimorphic systems, gonadal secretions do not explain sex differences in the morphology of the telencephalic song control nuclei of zebra finches. It is important to understand whether a novel mechanism for controlling structure is restricted to the forebrain regions specialized for song, and whether other areas respond more typically to gonadal steroids. Therefore, the effects of sex and adult androgen manipulation on the neuromuscular end of the song control system (tracheosyringeal portion of the hypoglossal nucleus, nXIIts, and the syrinx, or vocal organ) were investigated. Because lesion and axotomy experiments indicate a right-side bias in the functional control of song, asymmetry in the motor nucleus and in the ventralis and dorsalis syrinx muscles was also tested. Male-biased dimorphisms existed in the volume of nXIIts, and in syrinx mass and size of muscle fibers, but not in motoneuron number or size. Asymmetries favoring the right side were detected in nXIIts volume and motoneuron number in males, as well as in ventralis and dorsalis fiber size in both sexes. Hormone manipulations had no effect on nXIIts size, neuron size, or number. Testosterone treatment of adult females increased all of the syringeal measures, but the only effect of flutamide in males was to decrease syrinx weight. Thus, male-biased sexual dimorphisms and right side dominance in both nXIIts and the syrinx may facilitate singing behavior. Adult androgen exposure can induce partial masculinization of the syrinx, but other factors must be important in mediating the sex differences in both that structure and the volume of nXIIts.     

Central control of song in the canary, Serinus canarius.

We have traced central nervous pathways controlling bird song in the canary using a combination of behavioral and anatomical techniques. Unilateral electrolytic brain lesions were made in adult male canaries whose song had been previously recorded and analysed on a sound spectrograph. After several days of postoperative recording, the birds were sacrificed and their brains processed histologically for degeneration staining with the Fink-Heimer technique. Although large lesions in the neostriatum and rostral hyperstriatum had no effect on song, severe song deficits followed damage to a discrete large-celled area in the caudal hyperstriatum ventrale (HVc). Degenerating fibers were traced from this region to two other discrete nuclei in the forebrain: one in the parolfactory lobe (area X, a teardrop-shaped small-celled nucleus); and a round large-celled nucleus in the archistriatum (RA). Unilateral lesion of X had no effect on song; lesions of RA, however, caused severe song deficits. Degenerating fibers from RA joined the occipitomesencephalic tract and had widespread ipsilateral projections to the thalamus, nucleus intercollicularis of the midbrain, reticular formation, and medulla. It is of particular interest that direct connections were found onto the cells of the motor nucleus innervating the syrinx, the organ of song production. Unilateral lesions of n. intercollicularis (previously implicated in the control of vocal behavior) had little effect on song. One bilateral lesion of HVc resulted in permanent (9 months) and complete elimination of the audible components of song, although the bird assumed the posture and movements typical of song. Preliminary data suggest that lesions of the left hemisphere result in greater deficits than lesions of the right one. This finding is consistent with earlier reports that the left syrinx controls the majority of song components. Results reported here suggest a localization of vocal control in the canary brain with an overlying left hemispheric dominance.     

Respiratory gating of activity in the avian vocal control system

Electrical stimulation of brain nuclei within the vocal control pathway of budgerigals (Melopsittacus undulatus) or zebra finches (Poephila guttata) evoked neural activity in the hypoglossal nerves innervating the vocal organ, the syrinx. This activity was gated within the hypoglossal motor nucleus such that stimulation recruited more hypoglossal motor neurons to fire during expiration than during inspiration. This respiratory gating mechanism represents a novel solution to the problem of shared control of motor neurons during different behaviors, and may contribute to the patterning of neural activity during song production.     

Relation of medullary motor nuclei to nerves supplying the vocal tract of the budgerigar (Melopsittacus undulatus)

The locations of brainstem motor nuclei supplying efferents to organs of the vocal tract of the budgerigar (Melopsittacus undulatus) were determined as a first step in describing brain pathways for vocal control in this species. We identified four major nerves supplying the muscles of the syrinx, larynx, and tongue, and separately treated each of these with horseradish peroxidase. Nerves supplying efferents to the larynx and tongue arise from ipsilateral cells in the nucleus ambiguus and hypoglossal nucleus, respectively. Each right and left half of the syrinx is supplied by both right and left halves of the hypoglossal nucleus, via axons crossing in the common anastomosis of the tracheosyringeal nerves. This bilateral innervation of the syrinx will allow testing for central asymmetries of dominance for vocal control in the absence of any underlying anatomical laterality in the periphery.     

Androgen receptors in the embryonic zebra finch hindbrain suggest a function for maternal androgens in perihatching survival

Bird embryos are exposed to maternal androgens deposited in the egg, but the role of these hormones in embryonic development and hatchling survival is unclear. To identify possible target organs, we used in situ hybridization to study the distribution of androgen receptor (AR) RNA in the developing zebra finch brain. The first brain expression domain of AR mRNA is in the hindbrain. From embryonic day 7 (E7) onward, when the hypoglossal motor nucleus (nXII) has just formed, there was AR mRNA expression in both its lingual (nXIIl) and its tracheosyringeal (nXIIts) parts, and this was the major site of hindbrain expression at all embryonic stages and in both sexes. From E8 onward, we also found AR mRNA in the supraspinal motor nucleus (nSSp), which innervates neck muscles. Furthermore, the syrinx, the target of the nXIIts, contained AR mRNA by E10, localized principally in the perichondria. Muscle was first evident in the syringeal region at E9, but no AR was detected in syringeal muscles until after hatching. The expression pattern of AR in the zebra finch embryo suggests that maternal androgens act via AR in the brainstem and syrinx to influence hatching as well as acoustic and visual components of food-begging behavior. Maternal androgens seem unlikely to function in the development of sexual dimorphisms in the zebra finch nXIIts and syrinx, insofar as these are not evident until between 10 and 20 days posthatching.     

Clinical and neuroradiological features of syringomyelia associated with Chiari malformation

The clinical presentation and radiological features were analyzed in 30 cases of syringomyelia associated with Chiari malformation. None of the patients had spinal dysraphism. The age on admission ranged from 6 to 59 years with a mean of 27 years. Syringomyelia was diagnosed by CT myelography and or MRI from 1982 to 1988. The initial symptoms were skeletal abnormality (43%) such as scoliosis (12 cases) or pes cavus (one case), unilateral pain or numbness (40%) and unilateral motor weakness (17%). Frequently seen signs on admission were sensory deficit (100%), scoliosis (57%), muscle weakness (57%), muscle atrophy (37%) and lower cranial nerve palsy (40%). The neurological findings were asymmetrical in all patients. The characteristic neurological findings in the cases presenting under 20 years of age were unilateral sensory and motor deficits (61%) with decreased or absent deep tendon reflex on the same side. The localization of the syrinx in axial section varied according to the level even in the same case. In 15 cases with unilateral sensory disturbance or unilateral sensory and motor deficit, the syrinx was located in the region corresponding to the posterolateral portion on the same side as that of sensory disturbance in the cervical or thoracic level. On the other hand, in 15 cases with bilateral sensory and motor deficit, the syrinx was located in the central portion and extended into the posterolateral portion of the more affected side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuronal control of bird song production

Bird song represents a powerful model system for many of the important problems in behavioral neurobiology, offering both easily measured sensory and motor patterns and a discrete neural effector system. Methods were developed to record the discharge of neurons in singing birds to examine the functions of nuclei in the song control pathway previously implicated anatomically. In several cases, lesions and other techniques were employed to test predictions derived from electrode recordings. Four major findings emerge from these studies. Single-unit recordings from telencephalic nucleus hyperstriatum ventrale, pars caudale (HVc) show several classes of neurons with apparently specialized roles in song production and/or sensorimotor interaction. The nucleus interfacialis (Nlf; Nottebohm, 1980), which provides an input to HVc and is anatomically the "highest" nucleus in the descending motor pathway, is uniquely placed among vocal control nuclei to be a generator of timing cues for song. Consistent with the unidirectional connections between nuclei of the descending pathway, Nlf, HVc, and nucleus robustus archistriatalis (RA) are activated sequentially prior to sound onset. Three other nuclei with connections to or from the descending tract do not show song-related activity in the adult. Bilateral HVc recordings and peripheral disruptions of the vocal apparatus suggest that both hemispheres and syringeal halves normally make similar contributions to most if not all song syllables. The latter finding casts doubt on the analogy between neural lateralization in bird song and in human speech.     

Evolution of vertebrate motor systems for acoustic and electric communication: peripheral and central elements

Among vertebrates, there are a number of different neuromuscular systems specialized for the production of acoustic and electric social communication signals. Each system involves distinct sets of striated muscles that are derived from paraxial mesodermal somites and are components of a peripheral effector or 'communication organ': the larynx and syrinx of tetrapods, the sonic swim bladder and electric organ of fishes. Each of these systems further exhibits species-typical characters ranging from the number of muscles involved to myofibrillar architecture. Given that communication uses striated muscle, its circuitry is seen to represent a modification of existing motor systems. Thus, it is not surprising that these systems share several features suggesting that common mechanical or developmental factors influence their evolution. Of prime importance in comparing central communication circuitry between fishes and tetrapods is that among tetrapods the sound-generating organs are a part of the ventilatory system and so their activity must be coordinated with other sets of neuromuscular units. Differences in the central pattern of circuitry are thus expected between fishes and tetrapods, as well as among tetrapods themselves, since ventilation mechanisms differ between taxa. Moreover, birds have independently evolved a vocal syrinx which is distinctly separate from their larynx.     

Compromised circadian function in Parkinson's disease: enucleation augments disease severity in the unilateral model

Like enucleation, lateral hypothalamic (LH) lesions sever the connection between the retina and the pineal thereby simulating ambient exposure to constant darkness. While LH lesions have been employed to study either circadian function or Parkinson's disease (PD) independently, the application of such lesions to study circadian involvement specifically in this disease has never been attempted. In the present study, unilateral lesions of the LH, which compromise nigro-striatal dopamine (NSD) function, were combined with enucleation ipsilateral or contralateral to the hemisphere where 6-hydroxydopamine was applied. In addition to the observation that hemi-enucleation produced patterns of motor function that were grossly atypical compared to visually intact rats, hemi-enucleation ipsilateral to the side of NSD system denervation produced impairment of horizontal movement, limb retraction, ambulation and spontaneous or l-dopa induced turning. This impairment was more severe than that seen in rats with unilateral 6-OHDA lesions alone. Furthermore, hemi-enucleation contralateral to the side of unilateral NSD system denervation resulted in significantly improved performance on several parameters. While the rate of mortality in rats receiving unilateral 6-OHDA plus ipsilateral enucleation was similar to that occurring after bilateral lesions, it was not accompanied by severe weight loss and wasting that typically occurs in the acute stages of experimental PD. These results demonstrate the importance of the visual and circadian systems in PD and are consistent with reports that identify impaired circadian involvement as a major component in a wide range of neurological and neuropsychiatric conditions.     

Midbrain regions involved in call production in Java sparrows

Calling has been evoked from the midbrain of Java sparrows by currents of less than 200 μA. Minimum threshold were found in the medial nucleus intercollicularis, higher threshold sites surrounded these. Additionally, more ventral sites were found. Calling evoked did not resemble natural calls, but was similar to that produced after bilateral denervation of the syrinx. These results indicate that the nucleus intercollicularis acts as a controller rather than an organizer of calling.     

Neuromagnetic fields accompanying unilateral and bilateral voluntary movements: topography and analysis of cortical sources.

Movement-related magnetic fields (MRMFs) accompanying left and right unilateral and bilateral finger flexions were studied in 6 right-handed subjects. Six different MRMF components occurring prior to, and during both unilateral and bilateral movements are described: a slow pre-movement readiness field (RF, 1-0.5 sec prior to movement onset); a motor field (MF) starting shortly before EMG onset; 3 separate "movement-evoked" fields following EMG onset (MEFI at 100 msec; MEFII at 225 msec; and MEFIII at 320 msec); and a "post-movement" field (PMF) following the movement itself. The bilateral topography of the RF and MF for both unilateral and bilateral movements suggested bilateral generators for both conditions. Least-squares fitting of equivalent current dipole sources also indicated bilateral sources for MF prior to both unilateral and bilateral movements with significantly greater strength of contralateral sources in the case of unilateral movements. Differences in pre-movement field patterns for left versus right unilateral movements indicated possible cerebral dominance effects as well. A single current dipole in the contralateral sensorimotor cortex could account for the MEFI for unilateral movements and bilateral sensorimotor sources for bilateral movements. Other MRMF components following EMG onset indicated similar sources in sensorimotor cortex related to sensory feedback or internal monitoring of the movement. The results are discussed with respect to the possible generators active in sensorimotor cortex during unilateral and bilateral movement preparation and execution and their significance for the study of cortical organization of voluntary movement.     

Patterns of palmar skin temperature alterations during transthoracic endoscopic T2 sympathectomy for palmar hyperhidrosis

Transthoracic endoscopic T2 sympathectomy has been widely applied to the treatment of a variety of sympathetically mediated disorders. Palmar hyperhidrosis is probably the most common indication for thoracic sympathectomy, especially in certain subtropical areas. Which sympathetic ganglion is to be ablated and how extensive such ablation is enough to eliminate palm sweating are two important issues. Intraoperative monitoring of palmar skin temperature (PST) is the most frequently used method for assessing the accuracy as well as adequacy of ablation of the target sympathetic ganglia. With continuous monitoring of bilateral PST during the operative course of T2 sympathectomy, it was possible to depict the alterations of bilateral PST in response to specific surgical procedures in a real-time manner. For each case, a PST graph was obtained, which represented the graphical expression of intraoperatively recorded bilateral PST data plotted against time. The PST graphs of 93 consecutive cases were analysed. Three types of PST graphs existed, reflecting different responses of bilateral PST to different surgical procedures during the operation. In Type I PST graph pattern, found in 58 cases, skin incision and intercostal muscle dissection caused dramatic bilateral PST drop; and unilateral T2 sympathectomy induced synchronous bilateral PST elevation. Twenty-four cases demonstrated Type II PST graph pattern, in which unilateral T2 sympathectomy caused only ipsilateral PST elevation, although the PST-depressing effect of skin incision and muscle dissection was as significant as in Type I graph pattern. In the 11 cases who showed Type III PST graph pattern, neither skin incision nor T2 sympathectomy induced any apparent changes of PST on either side, giving rise to two rather flat PST curves on the PST graphs. These findings implicate that reciprocal interactions between bilateral sympathetic activities exist in the majority of cases, and that crossover sympathetic modulation may play a role in the neural control of the sudomotor and vasomotor activities of the palms. This study also provides information regarding how PST would possibly change following specific surgical procedures during transthoracic endoscopic T2 sympathectomy, which may be of importance to those who use intraoperative PST monitoring as a guide in determining whether or not the correct sympathetic ganglia are ablated for adequate sympathetic denervation of the palms.     

Bilateral organization of the vocal control pathway in the budgerigar, Melopsittacus undulatus

Anatomical and electrophysiological methods were used to map the vocal control nuclei of the budgerigar, Melopsittacus undulatus. Beginning with the motor nucleus of the syrinx, nuclei were located using antidromic stimulation and then injected with horseradish peroxidase (HRP). Retrogradely transported HRP labeled afferents to the injected nucleus. This procedure was repeated at successively higher levels along the vocal pathway. Connections found using this strategy then were confirmed using anterograde transport of HRP and/or tritiated proline and orthodromic electrical stimulation. We found that the primary vocal control pathway consisted of (1) the motor nucleus innervating the trachea and syrinx, nXIIts; (2) an archistriatal nucleus, RA; and (3) a neostriatal nucleus, "HVc." These nuclei correspond to similar, possibly homologous, nuclei in the vocal control pathway of the canary (Nottebohm, F., T. M. Stokes, and C. M. Leonard (1976) J. Comp. Neurol. 165: 457-486) but, because of differences in gross brain morphology, are displaced considerably in absolute position. Furthermore, the projection from RA to the motor nucleus is bilateral in the budgerigar, whereas the same connection is strictly ipsilateral in the canary. The projection of the motor nucleus to muscles of the vocal organ is also bilateral in the budgerigar (Manogue, K. R., and F. Nottebohm (1981) J. Compl. Neurol., in press) but ipsilateral in the canary. the possible significance of these species differences for lateralization of motor control is discussed.     

Nitric oxide synthase/nicotinamide adenine dinucleotide phosphate-diaphorase in the brainstem trigeminal nuclei after transection of the masseteric nerve in rats.

In this study, the responses of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal nitric oxide synthase (nNOS) activities were quantitatively analyzed at different times in both ipsilateral and contralateral sides of trigeminal nuclei, after unilateral trigeminal muscle nerve transection, in Sprague Dawley rats. In the control animals, both NADPH-d- and nNOS-positive neurons were constitutively distributed in the rostrolateral solitary tract nucleus, dorsomedial part of trigeminal nucleus oralis (Vo/Sn), and superficial layers (VcI/II) of the trigeminal nucleus caudalis (Vc). NADPH-d-positive neurons appeared in the trigeminal mesencephalic nucleus ipsilaterally at 5 days (mean +/- SEM: 30.5 +/- 5.6) and were maintained until 8 weeks (33 +/- 10.6) after the denervation. In the trigeminal motor nucleus, NADPH-d-positive neurons appeared transiently and bilaterally, peaking at 1 week (663.5 +/- 156.2, ipsilateral side; 687.5 +/- 118.6, contralateral side) after unilateral denervation of the masseteric nerve. In both Vo/Sn and Vc, the number of NADPH-d-positive neurons in the control animals showed a decrease at 3 days but significantly increased from 5 days to 1 week and gradually fell to the control values by 8 weeks after the denervation. There were no significant differences observed between the two sides in either Vo/Sn or Vc. nNOS-positive neurons were similarly distributed and the numbers of labeled neurons were similar to those of NADPH-d-positive neurons after the denervation, although the changes were delayed by approximately 1 week. In conclusion, after unilateral nerve transection, the peak NADPH-d activity occurs 1 week prior to nNOS activity.