Ultrastructure of synaptic input to medial olivocochlear neurons

Medial olivocochlear (MOC) neurons project from the brain to the cochlea to form the efferent limb of the MOC reflex. To study synaptic inputs to MOC neurons, we retrogradely labeled these neurons using horseradish peroxidase injections into the cochlea. Labeled neurons were identified in the ventral nucleus of the trapezoid body and documented with the light microscope before being studied with serial-section electron microscopy. MOC somata and dendrites were innervated by three different types of synapses, distinguished as either having: 1) large, round synaptic vesicles and forming asymmetric contacts; 2) small, round vesicles plus a few dense core vesicles and forming asymmetric contacts; or 3) pleomorphic vesicles and forming symmetric contacts. The first two types were the most frequent on somata. Acetylcholinesterase-stained material confirmed that the type containing large, round vesicles is most common on dendrites. We kept track of the synaptic terminals in serial sections and compiled them into three-dimensional swellings. Swellings with large, round vesicles formed up to seven synapses per swelling, were largest in size, and sometimes formed complex arrangements engulfing spines of MOC neurons. Swellings with small, round vesicles formed up to four synapses per swelling. The morphology of this type of synapse, and the moderate sizes of the swellings forming it, suggests that it originates from posteroventral cochlear nucleus stellate/multipolar neurons. This input may thus provide the sound-evoked input to MOC neurons that causes their reflexive response to sound.     

Urocortin-expressing olivocochlear neurons exhibit tonotopic and developmental changes in the auditory brainstem and in the innervation of the cochlea

The mammalian cochlea is under direct control of two groups of cholinergic auditory brainstem neurons, the medial and the lateral olivocochlear neurons. The former modulate the electromechanical amplification in outer hair cells and the latter the transduction of inner hair cells to auditory nerve fibers. The lateral olivocochlear neurons express not only acetylcholine but a variety of co-transmitters including urocortin, which is known to regulate homeostatic responses related to stress; it may also be related to the ontogeny of hearing as well as the generation of hearing disorders. In the present study, we investigated the distribution of urocortin-expressing lateral olivocochlear neurons and their connectivity and distribution of synaptic terminals in the cochlea of juvenile and adult gerbils. In contrast to most other rodents, the gerbil's audiogram covers low frequencies similar to humans, although their communication calls are exclusively in the high-frequency domain. We confirm that in the auditory brainstem urocortin is expressed exclusively in neurons within the lateral superior olive and their synaptic terminals in the cochlea. Moreover, we show that in adult gerbils urocortin expression is restricted to the medial, high-frequency processing, limb of the lateral superior olive and to the mid and basal parts of the cochlea. The same pattern is present in juvenile gerbils shortly before hearing onset (P 9) but transiently disappears after hearing onset, when urocortin is also expressed in low-frequency processing regions. These results suggest a possible role of urocortin in late cochlear development and in the processing of social calls in adult animals.     

Neuron-specific enolase-like immunoreactivity in inner hair cells but not outer hair cells in the guinea pig organ of Corti

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells. This is the first report of NSE-like immunoreactivity in a receptor cell. NSE-like immunoreactivity was also seen in efferent fibers and terminals and in both type I and type II spiral ganglion cells. The finding of NSE-like immunoreactivity in inner but not outer cells adds to the number of differences found between them and may be related to differences in function and action.     

Cholinergic and GABAergic neurons in the rat medial septum express muscarinic acetylcholine receptors

This study describes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) in the medial septum (MS), employing the monoclonal antibody M35 raised against purified mAChR-protein. mAChR-positive neurons are found throughout the MS, but are predominantly located in the midline area and in the lateral compartments. The labeled cell bodies are variable in shape and size (largest diameter ranging from 10–30 μm), while both soma and the associated dendritic processes are densely stained for mAChRs. Astrocytes immunoreactive for mAChRs were frequently found associated with the large blood vessels in the midline area. To study the neurotransmitter nature of the mAChR-positive cells, immunofluorescence double-labeling experiments were performed for mAChRs and GABAergic and cholinergic markers. GABAergic cells were identified immunocytochemically using antisera against glutamic acid decar☐ylase (GAD), parvalbumin (PARV) or calbindin protein (CaBP). The cholinergic transmitter nature of the mAChR-positive cells was studied using adjacent 8 μm thick serial sections stained immunocytochemically for choline acetyltransferase (ChAT), or histochemically for acetylcholinesterase (AChE). These experiments showed that approximately half (52.3%) of all mAChR-positive cells contain GAD, whereas the other half is cholinergic. Conversely, nearly all GABAergic (98.6%) and cholinergic (96.9%) cells are endowed with mACRs. GAD-positive terminals were found surrounding mAChR-positive perikarya which were either GAD-positive or GAD-negative, indicating GABAergic innervation on both GABAergic and cholinergic MS neurons. In general, the staining intensity for mAChRs appeared to be considerably higher in GABAergic than in cholinergic neurons, suggesting a stronger cholinergic impact upon the GABAergic neurons. The current anatomical findings contribute to the concept that the MS neurons form a firmly interconnected cell group, in which cholinergic neurotransmission mediated through mAChRs seems to play a significantly role.     

Fiber pathways and branching patterns of biocytin-labeled olivocochlear neurons in the mouse brainstem

Olivocochlear neurons have somata in the superior olivary complex in the brainstem and project fibers to the cochlea. The purpose of the present study was to demonstrate the fiber pathways and branching patterns of olivocochlear fibers within the brainstem. Olivocochlear fibers were labeled by extracellular injections of biocytin into the cochlea of mice. The injections labeled two populations of olivocochlear fibers. Thin olivocochlear fibers arose from small somata of the lateral olivocochlear group located ipsilaterally in the lateral superior olive. Thick olivocochlear fibers arose from larger somata of the medial olivocochlear group located bilaterally in the periolivary nuclei. The lateral olivocochlear and medial olivocochlear fibers had similar courses but differed in their branching patterns. Branches from lateral olivocochlear fibers terminated near their somata of origin in the lateral superior olive or in the lateral vestibular nucleus. Branches from medial olivocochlear fibers terminated in the inferior vestibular nucleus or in the cochlear nuclear complex. A few branches from medial olivocochlear fibers projected to the contralateral side. Although they project primarily to the cochlea, olivocochlear neurons also give off branches to a variety of nuclei in the brainstem, thus involving auditory and non-auditory nuclei in the olivocochlear reflex system. © 1993 Wiley-Liss,Inc.     

Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynaptically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.     

Bergmann glial cells form distinct morphological structures to interact with cerebellar neurons

It is well established that Bergmann glial cells closely interact with neuronal elements in the molecular layer of the cerebellum. We reconstructed dye-labeled Bergmann glial cells from electron microscopic serial sections and identified their contact sites with neurons as "glial microdomains" (Grosche et al. [1999] Nature Neurosci. 2:139-143). In the present paper we describe these structures in more detail, and show that 1) immature Bergmann fibers up to postnatal day 7 are smooth and lack appendages but contain several large mitochondria at sites where the first indications of growing side branches are observed; 2) Bergmann fibers from cerebella at postnatal day 30 form two types of outgrowths, short simple thorns and longer complex appendages; 3) each of the latter (i.e., a glial microdomain) is in contact with only a few synapses and nonsynaptic neuronal excrescences; 4) every given region of the neuropil is occupied by (at least) two interdigitating glial microdomains; 5) the synaptic clefts are entirely surrounded by glial protrusions, whereas the extrasynaptic surfaces and small axons are only partially covered; and 6) many small neuronal excrescenses without vesicles are completely ensheathed by glial caps, representing novel glial-neuronal structures of unknown function (glial thimbles). Computational modelling of the microdomains indicates that each is electrotonically independent of the stem process from which it arises, as well as of neighbouring domains. We assume that the glial microdomain is a morphological unit to compartmentalize ensembles of synapses, serving to synchronize local synaptic activity.     

Synapses formed by olivocochlear axon branches in the mouse cochlear nucleus

Cochlear nucleus branches of thick olivocochlear axons were labeled by injections of horseradish peroxidase into the spiral ganglion of the cochlear basal turn in mice. Six labeled axons were traced by light microscopy, and selected portions of seven branches were sectioned serially for electron microscopic examination. Axonal branches most frequently terminated near certain granule cell regions of the ventral cochlear nucleus. This article describes terminals, synapses, and postsynaptic elements of these olivocochlear branches. The olivocochlear branches had both terminal and en passant boutons that contained round vesicles and made asymmetric synapses with other neuronal processes. About a quarter of the synapses also possessed additional specializations, postsynaptic, or subjunctional bodies. Mossy terminals, a multisynaptic type of terminal commonly found in granule cell regions, were not found arising from any of the labeled branches. No somatic synapses were found, although contacts with cell bodies were occasionally observed. The predominant synaptic target of olivocochlear branches were what appeared to be dendrites of large diameter. At least some of these large dendrites received multiple synapses from a single labeled olivocochlear branch. The morphological characteristics of reconstructed dendrites suggest that multipolar cells might be predominant targets for the medial olivocochlear system in the cochlear nucleus. This was demonstrated in one case in which a large dendrite was followed to its cell body of origin.     

Neurochemically distinct classes of myenteric neurons express the mu-opioid receptor in the guinea pig ileum

The mu-opioid receptor (muOR), which mediates many of the opioid effects in the nervous system, is expressed by enteric neurons. The aims of this study were to determine whether 1) different classes of myenteric neurons in the guinea pig ileum contain muOR immunoreactivity by using double- and triple-labeling immunofluorescence and confocal microscopy, 2) muOR immunoreactivity is localized to enteric neurons immunoreactive for the endogenous opioid enkephalin, and 3) muOR immunoreactivity is localized to interstitial cells of Cajal visualized by c-kit. In the myenteric plexus, 50% of muOR-immunoreactive neurons contained choline acetyltransferase (ChAT) immunoreactivity, whereas about 43% of ChAT-immunoreactive neurons were muOR immunoreactive. Approximately 46% of muOR myenteric neurons were immunoreactive for vasoactive intestinal polypeptide (VIP), and about 31% were immunoreactive for nitric oxide synthase (NOS). MuOR immunoreactivity was found in about 68% of VIP-containing neurons and 60% of NOS-immunoreactive neurons. Triple labeling showed that about 32% of muOR neurons contained VIP and ChAT immunoreactivities. The endogenous opioid enkephalin (ENK) was observed in about 30% of muOR neurons; conversely, 48% of ENK neurons contained muOR immunoreactivity. MuOR was not detected in neurons containing calbindin, nor in interstitial cells of Cajal. MuOR-immunoreactive fibers formed a dense network around interstitial cells of Cajal in the deep muscular plexus. This study demonstrates that muOR is expressed by neurochemically distinct classes of myenteric neurons that are likely to differ functionally, is colocalized with the endogenous opioid ENK, and is not expressed by interstitial cells of Cajal.     

Generation patterns of immunocytochemically identified cholinergic neurons at autonomic levels of the rat spinal cord.

The time at which a neuron is "born" appears to have significant consequences for the cell's subsequent differentiation. As part of a continuing investigation of cholinergic neuronal development, we have combined ChAT immunocytochemistry and [3H]thymidine autoradiography to determine the generation patterns of somatic and autonomic motor neurons at upper thoracic (T1-3), upper lumbar (L1-3), and lumbosacral (L6-S1) levels of the rat spinal cord. Additionally, the generation patterns of two subsets of cholinergic interneurons (partition cells and central canal cluster cells) were compared with those of somatic and autonomic motor neurons. Embryonic day 11 (E11) was the first day of cholinergic neuronal generation at each of the three spinal levels studied, and it also was the peak generation day for somatic and autonomic neurons in the upper thoracic spinal cord. The peak generation of homologous neurons at upper lumbar and lumbosacral spinal levels occurred at E12 and E13, respectively. Somatic and autonomic motor neurons were generated synchronously, and their production at each rostrocaudal level was virtually completed within a 2-day period. Cholinergic interneurons were generated 1 or 2 days later than motor neurons at the same rostrocaudal level. In summary, the birthdays of all spinal cholinergic neurons studied followed the general rostrocaudal spatiotemporal gradient of spinal neurogenesis. In addition, the generation of cholinergic interneurons also followed the general ventrodorsal gradient. In contrast, however, autonomic motor neurons disobeyed the rule of a ventral-to-dorsal progression of spinal neuronal generation, thus adding another example in which autonomic motor neurons display unusual developmental patterns.     

Calpain activity in the amikacin-damaged rat cochlea

The principal aim of this study was to investigate the involvement of calpain in the degeneration of hair cells and ganglion neurons in the amikacin-poisoned rat cochlea. An antibody designed against fodrin-breakdown products (FBDP), which result exclusively from cleavage by calpain, was used. In addition, the involvement of both caspases and protein kinase C (PKC) was studied using, respectively, antibodies against activated caspase 3 and PKCgamma. The results demonstrate the accumulation of FBDP in the degenerating hair cells, in some supporting cells such as Deiters cells, and, later, in the affected ganglion neurons that had been deprived of their sensory targets. Activated caspase 3 was evidenced in a few dying hair cells and ganglion neurons. PKCgamma was highly expressed in all ganglion neurons, sometimes after the loss of hair cells. We conclude that calpain plays a role in the degradation of both the sensory cells and neurons after amikacin ototoxicity. In the poisoned hair cells, calpain and caspase 3 may have synergistic effects in the process of apoptosis. In the ganglion neurons deprived of their sensory elements, calpain may have a prominent role in cell degradation. By contrast, in these ganglion neurons PKCgamma may be implicated in a survival process. Finally, we suggest that calpain is involved in the remodeling of Deiters cells during the scarring process that follows hair cell loss.     

Cochlear-nucleus branches of thick (medial) olivocochlear fibers in the mouse: a cochleotopic projection

Olivocochlear neurons have somata in the superior olivary complex and provide an efferent innervation to the cochlea. One subgroup of olivocochlear neurons, medial olivocochlear neurons, sends fibers to innervate the cochlear outer hair cells. En route to the cochlea, medial olivocochlear fibers give off branches to the ventral cochlear nucleus, the first auditory center of the brain. This study examines the cochlear-nucleus branches of medial olivocochlear fibers, comparing those from fibers that innervate the cochlear base with those from fibers that innervate the cochlear apex. Basal fibers give off dorsal branches to the granule cell lamina and ventral branches to the auditory nerve root. Apical fibers give off few dorsal branches but many ventral branches that terminate rostrally to the nerve root. This cochleotopic mapping of medial olivocochlear branches corresponds in a general way to that of afferent fibers. Unlike afferent fibers, however, the branches terminate primarily along the edges of the cochlear nucleus. In the mouse, the particular edges of termination are (1) the medial border of the ventral cochlear nucleus where it meets the underlying vestibular nerve root, and (2) the border between the ventral cochlear nucleus and the granule cell lamina. Neurons and dendrites of these border regions may thus integrate efferent and afferent information in a frequency-specific manner.     

GABAergic neurons in the mammalian inferior olive and ventral medulla detected by glutamate decarboxylase immunocytochemistry.

Neurons containing glutamic acid decarboxylase (GAD) (presumed GABAergic neurons) were mapped by immunocytochemistry in the ventral medulla of rat, rabbit, cat, rhesus monkey, and human, with emphasis on the inferior olive. In all species, three categories of GABAergic neurons were identified: periolivary neurons in the gray matter and the white matter surrounding the inferior olive, internuclear neurons located in the white matter between the subnuclei of the inferior olive, and intranuclear neurons located within the olivary gray matter. The intranuclear GABAergic neurons of the inferior olive had a characteristic morphology which differed from non-GABAergic olivary neurons; they were usually smaller, and, wherever their processes were stained, they had radiating, sparsely branching dendrites. They were also usually distinguished from the other GABAergic neurons by their smaller size. The intraolivary GABAergic neurons constituted only a minor proportion of the total olivary neuronal population, but they were concentrated in regions of the olive that varied by species. In the rat, they were situated in the rostral tip of the medial accessory olive and in the caudal subdivision of the dorsal accessory olive, while in the rabbit, they were located in the caudal two-thirds of the medial accessory olive, in the dorsal cap, and in the ventral lateral outgrowth. Such neurons were extremely rare in the cat; only a few were found in the rostral parts of the principal olive, the medial accessory olive, and the dorsal accessory olive. In the rhesus monkey, the principal olive and the lateral region of the rostral medial accessory olive contained most of the intranuclear GABAergic neurons, but some were also present in the dorsal accessory olive. In the human, such neurons occurred in the principal olive, the dorsal accessory olive and the rostral medial accessory olive, but as in the rhesus monkey, most were observed in the principal olive.     

Medial olivocochlear reflex interneurons are located in the posteroventral cochlear nucleus: a kainic acid lesion study in guinea pigs

The medial olivocochlear (MOC) reflex arc is probably a three-neuron pathway consisting of type I spiral ganglion neurons, reflex interneurons in the cochlear nucleus, and MOC neurons that project to the outer hair cells of the cochlea. We investigated the identity of MOC reflex interneurons in the cochlear nucleus by assaying their regional distribution using focal injections of kainic acid. Our reflex metric was the amount of change in the distortion product otoacoustic emission (at 2f(1)-f(2)) just after onset of the primary tones. This metric for MOC reflex strength has been shown to depend on an intact reflex pathway. Lesions involving the posteroventral cochlear nucleus (PVCN), but not the other subdivisions, produced long-term decreases in MOC reflex strength. The degree of cell loss within the dorsal part of the PVCN was a predictor of whether the lesion affected MOC reflex strength. We suggest that multipolar cells within the PVCN have the distribution and response characteristics appropriate to be the MOC reflex interneurons.     

Distribution and neurochemical characterization of neurons expressing GIRK channels in the rat brain

G-protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G-protein-coupled receptors such as the serotonin 5-HT(1A) receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5-HT(1A) receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co-expression of GIRK subunits with the 5-HT(1A) receptor and cell markers of glutamatergic, gamma-aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double-label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double-labeling experiments show that GIRK subunits are present in most of the 5-HT(1A) receptor-expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases.     

Outer Hair Cell Glutamate Signaling through Type II Spiral Ganglion Afferents Activates Neurons in the Cochlear Nucleus in Response to Nondamaging Sounds

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels.SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels.     

The dual origins of the olivocochlear bundle in the albino rat

Recent studies of the origins and terminations of the olivocochlear bundle (OCB) in the cat provide evidence that separate efferent systems differentially innervate the two types of hair cells in the organ of Corti. To begin to test the generality of these separate olivocochlear systems, the cells of origin of the OCB were determined in the albino rat by using axonal transport of horseradish peroxidase. Our findings are that, as in the cat, two classes of olivocochlear (OC) neurons project to the cochlea. These neurons could be dichotomized according to their location in the superior olivary complex (lateral or medial), their size (small or large), and their preferred side of projection (ipsilateral or contralateral). All labeled OC neurons also exhibited a positive reaction for acetylcholinesterase. In the rat, however, lateral and medial OC neurons are each restricted to a single nucleus, and, furthermore, the lateral OC neurons project only ipsilaterally. The rat also has significantly fewer mean totals of both lateral (240 vs. 710) and medial (240 vs. 520) OC neurons than the cat. The present methods also demonstrated the course of axons of the OCB and axon collaterals entering the cochlear nuclear complex. Vestibular efferent neurons were also labeled bilaterally medial and lateral to the facial genu. Our results suggest that the general organizational plan of OC neurons in the rat may offer advantages over the situation in the cat for studies of connectional neuroanatomy, neurophysiology, and behavioral functions of the two olivocochlear systems.     

Differential regulation of the expression of neurotrophin receptors in rat extraocular motoneurons after lesion

Neurotrophins acting through high-affinity tyrosine kinase receptors (trkA, trkB, and trkC) play a crucial role in regulating survival and maintenance of specific neuronal functions after injury. Adult motoneurons supplying extraocular muscles survive after disconnection from the target, but suffer dramatic changes in morphological and physiological properties, due in part to the loss of their trophic support from the muscle. To investigate the dependence of the adult rat extraocular motoneurons on neurotrophins, we examined trkA, trkB, and trkC mRNA expression after axotomy by in situ hybridization. trkA mRNA expression was detectable at low levels in unlesioned motoneurons, and its expression was downregulated 1 and 3 days after injury. Expression of trkB and trkC mRNAs was stronger, and after axotomy a simultaneous, but inverse regulation of both receptors was observed. Thus, whereas a considerable increase in trkB expression was seen about 2 weeks after axotomy, the expression of trkC mRNA had decreased at the same post-lesion period. Injured extraocular motoneurons also experienced an initial induction in expression of calcitonin gene-related peptide and a transient downregulation of cholinergic characteristics, indicating a switch in the phenotype from a transmitter-specific to a regenerative state. These results suggest that specific neurotrophins may contribute differentially to the survival and regenerative responses of extraocular motoneurons after lesion.