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The trochlear projection is unique among the cranial nerves in that it exits the midbrain dorsally to innervate the contralateral superior oblique muscle in all vertebrates. Trochlear as well as oculomotor motoneurons uniquely depend upon Phox2a and Wnt1, both of which are downstream of Lmx1b, though why trochlear motoneurons display such unusual projections is not fully known. We used Pax2-cre to drive expression of ectopically activated Smoothened (SmoM2) dorsally in the midbrain and anterior hindbrain. We documented the expansion of oculomotor and trochlear motoneurons using Phox2a as a specific marker at E9.5. We show that the initial expansion follows a demise of these neurons by E14.5. Furthermore, SmoM2 expression leads to a ventral exit and ipsilateral projection of trochlear motoneurons. We compare that data with Unc5c mutants that shows a variable ipsilateral number of trochlear fibers that exit dorsal. Our data suggest that Shh signaling is involved in trochlear motoneuron projections and that the deflected trochlear projections after SmoM2 expression is likely due to the dorsal expression of Gli1, which impedes the normal dorsal trajectory of these neurons.

The LIM homeodomain transcription factor Lmx1a shows a dynamic expression in the developing mouse ear that stabilizes in the non-sensory epithelium. Previous work showed that Lmx1a functional null mutants have an additional sensory hair cell patch in the posterior wall of a cochlear duct and have a mix of vestibular and cochlear hair cells in the basal cochlear sensory epithelium. In E13.5 mutants, Sox2-expressing posterior canal crista is continuous with an ectopic "crista sensory epithelium" located in the outer spiral sulcus of the basal cochlear duct. The medial margin of cochlear crista is in contact with the adjacent Sox2-expressing basal cochlear sensory epithelium. By E17.5, this contact has been interrupted by the formation of an intervening non-sensory epithelium, and Atoh1 is expressed in the hair cells of both the cochlear crista and the basal cochlear sensory epithelium. Where cochlear crista was formerly associated with the basal cochlear sensory epithelium, the basal cochlear sensory epithelium lacks an outer hair cell band, and gaps are present in its associated Bmp4 expression. Further apically, where cochlear crista was never present, the cochlear sensory epithelium forms a poorly ordered but complete organ of Corti. We propose that the core prosensory posterior crista is enlarged in the mutant when the absence of Lmx1a expression allows JAG1-NOTCH signaling to propagate into the adjacent epithelium and down the posterior wall of the cochlear duct. We suggest that the cochlear crista propagates in the mutant outer spiral sulcus because it expresses Lmo4 in the absence of Lmx1a.

Npr2 (natriuretic peptide receptor 2) affects bifurcation of neural crest or placode-derived afferents upon entering the brain stem/spinal cord, leading to a lack of either rostral or caudal branches. Previous work has shown that early embryonic growth of cochlear and vestibular afferents is equally affected in this mutant but later work on postnatal Npr2 point mutations suggested some additional effects on the topology of afferent projections and mild functional defects. Using multicolor lipophilic dye tracing, we show that absence of Npr2 has little to no effect on the initial patterning of inner ear afferents with respect to their dorsoventral cochleotopic-specific projections. However, in contrast to control animals, we found a variable degree of embryonic extension of auditory afferents beyond the boundaries of the anterior cochlear nucleus into the cerebellum that emanates only from apical spiral ganglion neurons. Such expansion has previously only been reported for Hox gene mutants and implies an unclear interaction of Hox codes with Npr2-mediated afferent projection patterning to define boundaries. Some vestibular ganglion neurons expand their projections to reach the cochlear apex and the cochlear nuclei, comparable to previous findings in Neurod1 mutant mice. Before birth, such expansions are reduced or lost leading to truncated projections to the anteroventral cochlear nucleus and expansion of low-frequency fibers of the apex to the posteroventral cochlear nucleus.

RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels.

Several concepts developed in the nineteenth century have formed the basis of much of our neuroanatomical teaching today. Not all of these were based on solid evidence nor have withstood the test of time. Recent evidence on the evolution and development of the autonomic nervous system, combined with molecular insights into the development and diversification of motor neurons, challenges some of the ideas held for over 100 years about the organization of autonomic motor outflow. This review provides an overview of the original ideas and quality of supporting data and contrasts this with a more accurate and in depth insight provided by studies using modern techniques. Several lines of data demonstrate that branchial motor neurons are a distinct motor neuron population within the vertebrate brainstem, from which parasympathetic visceral motor neurons of the brainstem evolved. The lack of an autonomic nervous system in jawless vertebrates implies that spinal visceral motor neurons evolved out of spinal somatic motor neurons. Consistent with the evolutionary origin of brainstem parasympathetic motor neurons out of branchial motor neurons and spinal sympathetic motor neurons out of spinal motor neurons is the recent revision of the organization of the autonomic nervous system into a cranial parasympathetic and a spinal sympathetic division (e.g., there is no sacral parasympathetic division). We propose a new nomenclature that takes all of these new insights into account and avoids the conceptual misunderstandings and incorrect interpretation of limited and technically inferior data inherent in the old nomenclature.

The mammalian inner ear develops from a placodal thickening into a complex labyrinth of ducts with five sensory organs specialized to detect position and movement in space. The mammalian ear also develops a spiraled cochlear duct containing the auditory organ, the organ of Corti (OC), specialized to translate sound into hearing. Development of the OC from a uniform sheet of ectoderm requires unparalleled precision in the topological developmental engineering of four different general cell types, namely sensory neurons, hair cells, supporting cells, and general otic epithelium, into a mosaic of ten distinctly recognizable cell types in and around the OC, each with a unique distribution. Moreover, the OC receives unique innervation by ear-derived spiral ganglion afferents and brainstem-derived motor neurons as efferents and requires neural-crest-derived Schwann cells to form myelin and neural-crest-derived cells to induce the stria vascularis. This transformation of a sheet of cells into a complicated interdigitating set of cells necessitates the orchestrated expression of multiple transcription factors that enable the cellular transformation from ectoderm into neurosensory cells forming the spiral ganglion neurons (SGNs), while simultaneously transforming the flat epithelium into a tube, the cochlear duct, housing the OC. In addition to the cellular and conformational changes forming the cochlear duct with the OC, changes in the surrounding periotic mesenchyme form passageways for sound to stimulate the OC. We review molecular developmental data, generated predominantly in mice, in order to integrate the well-described expression changes of transcription factors and their actions, as revealed in mutants, in the formation of SGNs and OC in the correct position and orientation with suitable innervation. Understanding the molecular basis of these developmental changes leading to the formation of the mammalian OC and highlighting the gaps in our knowledge might guide in vivo attempts to regenerate this most complicated cellular mosaic of the mammalian body for the reconstitution of hearing in a rapidly growing population of aging people suffering from hearing loss.

Understanding the evolution of the neurosensory system of man, able to reflect on its own origin, is one of the major goals of comparative neurobiology. Details of the origin of neurosensory cells, their aggregation into central nervous systems and associated sensory organs and their localized patterning leading to remarkably different cell types aggregated into variably sized parts of the central nervous system have begun to emerge. Insights at the cellular and molecular level have begun to shed some light on the evolution of neurosensory cells, partially covered in this review. Molecular evidence suggests that high mobility group (HMG) proteins of pre-metazoans evolved into the definitive Sox [SRY (sex determining region Y)-box] genes used for neurosensory precursor specification in metazoans. Likewise, pre-metazoan basic helix-loop-helix (bHLH) genes evolved in metazoans into the group A bHLH genes dedicated to neurosensory differentiation in bilaterians. Available evidence suggests that the Sox and bHLH genes evolved a cross-regulatory network able to synchronize expansion of precursor populations and their subsequent differentiation into novel parts of the brain or sensory organs. Molecular evidence suggests metazoans evolved patterning gene networks early, which were not dedicated to neuronal development. Only later in evolution were these patterning gene networks tied into the increasing complexity of diffusible factors, many of which were already present in pre-metazoans, to drive local patterning events. It appears that the evolving molecular basis of neurosensory cell development may have led, in interaction with differentially expressed patterning genes, to local network modifications guiding unique specializations of neurosensory cells into sensory organs and various areas of the central nervous system.

Facial branchiomotor neurons (FBMs) of vertebrates typically develop in rhombomere 4 (r4), and in mammals and several other vertebrate taxa, migrate caudally into r6 and subsequently laterally and ventrally to the pial surface. How similar or dissimilar these migratory processes between species are at a molecular level remains unclear. In zebrafish and mouse, mutations in certain PCP genes disrupt normal caudal migration of FBMs. Zebrafish prickle1a (prickle-like 1a) and prickle1b, two orthologs of Prickle1, act non-cell-autonomously and cell-autonomously, respectively, to regulate FBM migration. Here, we show that, in Prickle1 (C251X/C251X) mice which have reduced Prickle1 expression, the caudal migration of FBMs is affected. Most FBM neurons do not migrate caudally along the floor plate. However, some neurons perform limited caudal migration such that the neurons eventually lie near the pial surface from r4 to anterior r6. FBMs in Prickle1 (C251X/C251X) mice survive until P0 and form an ectopic nucleus dorsal to the olivo-cochlear efferents of r4. Ror2, which modifies the PCP pathway in other systems, is expressed by the migrating mouse FBMs, but is not required for FBM caudal migration. Our results suggest that, in mice, Prickle1 is part of a molecular mechanism that regulates FBM caudal migration and separates the FBM and the olivo-cochlear efferents. This defective caudal migration of FBMs in Prickle1C251X mutants resembles Vangl2 mutant defects. In contrast to other developing systems that show similar defects in Prickle1, Wnt5a and Ror2, the latter two only have limited or no effect on FBM caudal migration.

Reconstructing a functional organ of Corti is the ultimate target towards curing hearing loss. Despite the impressive technical gains made over the last few years, many complications remain ahead for the two main restoration avenues: in vitro transformation of pluripotent cells into hair cell-like cells and adenovirus-mediated gene therapy. Most notably, both approaches require a more complete understanding of the molecular networks that ensure specific cell types form in the correct places to allow proper function of the restored organ of Corti. Important to this understanding are the basic helix-loop-helix (bHLH) transcription factors (TFs) that are highly diverse and serve to increase functional complexity but their evolutionary implementation in the inner ear neurosensory development is less conspicuous. To this end, we review the evolutionary and developmentally dynamic interactions of the three bHLH TFs that have been identified as the main players in neurosensory evolution and development, Neurog1, Neurod1 and Atoh1. These three TFs belong to the neurogenin/atonal family and evolved from a molecular precursor that likely regulated single sensory cell development in the ectoderm of metazoan ancestors but are now also expressed in other parts of the body, including the brain. They interact extensively via intracellular and intercellular cross-regulation to establish the two main neurosensory cell types of the ear, the hair cells and sensory neurons. Furthermore, the level and duration of their expression affect the specification of hair cell subtypes (inner hair cells vs. outer hair cells). We propose that appropriate manipulation of these TFs through their characterized binding sites may offer a solution by itself, or in conjunction with the two other approaches currently pursued by others, to restore the organ of Corti.

The developing sensory neurons of the mammalian ear require two sequentially activated bHLH genes, Neurog1 and Neurod1, for their development. Neurons never develop in Neurog1 null mice, and most neurons die in Neurod1 null mutants, a gene upregulated by Neurog1. The surviving neurons of Neurod1 null mice are incompletely characterized in postnatal mice because of the early lethality of mutants and the possible compromising effect of the absence of insulin on peripheral neuropathies. Using Tg(Pax2-cre), we have generated a conditional deletion of floxed Neurod1 for the ear; this mouse is viable and allows us to investigate ear innervation defects of Neurod1 absence only in the ear. We have compared the defects in embryos and show an ear phenotype in conditional Neurod1 null mice comparable with the systemic Neurod1 null mouse. By studying postnatal animals, we show that Neurod1 not only is necessary for the survival of most spiral and many vestibular neurons, but is also essential for a segregated central projection of vestibular and cochlear afferents. In the absence of Neurod1 in the ear, vestibular and cochlear afferents enter the cochlear nucleus as a single mixed nerve. Neurites coming from vestibular and cochlear sensory epithelia project centrally to both cochlear and vestibular nuclei, in addition to their designated target projections. The peripheral innervation of the remaining sensory neurons is disorganized and shows collaterals of single neurons projecting to multiple endorgans, displaying no tonotopic organization of the organ of Corti or the cochlear nucleus. Pending elucidation of the molecular details for these Neurod1 functions, these data demonstrate that Neurod1 is not only a major factor for the survival of neurons but is crucial for the development of normal ear connections, both in the ear and in the central nervous system.

A striking feature of vestibular hair cells is the polarized arrangement of their stereocilia as the basis for their directional sensitivity. In mammals, each of the vestibular end organs is characterized by a distinct distribution of these polarized cells. We utilized the technique of post-fixation transganglionic neuronal tracing with fluorescent lipid soluble dyes in embryonic and postnatal mice to investigate whether these polarity characteristics correlate with the pattern of connections between the endorgans and their central targets; the vestibular nuclei and cerebellum. We found that the cerebellar and brainstem projections develop independently from each other and have a non-overlapping distribution of neurons and afferents from E11.5 on. In addition, we show that the vestibular fibers projecting to the cerebellum originate preferentially from the lateral half of the utricular macula and the medial half of the saccular macula. In contrast, the brainstem vestibular afferents originate primarily from the medial half of the utricular macula and the lateral half of the saccular macula. This indicates that the line of hair cell polarity reversal within the striola region segregates almost mutually exclusive central projections. A possible interpretation of this feature is that this macular organization provides an inhibitory side-loop through the cerebellum to produce synergistic tuning effects in the vestibular nuclei. The canal cristae project to the brainstem vestibular nuclei and cerebellum, but the projection to the vestibulocerebellum originates preferentially from the superior half of each of the cristae. The reason for this pattern is not clear, but it may compensate for unequal activation of crista hair cells or may be an evolutionary atavism reflecting a different polarity organization in ancestral vertebrate ears.

Neurod1 is a crucial basic helix-loop-helix gene for most cerebellar granule cells and mediates the differentiation of these cells downstream of Atoh1-mediated proliferation of the precursors. In Neurod1 null mice, granule cells die throughout the posterior two thirds of the cerebellar cortex during development. However, Neurod1 is also necessary for pancreatic beta-cell development, and therefore Neurod1 null mice are diabetic, which potentially influences cerebellar defects. Here, we report a new Neurod1 conditional knock-out mouse model created by using a Tg(Atoh1-cre) line to eliminate Neurod1 in the cerebellar granule cell precursors. Our data confirm and extend previous work on systemic Neurod1 null mice and show that, in the central lobules, granule cells can be eradicated in the absence of Neurod1. Granule cells in the anterior lobules are partially viable and depend on as yet unknown genes, but the Purkinje cells show defects not previously recognized. Interestingly, delayed and incomplete Tg(Atoh1-cre) upregulation occurs in the most posterior lobules; this leads to near normal expression of Neurod1 with a concomitant normal differentiation of granule cells, Purkinje cells, and unipolar brush cells in lobules IX and X. Our analysis suggests that Neurod1 negatively regulates Atoh1 to ensure a rapid transition from proliferative precursors to differentiating neurons. Our data have implications for research on medulloblastoma, one of the most frequent brain tumors of children, as the results suggest that targeted overexpression of Neurod1 under Atoh1 promoter control may initiate the differentiation of these tumors.

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a (dr)) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a "cochlear-gravistatic" endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants.

Many elderly people worldwide lose the neurosensory part of their ear and turn deaf. Cochlear implants to restore some hearing after neurosensory hearing loss are, at present, the only therapy for these people. In contrast to this therapy, replacement of hair cells via stem cell therapies holds the promise for a cure. We review here current insights into embryonic, adult, and inducible stem cells that might provide cells for seeding the cochlea with the hope of new hair cell formation. We propose a two-step approach using a first set of transcription factors to enhance the generation of inducible pluripotent stem (iPS) cells and a second set of factors to initiate the differentiation of hair cells. Recent evidence regarding ear development and stem cell research strongly suggest that microRNAs will be an important new regulatory factor in both iPS cell formation and differentiation to reprogram cells into hair cells. In addition, we highlight currently insurmountable obstacles to the successful transformation of stem cells into hair cell precursors and their injection into the cochlear canal to replace lost hair cells.

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ( tm1Kjn )) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of beta-galactosidase activity in Tmhs ( tm1Kjn ) heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.

Vibrissae are a unique sensory system of mammals that is characterized by a rich and diverse innervation involved in numerous sensory tasks with the potential for species-specific differences. In the present study, indocarbocyanine dyes (DiI and PTIR271) and confocal microscopy were combined to study the innervation of the mystacial vibrissae and vibrissa-specific sensory neuron distribution in the maxillary portion of the trigeminal ganglion of the mouse. The deeper regions of the vibrissa cavernous sinus (CS) contained a dense plexus of free nerve endings, possibly of autonomic fibers. The superficial part of this sinus displayed a massive array of corpuscular endings. Innervation in the region of the ring sinus consisted of Merkel endings and different morphological variances of lanceolate endings. The region of the inner conical body had a circular plexus of free nerve endings. In addition to confirming previous observations obtained by a variety of other techniques and ultrastructural studies, our studies revealed denser terminal receptor endings in a different distribution pattern than previously demonstrated in studies using the rat. We also revealed the distribution of sensory neurons in the trigeminal ganglion using retrograde tracing with fluorescent tracers from two nearby vibrissae. We determined that the populations of sensory neurons innervating the two vibrissae were largely overlapping. This suggests that the somatotopic maps of vibrissal projections reported at the different levels in the neuraxis are not faithfully reproduced at the level of the ganglion.

We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

The development of the oculomotor nucleus in five species of salamanders and one anuran species was investigated with tracing techniques. The data presented support the hypothesis that oculomotor motoneurons innervating the superior rectus muscle migrate across the midline. In the salamander Pleurodeles waltl, only ipsilateral oculomotor motoneurons are labeled in early development. Later, these neurons extend dendrites toward the contralateral side into the ventral tegmental neuropil, after which there is displacement of their nuclei (neuronal somata) across the midline. Cell bodies can be observed directly at the midline. In adult Salamandra salamandra, motoneurons innervating the superior rectus muscle are seen occasionally at the midline and on the ipsilateral side, with dendrites toward the contralateral side. Motoneurons on the ipsilateral side do not display these features. In Pleurodeles, developmental brain processes are slowed down, and the sequence of development of the contralateral subnucleus, which can be clearly observed, supports the migration hypothesis. In Xenopus laevis and most other species of salamanders this process is accelerated.

The retinal projections to the brain were studied in three species of European Salamandridae using anterograde transport of horseradish peroxidase and autoradiography. The results obtained were basically identical for all species and confirmed earlir findings on the fiber supply to the preoptic nucleus and the basal optic neuropil. In the anterior thalamus projections to three distinct terminal fields are clearly visible: (i) the diffusely stained corpus geniculatum thalamicum, (ii) the neuropil of Bellonci, pars lateralis, and (iii) a dorsomedial terminal field, the neuropil of Bellonci, pars medialis. Caudal to these terminal fields is an almost terminal-free region, the lateral neuropil. In the posterior thalamus a medial terminal field, the uncinate field, and a laterally located terminal field, the posterior thalamic neuropil, are distinguishable. The tectum opticum displays as many as four dense layers of retinofugal fibers and terminals in the rostral part and, in addition, a more densely stained strip of neuropil running from rostral to caudal over the tectum. The extent of ipsilateral fibers is greater than previously reported in other urodele species. They supply the medial and the lateral parts of the neuropoil of Bellonci, the uncinate field, and reach the tectum opticum via the medial optic tract. Further, they form terminals in the innermost optic fiber layer throughout the rostral half of the ipsilateral tectum. A small proportion of ipsilateral fibers contributes very sparsely to all other thalamic terminal fields, leaving only the caudal part of the tectum and several layers of the rostral tectum completely free of a direct retinofugal fiber supply.

The effect of vestibular afferent deprivation on the synaptogenesis of the nodular molecular layer has been studied quantitatively. No detectable effect on the time sequence of the development of the molecular layer and the external granular layer was found. Only around hatching a significantly reduced synaptic profile density was found in otocyst-deprived chickens on both halves of the nodulus. This effect can most easily be explained by the assumption of an anterograde transient transneuronal influence of vestibular afferents on the ability of parallel fibers to form synapses.

The cells of origin of the trochlear nerve of urodeles, anurans and gymnophionans were labelled with HRP in order to compare the location and morphology of trochlear motoneurons and to find evidence for sensory fibers in the trochlear nerve of amphibians. Trochlear motoneuron perikarya were found in a ventral tegmental position predominantly on the contralateral side, but an ipsilateral cell was present in some specimens of urodeles and anurans. About 19 motoneurons were labelled in Ambystoma, about 60 motoneurons in Xenopus, and a maximum of 7 cells in Ichthyophis. Decussation of trochlear nerve fibers showed only in Xenopus a highly variable pattern. In urodeles, selective filling of the trochlear nerve labelled in addition to trochlear motoneurons a caudo-medical tectal group of about 20 neurons of the nucleus of the mesencephalic root of the trigeminal nerve. Gymnophionans showed also labelled cells of the mesencephalic trigeminal root in the caudal midbrain close to the trochlear nerve root. In some frogs, a few cells of the mesencephalic trigeminal root were labelled in the caudal tectum and occasionally in the velum medullare anterius. Comparison of the numbers of trochlear nerve fibers with HRP-labelled motoneurons revealed in Xenopus a proportion of 1.2:1, but of 2.7:1 in Ambystoma. However, counting both labelled motoneurons and cells of the mesencephalic trigeminal root resulted in a trochlear nerve fiber to labelled neuron proportion of 1.3:1 in Ambystoma much like in Xenopus. The numbers of superior oblique muscle fibers and of trochlear nerve fibers, but not of HRP-labelled motoneurons, increased significantly with size in Xenopus laevis. We suggest that increased peripheral branching of individual fibers within the trochlear nerve with size rather than differentiation of additional motoneurons takes place in growing postmetamorphic Xenopus. In contrast to other vertebrates studied so far, the trochlear nerve is a mixed nerve in Ambystoma and perhaps in Ichthyophis. Whether this reflects a primitive or a derived condition is at present unclear.

The cells of origin of the trochlear nerve of Lampetra fluviatilis have been labelled with horseradish peroxidase (HRP) in order to compare the location and morphology of trochlear motoneurons with those of other vertebrates and to gain insight into the phylogenetic changes of the trochlear system. About 126 bipolar and tripolar trochlear motoneuron perikarya are found in a dorsal tegmental position close to the trochlear root. Only approximately 16% of the labelled cells are on the ipsilateral side of the brain, i.e. they lie predominantly contralateral as in gnathostome vertebrates. Dorsally directed dendrites reach the area of lateral-line and retinofugal fibres, and may establish functional contacts. In addition, each motoneuron has a ventral dendrite that extends towards the fasciculus longitudinalis medialis and to the ventral tegmentum. The dendrites branch close to the oculomotor root. Lampreys show a low muscle fibre to motoneuron ratio (4.5:1), i.e., they resemble amniotic vertebrates more than other anamniotic vertebrates. These data demonstrate both closer resemblance and larger differences of cyclostome and gnathostome trochlear motoneurons than previously suggested.

The organization of posterior and anterior afferents of the lateral-line system was studied in several species of urodeles by means of transganglionic transport of horseradish peroxidase. The afferents of each lateral-line nerve form distinct fascicles in the medullary alar plate. Each of the two branches of the anterior lateral-line nerve is organized in two long and one short fascicles. The posterior lateral-line afferents form only two long fascicles. Each ordinary neuromast is supplied by only two afferents, which run in the two ventral medullary fiber bundles. It is suggested that afferents to hair cells displaying one type of polarity form together one bundle, but those contacting hair cells polarized in the opposite way form the second ventral bundle of one lateral-line branch. Thus, the lateral-line afferents may be organized in a directotopic fashion. The short dorsal fascicle formed only by the anterior lateral-line afferents receives fibers exclusively from small pit organs. Each pit organ is supplied by only one afferent. Anatomically, these pit organs resemble in many respects the electroreceptive ampullary organs of certain fish. Neurons labeled retrogradely via the anterior lateral-line nerve afferents have been attributed to the nervus trigeminus or facialis. In addition to the posterior lateral-line afferents, only few centrifugally projecting neurons were labeled. These neurons are discussed as efferents to the posterior lateral-line neuromasts.

The metamorphic loss of lateral-line organs, lateral-line nerves and second order lateral-line neurons was examined in two Anuran species. At the onset of metamorphic climax, terminals within the lateral-line neuropil showed accumulation of glycogen-like granules. Neither the lateral-line nerve nor the organs or the nerve terminals inside the organs displayed any sign of degeneration at this stage. A few second order neurons exhibited accumulations of chromatin into conspicuous masses. These cells were partially or completely engulfed by phagocytes. At mid-metamorphosis all lateral-line organs were lost. The proximal parts of the lateral-line nerve fibers entering the rhombencephalic alar plate showed signs of degeneration. Within the lateral-line neuropil, pre- and some postsynaptic elements exhibited the flocculent type of degeneration or, to a lesser extent, the dark type of degeneration. Second order lateral-line neurons underwent an electron-dense or electron-lucent type of degeneration and were taken up by phagocytes. At the end of metamorphic climax the distal parts of the lateral-line nerves showed numerous dark degenerating fibers inside an intact myelin sheath. Within the lateral-line neuropil, numerous dark degenerating presynaptic elements were found next to some elements showing flocculent degeneration. Fewer degenerating second order neurons were found in the alar plate. They showed predominantly the dark type of degeneration. In contrast to earlier reports, our data suggest that the degenerative metamorphic changes observed in the present study are initiated in all parts of the lateral-line system simultaneously, and lead to the complete loss of all lateral-line organs and nerves and presumably all second order lateral-line neurons as well.

The development of the retinofugal projection areas of the brain has been studied in larvae of Triturus alpestris by means of anterograde transported horseradish peroxidase. The optic tract establishes contacts with the optic tectum prior to the onset of robust terminal formation in the diencephalon. The tectum becomes covered by the retinofugal projection in a rostro-caudal direction. The basal optic neuropil develops synchronously with the oculomotor neurons. Their dendrites extend into this neuropil area. A small amount of uncrossed label occurs long before metamorphosis. Around metamorphotic climax this ipsilateral label increases but does not attain the adult pattern even three monts postmetamorphosis. The data are compared with the onset of visual induced behaviour.

The cerebellar noduli of 18 neonatal mice and 13 young chickens were studied by light and electron microscopy. Beginning with postnatal day 10 in mice and incubation day 17 in chickens some Purkinje cells of the nodulus show degenerative changes. The nuclei and cytoplasm of these cells are darker than the neighbouring tissue due to an accumulation of large amounts of ribosomelike particles and an increased stainability of the cytoplasm. Mitochondria and endoplasmic reticulum undergo a moderate swelling, the Golgi apparatus hypertrophies whereas the somata and nuclei atrophy. The percentage of degenerating Purkinje cells reaches a peak of about 45% around postnatal day 14 in mice and the 4th day after hatching in chickens. In 26 otocyst-deprived chickens a smaller proportion of Purkinje cells with degenerative changes is found. The way in which the otocyst removal influences the neonatal Purkinje cell degeneration is not clear.

The system of lateral-line organs in urodeles was examined by the use of various light- and electron-microscopical techniques. The results show that, in addition to the well-known mechanoreceptive neuromast organs, a second type of receptor can be identified. This second type of organ was presumably seen by earlier workers, but they seemingly failed to point out the distinction between the two organs. The presently described organs are anatomically similar to the ampullary organs of various anamniotic species such as Brachiopterygii, sturgeons, lungfish, and silurids. In all these species the ampullary organs display only one afferent fiber but no efferent innervation and are situated around an ampullary enlargement in or below the epidermis as in urodeles. All ampullary receptors including those of urodeles are very sensitive to weak electrical fields. Similar to the situation in teleosts, the ampullae of urodeles show numerous microvilli but no kinocilia. All other nonteleostean ampullary receptors appear to possess only kinocilia as apical specializations but no microvilli. Current evidence suggests that the electroreceptive ampullary organs are as phylogenetically old as all other vertebrate sensory systems; they are now known to be relatively common among anamniotic vertebrates. Since all ampullary receptors share many common characteristics, it is assumed that they were derived from one phylogenetic precursor but have evolved certain peculiarities in each species not shared by other ampullary receptors.