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In the vertebrate central nervous system, groups of functionally related neurons, including cranial motor neurons of the brainstem, are frequently organised as nuclei. The molecular mechanisms governing the emergence of nuclear topography and circuit function are poorly understood. Here we investigate the role of cadherin-mediated adhesion in the development of zebrafish ocular motor (sub)nuclei. We find that developing ocular motor (sub)nuclei differentially express classical cadherins. Perturbing cadherin function in these neurons results in distinct defects in neuronal positioning, including scattering of dorsal cells and defective contralateral migration of ventral subnuclei. In addition, we show that cadherin-mediated interactions between adjacent subnuclei are critical for subnucleus position. We also find that disrupting cadherin adhesivity in dorsal oculomotor neurons impairs the larval optokinetic reflex, suggesting that neuronal clustering is important for co-ordinating circuit function. Our findings reveal that cadherins regulate distinct aspects of cranial motor neuron positioning and establish subnuclear topography and motor function., Competing Interests: AK, GD, GW, JC, SG No competing interests declared, (© 2020, Knüfer et al.)

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 Isl MN -GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

Infantile strabismus is characterized by numerous visual and oculomotor abnormalities. Recently nonhuman primate models of infantile strabismus have been established, with characteristics that closely match those observed in human patients. This has made it possible to study the neural basis for visual and oculomotor symptoms in infantile strabismus. In this review, we consider the available evidence for neural abnormalities in structures related to oculomotor pathways ranging from visual cortex to oculomotor nuclei. These studies provide compelling evidence that a disturbance of binocular vision during a sensitive period early in life, whatever the cause, results in a cascade of abnormalities through numerous brain areas involved in visual functions and eye movements., (Copyright © 2017 the American Physiological Society.)

Background: Oculomotor neurons develop initially like typical motor neurons, projecting axons out of the ventral midbrain to their ipsilateral targets, the extraocular muscles. However, in all vertebrates, after the oculomotor nerve (nIII) has reached the extraocular muscle primordia, the cell bodies that innervate the superior rectus migrate to join the contralateral nucleus. This motor neuron migration represents a unique strategy to form a contralateral motor projection. Whether migration is guided by diffusible cues remains unknown., Methods: We examined the role of Slit chemorepellent signals in contralateral oculomotor migration by analyzing mutant mouse embryos., Results: We found that the ventral midbrain expresses high levels of both Slit1 and 2, and that oculomotor neurons express the repellent Slit receptors Robo1 and Robo2. Therefore, Slit signals are in a position to influence the migration of oculomotor neurons. In Slit 1/2 or Robo1/2 double mutant embryos, motor neuron cell bodies migrated into the ventral midbrain on E10.5, three days prior to normal migration. These early migrating neurons had leading projections into and across the floor plate. In contrast to the double mutants, embryos which were mutant for single Slit or Robo genes did not have premature migration or outgrowth on E10.5, demonstrating a cooperative requirement of Slit1 and 2, as well as Robo1 and 2. To test how Slit/Robo midline repulsion is modulated, we found that the normal migration did not require the receptors Robo3 and CXCR4, or the chemoattractant, Netrin 1. The signal to initiate contralateral migration is likely autonomous to the midbrain because oculomotor neurons migrate in embryos that lack either nerve outgrowth or extraocular muscles, or in cultured midbrains that lacked peripheral tissue., Conclusion: Overall, our results demonstrate that a migratory subset of motor neurons respond to floor plate-derived Slit repulsion to properly control the timing of contralateral migration.

Paediatric cerebral malaria is the most serious complication of Plasmodium falciparum infection. While the majority recover, long-term cognitive impairment has been highlighted as a significant and neglected problem. Persistent or serious deficits in processes such as attention or behavioural inhibition should be manifest in changes to performance on oculomotor tasks. Therefore we investigated the impact of cerebral malaria on the development of reflexive pro-saccades and antisaccades. In a longitudinal study, 47 children previously admitted with retinopathy-confirmed cerebral malaria (mean age at admission 54 months), were compared with 37 local healthy controls (mean ages at first study visit 117 and 110 months respectively). In each of three or four test sessions, over a period of up to 32 months, participants completed 100 prosaccade tasks and 100 antisaccade tasks. Eye movements were recorded using infrared reflectance oculography; prosaccade, correct antisaccade and error prosaccade latency, and antisaccade directional error rate were calculated. Hierarchical linear modelling was used to investigate the effect of age and the influence of cerebral malaria on these parameters. Data were also collected from an independent, older group (mean age 183 months) of 37 local healthy participants in a separate cross-sectional study. Longitudinal data exhibited the expected decrease in latency with age for all saccade types, and a decrease in the antisaccade directional error rate. Hierarchical linear modelling confirmed that age had a statistically significant effect on all parameters (p< = 0.001). However, there were no statistically significant differences between the cerebral malaria and control groups. Combining groups, comparison with the literature demonstrated that antisaccade directional error rate for the Malawi sample was significantly higher than expected, while latencies for all saccade types were indistinguishable from published. The high directional error rate was also confirmed in the older, healthy Malawian participants from the cross sectional study. Our observation of similar oculomotor performance in cerebral malaria and control groups at long follow-up periods suggests that cerebral malaria survivors are not at a generally increased risk of persistent cognitive deficits. Our data raise questions about the prevailing hypothesis that cerebral malaria has gross impacts on the development of processes such as attention and behavioural inhibition. More importantly, our novel finding of a clear difference in antisaccade performance between all of the Malawi participants and published data suggests that the Malawian paediatric population as a whole faces serious challenges to cognitive development beyond cerebral malaria., Competing Interests: The authors have declared that no competing interests exist.

Background: The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points., Results: We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes., Conclusions: We define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.

Background: The trochlear nucleus, the smallest of the extraoculomotor nuclei, is unique or even curious, because the nerve roots emerge dorsally from the superior medullary velum after decussation. Little information is available on the developmental anatomy of this nucleus in humans., Design/subjects: We examined serial brain sections from 10 premature infants aged 20-39 weeks of gestation to document the histology and morphometry., Results: The trochlear nucleus was composed of three parts: the rostral tip, the main body, and the caudal division. The rostral tip was a rostral continuation of the main body, being closely related to the oculomotor nucleus; the main body was enveloped by a fibrous capsule; the caudal division was a small separate cluster of neurons in the medial longitudinal fasciculus or the root fibers with individual variations. Tigroid Nissl bodies first appeared at 28 weeks in presumed motoneurons. Various sizes of motoneurons were recognized; medium-sized to small motoneurons were preferentially accumulated in the rostral tip. Among the motoneurons, presumed non-motor neurons were infrequently scattered. Morphometric analysis showed that the nuclear volume exponentially increased with age, about 15 fold over 20-39 weeks, while the average profile area of the neurons linearly increased. Statistical analysis confirmed that cell area was smallest in the rostral tip among the three parts., Conclusion: Although the sample number is small in this study, it suggests that the human trochlear nucleus can be divided into three parts, and that the overall growth may be accelerated at about 30 weeks of gestation., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Changes in the electrophysiological and morphological characteristics of motoneurons (Mns) of the oculomotor nucleus during postnatal development have been reported, however synaptic modifications that take place concurrently with postnatal development in these Mns are yet to be elucidated. We investigated whether cholinergic inputs exert different effects on the recruitment threshold and firing rate of Mns during postnatal development. Rat oculomotor nucleus Mns were intracellularly recorded in brain slice preparations and separated in neonatal (4-7 postnatal days) and adult (20-30 postnatal days) age groups. Stimulation of the medial longitudinal fasciculus evoked a monosynaptic excitatory potential in Mns that was attenuated with atropine (1.5 μM, a muscarinic antagonist). Mns were silent at their resting membrane potential, and bath application of carbachol (10 μM, a cholinergic agonist) induced depolarization of the membrane potential and a sustained firing rate that were more pronounced in adult Mns. Pharmacological and immunohistochemical assays showed that these responses were attributable to muscarinic receptors located in the membrane of Mns. In addition, compared to control Mns, carbachol-exposed Mns exhibited a higher firing rate in response to the injection of the same amount of current, and a decrease in the current threshold required to achieve sustained firing. These latter effects were more pronounced in adult than in neonatal Mns. In conclusion, our findings suggest that cholinergic synaptic inputs are already present in neonatal Mns, and that the electrophysiological effects of such inputs on recruitment threshold and firing rate are enhanced with the postnatal development in oculomotor nucleus Mns. We propose that cholinergic input maturation could provide a greater dynamic range in adult Mns to encode the output necessary for graded muscle contraction., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

The oculomotor nerve (ON) provides motor innervation to the eyeball and exhibits alterations in various physiological and pathological conditions, which may result in abnormal ocular movement. Although the nerve has been studied in detail, very few data are available regarding its morphology and changes with aging. Hence, in the present investigation, the neural and connective tissue organizations of the pre-cavernous part of the ON were studied in order to provide sequential data regarding age-related morphological changes. Thirty-eight ON from cadavers aged from 40 post-natal days to 78 years were studied. Cross-sections of the nerve revealed a poorly defined multifascicular arrangement with predominant myelinated fibres of various calibres randomly intermingled with unmyelinated fibres. Small- and medium-sized fibres (probably parasympathetic) were mainly located at the junction of the central and the paracentral zones of the nerves. Using unbiased stereological techniques, the total number of the axons, the area of the myelinated fibres and myelin sheath thickness were estimated. The cross-sectional area of the nerve increased significantly up to the third decade. The minimal and maximal total number and area of myelinated axons varied from 17,000 to 21,000 and from 8.95 to 14.02 microm(2), respectively. There was a significant increase in the myelin thickness of axons with age. Connective tissue gradually increased in later decades and was more pronounced in the eighth decade. The present study provides novel baseline morphometric data on the ON that would be of help to future studies.

This work investigates the somatodendritic shaping of rat oculomotor nucleus motoneurons (Mns) during postnatal development. The Mns were functionally identified in slice preparation, intracellularly injected with neurobiotin, and three-dimensionally reconstructed. Most of the Mns (approximately 85%) were multipolar and the rest (approximately 15%) bipolar. Forty multipolar Mns were studied and grouped as follows: 1-5, 6-10, 11-15, and 21-30 postnatal days. Two phases were distinguished during postnatal development (P1-P10 and P11-P30). During the first phase, there was a progressive increase in the dendritic complexity; e.g., the number of terminals per neuron increased from 26.3 (P1-P5) to 47.7 (P6-P10) and membrane somatodendritic area from 11,289.9 microm(2) (P1-P5) to 19,235.8 microm(2) (P6-P10). In addition, a few cases of tracer coupling were observed. During the second phase, dendritic elongation took place; e.g., the maximum dendritic length increased from 486.7 microm (P6-P10) to 729.5 microm in adult Mns, with a simplification of dendritic complexity to values near those for the newborn, and a slow, progressive increase in membrane area from 19,235.8 microm(2) (P6-P10) to 24,700.3 microm(2) (P21-P30), while the somatic area remained constant. In conclusion, the electrophysiological changes reported in these Mns with maturation (Carrascal et al. [2006] Neuroscience 140:1223-1237) cannot be fully explained by morphometric variations; the dendritic elongation and increase in dendritic area are features shared with other pools of Mns, whereas changes in dendritic complexity depend on each population; the first phase paralleled the establishment of vestibular circuitry and the second paralleled eyelid opening.

The temporal sequence of changes in electrophysiological properties during postnatal development in different neuronal populations has been the subject of previous studies. Those studies demonstrated major physiological modifications with age, and postnatal periods in which such changes are more pronounced. Until now, no similar systematic study has been performed in motoneurons of the oculomotor nucleus. This work has two main aims: first, to determine whether the physiological changes in oculomotor nucleus motoneurons follow a similar time course for different parameters; and second, to compare the temporal sequence with that in other neuronal populations. We recorded the electrophysiological properties of 134 identified oculomotor nucleus motoneurons from 1 to 40 days postnatal in brain slices of rats. The resting membrane potential did not significantly change with postnatal development, and it had a mean value of -61.8 mV. The input resistance and time constant diminished from 82.9-53.1 M omega and from 9.4-4.9 ms respectively with age. These decrements occurred drastically in a short time after birth (1-5 days postnatally). The motoneurons' rheobase gradually decayed from 0.29-0.11 nA along postnatal development. From birth until postnatal day 15 and postnatal day 20 respectively, the action potential shortened from 2.3-1.2 ms, and the medium afterhyperpolarization from 184.8-94.4 ms. The firing gain and the maximum discharge increased with age. The former rose continuously, while the increase in maximum discharge was most pronounced between postnatal day 16 and postnatal day 20. We conclude that the developmental sequence was not similar for all electrophysiological properties, and was unique for each neuronal population.

To further our understanding of the role that voltage-activated Ca2+ channels play in the development, physiology and pathophysiology of motoneurones (MNs), we used whole-cell patch-clamp recording to compare voltage-activated Ca2+ currents in oculomotor (III) and hypoglossal (XII) MNs of neonatal [postnatal day (P)1-5] and juvenile (P14-19) rats. In contrast to III MNs that innervate extraocular muscles, XII MNs that innervate tongue muscles mature more rapidly, fire bursts of low frequency action potentials and are vulnerable to degeneration in amyotrophic lateral sclerosis. In neonates, low voltage-activated (LVA) Ca2+ current densities are similar in XII and III MNs but high voltage-activated (HVA) Ca2+ current densities are twofold higher in XII MNs. The HVA Ca2+ channel antagonists (nimodipine and nifedipine for L-type, omega-agatoxin-TK for P/Q-type and omega-conotoxin-GVIA for N-type) revealed that, while N- and P/Q-type HVA Ca2+ channels are present in both MN pools, a 3.5-fold greater P/Q-type Ca2+ current in XII MNs accounts for their greater HVA Ca2+ currents. Developmentally, LVA and HVA Ca2+ current densities decrease in III MNs but remain unchanged in XII MNs. Thus, the differences between these MN pools increase developmentally so that, in juveniles, the LVA Ca2+ current density is twofold greater and the HVA Ca2+ current density is threefold greater in XII compared with III MNs. We propose that this differential expression of LVA and HVA Ca2+ channels in XII and III MNs during development contributes to their distinct physiology and may also be a factor contributing to the greater susceptibility of XII MNs to degeneration as seen in amyotrophic lateral sclerosis.

We describe three patients with aberrant regeneration of the third nerve secondary to traumatic brain injury. The full blown features of the syndrome include horizontal gaze-eyelid synkinesis, pseudo-Graefe sign, limitation of elevation and depression of the eye with retraction of the globe on attempted vertical movements, adduction of the involved eye on attempted elevation or depression, pseudo-Argyll Robertson pupil and absent vertical optokinetic response. The 'misdirection' incidence in our study is 15%.

We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences to Islet-1 promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.

Mice with a nontargeted transgene insertion at the motor endplate disease (med) locus (med(tg)) contain a deletion of a novel gene encoding a neuronal voltage-gated sodium channel, designated Scn8a. We characterized severe skeletal muscle atrophy beginning by Postnatal Day 10 (P10) and death by P20 in the med(tg) mouse. Denervation was functional, rather than structural, since the Scn8a mutation was not accompanied by retraction of neuromuscular contacts, motoneuron death, or decreased motoneuron soma diameter. Although pathology consistent with denervation was seen in both hindlimb and forelimb musculature, the postnatal maturation of the extraocular muscles was not altered. The onset of paralysis is likely coincident with the time that the Scn8a sodium channel normally assumes a critical role in the initiation and/or propagation of action potentials in spinal motoneurons. By contrast, the lack of consequences for extraocular muscle suggests that the Scn8a voltage-gated sodium channel may be of relatively minor importance for oculomotor motoneurons.

The electrophysiological properties of oculomotor neurons were studied in neonatal rats aged 1-15 days. Action potentials were recorded from brainstem slices (frontal section) using the intracellular recording method at 35 degrees C. No significant age-dependent differences were detected in the resting potential (around -55 mV) and in the amplitude of the action potential (approximately 60 mV). However, the input resistance of oculomotor neurons declined with age from a mean of 60.8 M omega for rats 1-3 days old to 17.0 M omega for rats 14-15 days old. In addition, the duration of the action potential measured at the half-amplitude gradually decreased from 0.74 ms to 0.34 ms with increasing age. Increases were detected in the maximum rate of rise (from 117 V/s to 181 V/s) and the maximum rate of fall (from -67 V/s to -103 V/s) of the action potential. When long-lasting (500 ms) depolarizing current pulses were applied to oculomotor neurons, some neurons exhibited continuous repetitive discharge (i.e. tonic firing) while others showed transient discharge (phasic firing). The proportion of tonic-type neurons increased with age: the value was 9% for rats 1-5 days old, 37% for rats 6-10 days old and 54% for rats 11-15 days old. Concomitantly, the number of neurons showing a time-dependent inward rectification increased and the average maximum frequency of the discharge rose from 150 to 420 Hz, approximately, with age. Furthermore, it was found that the electrophysiological properties of oculomotor neurons of rats 14-15 days old were similar to those in adult rats. It is concluded that oculomotor neurons in neonatal rats show rapid alterations in their electrophysiological properties and that the ratio of tonic-type to phasic-type neurons changes during the early stages of development.

Retrograde transport of horseradish peroxidase (HRP) after its application into the orbit was used to investigate the development of the different ocular motor nuclei in larvae of the sea lamprey (Petromyzon marinus) and to identify their regions of origin. In the smallest larvae studied (10-19 mm in length), the oculomotor and abducens neurons were ipsilateral to the site of HRP application, whilst trochlear neurons were contralateral. These motoneurons did not have dendritic processes. In larvae more than 19 mm in length, both ipsilateral and contralateral components were found in the oculomotor and trochlear nuclei; dendrites were present, and their length and branching increased with larval age. An adult-like pattern of topographic organization and dendritic arborization was reached in larvae of about 45-60 mm in length. In oculomotor neurons, medial dendrites appear first, then dorsolateral dendrites, and finally ventral dendrites. Similarly, in trochlear neurons ventral and ventrolateral dendrites develop first, followed by dorsal dendrites that course either to the caudal optic tectum or to the terminal fields of the octaval and lateral line nerves in the cerebellar plate. Dorsal and ventral dendrites of the abducens neurons arise at the same time, but dorsal dendrites attain an adult-like morphology earlier. A few motoneurons showed ventricular attachments in larvae longer than 40 mm. The significance of these processes and their possible usefulness as a marker for the regions of origin of the ocular motor nuclei are discussed. Finally, the results presented here indicate that differentiation of the ocular motor nuclei in larval lampreys precedes and is independent of the maturation of the eye at transformation.

The development of the ocular motor system was studied in 3- to 6-year old larval lampreys with two different retrograde tracers. Motor neurons of the oculomotor and trochlear nuclei are situated closely to one another in younger larvae. Cases in which only trochlear neurons were labelled revealed trochlear motor neurons scattered from the midbrain tegmentum through the anterior medullary velum. We believe this distribution reflects the place of final mitosis (midbrain tegmentum) and subsequent migration (anterior medullary velum) of lamprey trochlear motor neurons. Evidence is also presented for contralateral migration of oculomotor motor neurons and for ventrolateral migration of abducent motor neurons. The distances covered by migrating ocular motor neurons range from 100 to 150 microns in small larvae; these are distances that could be covered easily during the several years duration of larval development in lampreys.

In guinea-pigs the oral administration of L-acetylcarnitine (L-AC) markedly favours the process of reinnervation of the oculomotor nerve sectioned at intracranial level. The gains of the horizontal and vertical vestibulo-ocular reflexes (HVOR, VVOR) were taken into consideration in testing the functional recovery of the nerve. As a consequence of the drug administration, 24 weeks after the operation the gains of the treated animals were higher than those of the controls. Reduction of misalignments of the stimulus-response orientation was also observed in treated animals as compared to the controls. This suggests that L-AC potentiates motor reinnervation by enhancing the nerve-growing processes and favouring a better consolidation of the appropriate neuromuscular synapses. The increased gain, and the improvement of the alignment in ocular responses, due to L-AC would allow for an increase of visual function during head movement by optimizing gaze stability.

Development of the vegetative component of the oculomotor nerve nuclei was studied in the main mammalia orders and in human ontogenesis. In phylogenesis parasympathetic nuclei of Jakubovitch--Edinger--Westphal and those of Perlia are formed later than somatic nuclei of the oculomotor nerve. The animals studied have, together with well developed somatic components of the oculomotor nerve nuclei, gomologues of parasympathetic nuclei, which are rather well developed in carnivores and monkeys. In human prenatal ontogenesis, somatic nuclei are formed first and then parasympathetic ones. Both parts of the oculomotor nerve nuclei are fully developed in adult man.

The effects on rat cranial nerve growth of removing various amounts of extraocular muscle was studied using morphometric techniques. Growth in the third cranial nerves was found to be severely retarded when most of the muscle tissue was removed. By contrast, removal of the eye alone, leaving extraocular muscles relatively intact, was found to have little or no effect on the subsequent growth of third and sixth cranial nerve fibers and of extraocular muscle fibers. This conclusion could be drawn only through the application of statistical methods which take into account several generally unrecognized facts: frequency distributions of axon circumference and myelin sheath thickness are highly variable from nerve to nerve even in normal rats, which often have more large fibers in left than in right nerves. The bimodal nature of peripheral nerve fiber distributions precludes the use of such parametric tests as the commonly and inappropriately used t-test, but a non-parametric test such as the Kolmogorov-Smirnov test, extensively used in these studies, is inadequate for data comprising several sets of distributions to be compared. The application of the analysis of variance to some of the data and the merits of the procedure are discussed.

Quantitative light-microscopic studies on the ontogenetic development of 11 visual brain regions of Tupaia belangeri were done. The fresh volumes of the visual brain regions showed a heterochronical and heterotypical development. Brain regions with overshooting or monotonously increasing growth could be found using computer calculations of logistic curves. Studies on the development of the number of nerve cells and glial cells showed an overshooting growth of the nerve cell quantity and a monotonously increasing growth of the glial cell quantity in the nuclei nn. oculomotorii et trochlearis. A compartment analysis with computer aided stereological procedures was done for the nucl. n. oculomotorii. The present findings as well as those from proceeding investigations serve as the basis for a hypothesis describing the significance of the growth curve for fresh volumes, and especially for the spontaneous degeneration of neurons in the formal genesis of the nervous system.

A developmental study of the Telencephalon of the trout (Salmo irideus) has been done. The stages of fixation were 18 days after fecondation, hatching; 5 days, 1, 2, and 3 months, and 1 year after hatching. The different cell-masses are summarized in table 1. In the young trout, eversion is not important. Just, 2 olfactory bulbs are evaginated. In front of the commissura anterior, we can see: the Nucleus ventroventralis and the Nucleus ventrodorsalis, on the one hand; a voluminous dorsal area which includes: the Nucleus dorsolateralis, the Nucleus dorsomedialis, the Nucleus dorsocentralis and the dorso and ventro-lateral groups, on the other hand. The different Nuclei of the dorsal area are differentiated from a primordial territory which is the area intermedius at the hatching stage. On the hemispheric wall at the level of the tela, we can see the Nucleus teniae. Behind the plan of the commissura anterior, the Nucleus posterior is already seeing at the end of the first month after the hatching. A Golgi-Cox study showed some aspects of different kinds of neurons, and an important neuropil at the level of the Nucleus dorsocentralis too.

It was investigated when and how the four examined sensory systems and the behaviour performances depending on them are affected by metamorphosis and whether they develop at an uniform rate from hatching up to metamorphosis. The following conclusions were reached on the basis of the results: -- The central nervous system starts to metamorphose earlier than the habitus and the viscera. -- Motor reactions based on the new capacities of the metamorphosed sensory systems only set in after metamorphosis. -- During metamorphosis the threshold of general sensitivity for the release of motor reactions is drastically raised. -- The sense of taste does not begin to function until after metamorphosis. -- The olfactory sense is the only one of the four tested systems that functions immediately after hatching. -- The level of development of the sensory systems and motor reactions is low in the early larva. The lack of taxis is characteristic of this. -- There is a phase of accelerated development of the sensory systems and motor reactions between the early and late larval stages. -- By comparison with the situation after metamorphosis the sensory systems of the old larva are fairly simple in their structure, but the motor reactions they permit are sufficient for the larva to cope with its aquatic biotope. -- Not until after the morphological structures have attained a new level of development do the relevant motor reactions show a corresponding advance.

Unlabelled: In this paper we analyze long-term changes, extending into adulthood, of the number of axons in the oculomotor nerve of Xenopus laevis. We counted the number of axons in that nerve in normal control (nor) animals, and on the operated (ipsilateral, ip) and contralateral (co) sides of enucleated animals at premetamorphosis, metamorphic climax, juvenile (3 and 7 months) and adult (16.5 months) stages. The experimental animals had had one of their ocular primordia removed at hatching. In the nor-nerves there is a loss of 23% of the axons between premetamorphosis and climax, then a further drop of 35% by the 3-month stage, followed by a gain of 39% on reaching adulthood. At premetamorphosis the ip-nerves already contain less than the normal number of axons, which is reduced a further 73% by 3 months; there is no subsequent increase in adulthood. The co-nerves lose no axons from premetamorphosis to climax; they therefore have 30% more axons than normals; this excess is kept until 7 months; from then to sexual maturity the increase is only 11%., Conclusions: (1) unilateral target removal affects also the nerve innervating the contralateral target: the increased loss of axons on the operated side is accompanied by reduced loss on the other; (2) the contralateral side displays for some time an excess of axons compared to normal, but this is finally cancelled out by the addition of many axons in the adult nor animals; (3) adjustment of axon number is not restrained to embryonic stages but continues at least until sexual maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

The spontaneous degeneration of neurons of the Nucl. tractus mesenceph. n. trigemini of 33 male NMRI-mice between 19 and 60 days of ontogenesis has been analyzed fitting the exponential function (see article). The number of neurons decreases from 1500 at birth to 1200 at the adult level. The damping of the curve is 4.8. This nucleus is compared with other nuclei of the albino mouse (Nucl. n. oculomotorii, Nucl. cochleares dorsalis et ventralis). The spontaneous reduction of the number of neurons seems to be a common phenomen during the perinatal time of the mammals. There is a heterochronous reduction for the different analyzed nuclei.

The Edinger-Westphal (EW) nucleus, also known as the accessory oculomotor nucleus in the chick, provides the cholinergic preganglionic input to the parasympathetic ciliary ganglion. In addition to acetylcholine, many EW neurons have been shown to contain enkephalin-like and/or substance P-like immunoreactivity. Establishment of EW neurons in culture would make possible study of their interactions with ciliary ganglion neurons in vitro and in addition would provide a valuable system for studying cholinergic/peptidergic neurons of the vertebrate central nervous system. We describe here dissociated cell cultures established from midbrain tissue containing the EW nucleus. In these cultures, 86% of the cells with neuronal morphology were positive for intracellular acetylcholinesterase activity, 54% were positive for enkephalin-like immunoreactivity, and 4% were positive for substance P-like immunoreactivity. The proportions of neurons that scored as labeled were even higher if the number of positive cells was compared to the number of cells in sister cultures immunoreactive for the large neurofilament protein polypeptide. When the cultures were stained simultaneously for acetylcholinesterase activity and enkephalin-like immunoreactivity, 34% of the cells with neuronal morphology were positive for both. In cultures derived from adjacent tissue regions very few cells expressed both activities. These results suggest that the cells expressing both acetylcholinesterase activity and enkephalin-like immunoreactivity in culture are EW neurons. The putative EW neurons survive for weeks in vitro in the absence of their normal target, the ciliary ganglion.

1. The activities of the transmitter enzymes acetylcholine esterase (AChE), monoamine oxidase (MAO) and gamma-aminobutyrate transaminase (GABA-TR) were investigated in the nucleus nervi oculomotorii of Salmo irideus (Teleostei) during biomorphosis. 2. AChE as well as MAO-activity could be demonstrated already before the time of hatching, whereas GABA-TR occured from the 10th day post hatching. 3. In earliest stages the AChE activity was found in the neuronal pericarya of the nucleus n. III, but later on a strong increase occured in vestibular presynaptic terminals establishing synaptic contacts with the lateral surface of the large pericarya, especially in the medial part of the dorsal subnucleus and the ventral subnucleus. In adults the enzyme activity was localized in vestibular nerve fiber terminals exclusively due to the loss of enzyme activity in the pericarya. 4. The activity of MAO was found surrounding the neurons of the nucleus nervi oculomotorii with stronger deposits of reaction product at the medial surface that is not occupied by cholinergic terminals. 5. Besides non reacting neurons in all parts of the nucleus some cells in the transition zone between the medial and lateral area of the dorsal subnucleus, exhibited high cytoplasmatic GABA-TR activity. 6. The different ways of chemical transmission between nerve terminals and the neurons of the nucleus nervi oculomotorii are discussed.

The number of axons in the oculomotor (OM) nerve of Xenopus laevis tadpoles was counted in unoperated and in unilaterally enucleated animals, raised in 0.4 g/l thiourea (TU), a thyroid-blocking agent, which arrested their development at premetamorphosis. In unoperated animals the number of axons starts to decrease with metamorphosis. When raised in TU, the tadpoles do not metamorphose and show no axon loss; rather, there is a moderate increase in axon number (13%) after 6 months of thiourea-treatment. Thus metamorphosis is necessary for the initiation of axon loss. In unilaterally enucleated tadpoles, increased axon loss occurs at metamorphosis in the OM nerve of the operated side, but the contralateral OM nerve shows no loss at all. When these animals are treated with TU, there is, as compared with the effects of the TU-treatment in unoperated animals, an increase of 7% in the ipsilateral side and of 28% in the contralateral one. Thus thyroxine blockade prevents the effects of unilateral enucleation and induces an excessive number of axons during the period observed. We postulate that through blockade of metamorphosis, axon elimination is arrested, while their production continues, and that these effects are accentuated by manipulation of the axonal target.