The development of the isthmo-optic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons.

The development of the centrifugal projection to the chick retina in the isthmo-optic tract (IOT) has been studied by the retrograde transport of the enzyme marker horseradish peroxidase (HRP) injected into the eye at various times during the incubation period. Some neurons in the isthmo-optic nucleus (ION--the cells of origin of the IOT) can first be labeled from the eye following injections on the tenth day of incubation; after injections on the twelfth day or later, about 95% of the neurons can be so labeled. It follows from this that the axons of virtually all the neurons in the ION (including the 60% which normally degenerate between the thirteenth and seventeenth days of incubation) reach the contralateral eye. Since in 12-day old embryos the IOT is between six and seven millimeters in length and HRP can be identified in the perikarya of ION neurons within three and one-half hours, the rate of retrograde transport in the system must be of the order of 48 mm/day. A similar time is required for HRP to appear in the perikarya of ION neurons in post-hatched chicks in which the length of the IOT is estimated to be about 14 mm. This suggests that at some time during the latter half of the incubation period there is a significant acceleration in the rate of retrograde transport, similar to that found for anterograde axonal transport in the chick and rabbit visual systems.     

The development of the isthmo-optic nucleus.

The nucleus of origin of centrifugal fibers to the retina (the so-called isthmo-optic nucleus - ION) has been used as a model for the study of the major features of neural development, from the period of cell proliferation until after the formation of its afferent and efferent connections. 3H-thymidine autoradiography has established that in the chick cells of the ION are generated (i.e., become post-mitotic) between the middle of the 5th and the end of the 7th days of incubation. The first-formed cells are found in the ventrolateral part of the nucleus, while those that are generated at successively later stages come to occupy progressively more medial and dorsal positions within the nucleus. The anlage of the ION can be identified on the 8th day of incubation, and by the 11th day, when it is numerically complete, it occupies a prominent position in the caudo-dorsal part of the midbrain tegmentum at the level of the IVth nerve nucleus. At this stage the nucleus contains about 22,000 neurons, and shows no signs of cytoarchitectonic differentiation. Between the 13th and 17th days of incubation, about 60% of the neurons in the nucleus degenerate; as a result of this degeneration, the arrival of afferent fibers, and the growth of the cells' processes, the nucleus comes to have its characteristic adult form of a complex, folded, bilaminar sheet, in which each part of the retina is precisely represented. Experiments based on the retrograde transport of horseradish peroxidase (HRP) from the eye indicate that the first centrifugal fibers, in the isthmo-optic tract (IOT), reach the retina on the 10 day of incubation, and by the 12th day all but about 5% of the neurons in the ION can be retrogradely labeled in this way...     

Dendritic development in the isthmo-optic nucleus of chick embryos

In the mature isthmo-optic nucleus, the neuronal perikarya are arranged in a convoluted lamina surrounding the neuropil, and the primary dendrites of each neuron project perpendicular to the lamina into the neuropil. Thus, the neurons are highly polarized. It was previously believed that this mature morphology was the result of remodelling during the last week of embryogenesis, and that the neurons were initially multipolar and nonpolarized. We here show that as early as embryonic day 12 the neurons are already polarized, with a morphology resembling that in older embryos and hatched chicks. However, the neurons in the core of the isthmo-optic nucleus are almost all polarized ventrally or ventromedially at this age, whereas they later take on many different orientations. In contrast, most neurons near the border of the nucleus already have inwardly polarized dendrites, as in hatched chicks.     

Postnatal development of the ipsilaterally projecting retinal ganglion cells in normal rats and rats with neonatal lesions

The postnatal development of normal and anomalous uncrossed retinofugal projections in albino rats was studied using horseradish peroxidase and lectin-conjugated horseradish peroxidase as retrograde neuronal tracers. In normal rats, the number of ipsilaterally projecting retinal ganglion cells (IPRGCs) decreased continuously from more than 3000 cells on the day of birth (day 0) to slightly more than 1000 on postnatal day 5. In contrast, the number of IPRGCs in rats which received either unilateral eye enucleation or thalamectomy at birth increased abruptly to more than 4000 after 24 h on postnatal day 1, thereafter the number decreased rapidly reaching an adult level of slightly more than 2000 on postnatal day 5. The overall pattern of changes of the number, density and distribution of the IPRGCs was similar in rats which received eye enucleation or thalamectomy, although minor differences regarding the number of cells labelled in the temporal and nasal parts of retinas in rats with different lesions were detected. These findings imply that a neonatal lesion such as monocular enucleation or unilateral thalamectomy may not only result in retention of normally transient IPRGCs but also cause an increased number of IPRGCs by misrouting, rerouting or collateral sprouting of optic axons at the optic chiasma that could significantly affect the developing optic pathway. Furthermore, the differential effects of the two types of neonatal lesions upon the IPRGCs provide further evidence to previous findings that different mechanisms might be involved in generating the anomalously enlarged uncrossed retinofugal projections in rodents.     

Serotonin immunoreactivity in the retinal projecting isthmo-optic nucleus and evidence of brainstem raphe connections in the pigeon

Serotonin (5-HT) immunoreactive (-ir) profiles within the isthmo-optic nucleus (ION) of the centrifugal visual system (CVS) were studied in the pigeon using light microscopic immunohistofluorescent and electron microscopic immunocytochemical pre-embedding techniques. The brainstem origin of the 5-HT input upon the ION was determined by combining 5-HT immunohistofluorescence (FITC) and retrograde transneuronal tracing after intraocular injection of Rhodamine beta-isothiocyanate. The light microscopic results showed that 5-HT endings were mainly localised within the neuropillar zones of the ventral ION. The 5-HT-ir cell bodies, belonging to a lateral extension of the dorsal raphe system, were observed within the same region as the centrifugal ectopic neurons (EN) underlying the ION and some displayed dendritic processes which penetrated the nucleus. Double-labeled neurons, representing 5-HT-ir afferents to the ION, were identified only within the n. linearis caudalis region of the ventral raphe. The electron microscopic results confirmed the presence of 5-HT-ir dendritic processes within the ventral part of the nucleus and showed that they were contacted by axon terminals belonging to intrinsic interneurons. The functional organisation of the ION and the possible contribution of serotonergic raphe afferents and efferents are discussed in relation to present hypotheses linking the avian CVS to mechanisms of visual attention.     

Cell groups in the lower brain stem of the rabbit projecting to the spinal cord, with special reference to catecholamine-containing neurons

Two groups of experiments were carried out in rabbits. In the first groups, the distribution of cell bodies within the pons and medulla projecting ipsilaterally and contralaterally to the thoracic or lumbar spinal cord was studied using the horseradish peroxidase (HRP)/tetramethylbenzidine (TMB) procedure. In the second group, both a previously described double-labeling technique and a new modification of it were used to determine the location of catecholamine (CA)-fluorescent pontomedullary cells projecting to the spinal cord. The results demonstrate that the catecholamine (probably norepinephrine)-containing neurons which innervate the thoracic spinal cord are confirmed almost exclusively to the pons where they were found within the A5, A7 and subcoeruleus groups, as well as the ventral portion of the principal part of the locus coeruleus and the more caudal locus coeruleus, including the A4 cell group. Within the medulla oblongata no doubly labeled A2 cells were observed and the few double labeled A1 cells which were observed were confined to the rostral portion of this group. A dense group of HRP-positive but non-fluorescent cells was found rostral to the A1 area in the ventrolateral reticular formation. These cells, which correspond in position to PNMT-containing cells in the rat, appear to project to both thoracic and lumbar segments of the spinal cord. In contrast, spinally projecting neurons within the nucleus tractus solitarius originated from different subnuclei according to their segmental destination. New information about the organization of medial reticulospinal and vestibulospinal pathways was also obtained.     

Electronmicroscopic study of somatostatin-containing neurons in rat arcuate nucleus with special reference to neuronal regulation

After an intraventricular administration of colchicine, the arcuate nucleus of rat hypothalamus was examined light and electron microscopically by pre-embedding immunohistochemistry for somatostatin. The arcuate nucleus exhibited numerous immunoreactive cell bodies and dense networks of immunoreactive fibers. The fibers appeared to surround immunonegative cell bodies. The immunoreactive cell bodies were multipolar in shape and projected immunoreactive processes to some extent. The immunoreactive cell bodies and fibers received synaptic contacts by immunonegative fiber terminals containing a large number of synaptic clear vesicles. Similarly, immunoreactive somatostatin fibers appeared to terminate upon other immunonegative cell bodies and fibers. The immunoreactive presynaptic terminals contain several labeled granules and numerous synaptic vesicles. In close proximity to these immunolabeled terminals, non-labeled presynaptic fibers. This suggests that in the arcuate nucleus neurons regulated by somatostatin neurons are also under the control of other types of neurons.     

Peptidergic and aminergic innervation of the facial nucleus of the rat with special reference to ontogenetic development

The distribution and ontogenetic development of several neuropeptides such as enkephalin, substance P, somatostatin, neuropeptide Y, and of monoamines such as serotonin and catecholamines in the facial nucleus of the rat were investigated with immunocytochemistry. The neuropeptides were concentrated in certain subnuclei. Enkephalin-immunoreactive fibers were distributed in the medial and dorsal subnuclei, substance P in the intermediate and dorsal subnuclei, somatostatin in the intermediate subnucleus, and neuropeptide Y in the dorsal subnucleus. The amines were distributed evenly throughout the nucleus. These distribution patterns suggest that peptidergic fibers are closely related to the functions of different subnuclei, while fibers containing monoamines are more basic--not specific to individual muscles. Few of these fibers were observed in the prenatal stage of the rat, but they increased markedly in number during the first postnatal week, and had established their innervation pattern by the tenth postnatal day, which coincides with the establishment of nerve-muscle innervation. The present study further showed that fibers containing serotonin are supplied mainly from the raphe nucleus, that catecholamine fibers are from neurons containing catecholamine surrounding the facial nucleus, and that fibers containing neuropeptide Y are from the lateral part of the caudal medullary reticular formation. These findings suggest that catecholamine and neuropeptide Y are not both present in the single neurons projecting to the facial nucleus.     

Properties of velocity-mechanosensitive neurons of the cat ventrobasal thalamic nucleus with special reference to the concept of convergence

Neurons in the ventrobasal (VB) thalamic nucleus of lightly anesthetized cats were studied in order to analyze their discharge properties in response to controlled mechanical stimuli. Properties of the vast majority of the neuronal population largely resemble those of peripheral sensitive mechanoreceptors in their response to the velocity and, to a more limited extent, the amplitude component, of skin and hair displacement within restricted receptive fields. Detailed examination reveals some ‘complex’ characteristics suggestive of central integration in about 11% of VB neurons. Complex properties appear to indicate convergent input reflecting receptive field organization, the variety of velocity-related discharges and patterns of inhibition. The rarity of isolated ‘surround’ inhibition and the definition of what constitutes ‘lemniscal’ properties are discussed in the context of these and other related findings.     

Visual pretectal neurons projecting to the dorsal lateral geniculate nucleus and pulvinar nucleus in the cat

We observed morphological subtypes of visual pretectal neurons ascending to the dorsal thalamus, following injections of wheat germ agglutinin conjugated to horseradish peroxidase into the dorsal lateral geniculate nucleus (LGNd) or the pulvinar nucleus. These neurons are composed of fusiform cells and small-sized multipolar cells in the olivary pretectal nucleus, superficial horizontal cells, fusiform cells, small-, medium- and large-sized multipolar cells in the optic tract nucleus, and small- and medium-sized multipolar cells in the posterior pretectal nucleus. When somal size of the neurons projecting to the LGNd was compared to the size of neurons projecting to the pulvinar, the neuronal groups were not identical.     

Thalamic innervation of striatal and subthalamic neurons projecting to the rat entopeduncular nucleus

The present study analyses the anatomical arrangement of the projections linking the Wistar rat parafascicular thalamic nucleus (PF) and basal ganglia structures, such as the striatum and the subthalamic nucleus (STN), by using neuroanatomical tract-tracing techniques. Both the thalamostriatal and the striato-entopeduncular projections were topographically organized, and several areas of overlap between identified circuits were noticed, sustaining the existence of up to three separated channels within the Nauta-Mehler loop. Thalamic afferents arising from dorsolateral PF territories are in register with striatofugal neurons located in dorsolateral striatal areas, which in turn project to dorsolateral regions of the entopeduncular nucleus (ENT). Medial ENT regions are innervated by striatal neurons located within medial striatal territories, these neurons being the target for thalamic afferents coming from medial PF areas. Finally, afferents from neurons located in ventrolateral PF areas approached striatal neurons in ventral and lateral striatal territories, which in turn project towards ventral and lateral ENT regions. Efferent STN neurons projecting to ENT were found to be the apparent postsynaptic target for thalamo-subthalamic axons. The thalamo-subthalamic projection was also topographically organized. Medial, central and lateral STN territories are innervated by thalamic neurons located within medial, ventrolateral and dorsolateral PF areas, respectively. Thus, each individual PF subregion projects in a segregated fashion to specific parts of the striato-entopeduncular and subthalamo-entopeduncular systems. These circuits enabled the caudal intralaminar nuclei to modulate basal ganglia output.     

Responses of subfornical organ neurons projecting to the hyphotalamic paraventricular nucleus to hemorrhage

The activity of subfornical organ (SFO) neurons that were antidromically identified by electrical stimulation of the rat hypothalamic paraventricular nucleus (PVN) was tested for a response to microiontophoretic application of angiotensin II (ANG II) or hemorrhage (10 ml/kg b.v.t.). Microiontophoretically (MIPh) applied ANG II caused an increased excitability in 24 out of 28 neurons tested and the excitation was blocked by MIPh-applied saralasin (Sar), a specific ANG II antagonist. Of these neurons that responded to ANG II, 14 displayed an increase in neuronal firing in response to hemorrhage, while 10 were unresponsive. The excitatory response to hemorrhage in 5 out of 14 neurons tested was prevented by MIPh-applied Sar, whereas the response of the remaining neurons was not affected. These results show that part of SFO neurons projecting to the PVN may receive neural inputs from the peripheral baroreceptors, and suggest that the inputs may be partially attributable to the involvement of central angiotensinergic circuits.     

Activation of neurons projecting to the paraventricular hypothalamic nucleus by intravenous lipopolysaccharide

The central nervous system interacts with the immune system to coordinate several components of the acute phase response, although the specific neuroanatomical pathways that mediate these responses are still uncharacterized. However, neurons in both the autonomic and endocrine components of the paraventricular hypothalamic nucleus (PVH) are characteristically activated in different models of immune stimulation. In the current study, we have used intravenous administration of lipopolysaccharide (LPS; 5 or 125 μg/kg) to induce the acute phase response. We subsequently coupled immunohistochemistry for Fos (as a marker of neuronal activation) with retrograde transport of the neuroanatomical tracer cholera toxin-b from the PVH. Several of the activated cell groups directly projected to the paraventricular nucleus, including the visceromotor (infralimbic) cortex, median preoptic nucleus, ventrome-dial preoptic area, bed nucleus of the stria terminalis, parabrachial nucleus, ventrolateral medulla, and nucleus of the solitary tract. These findings indicate that immune system stimulation activates cell groups from multiple nervous system levels that project to the paraventricular nucleus. We hypothesize that the activation of specific autonomic and endocrine elements of the PVH may be due to the activity of distinct afferents that converge on the PVH from multiple components of the central autonomic control system. Our results are consistent with the hypothesis that the PVH plays a key role in integrating diverse physiological cues into the varied manifestations that constitute the cerebral component of the acute phase response. © 1996 Wiley-Liss, Inc.     

Anatomical and physiological properties of ipsilaterally projecting spinothalamic neurons in the second cervical segment of the cat's spinal cord

Anatomical and electrophysiological methods were used to investigate the projections and response properties of neurons in the second cervical (C2) spinal segment of the cat giving origin to a previously undescribed projection to the ipsilateral thalamus. The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify neurons in C2 giving rise to thalamic projections. Following large (3.0 μl) thalamic HRP injections, a large numbers of labeled neurons was observed in lateral laminae VII–VIII of C2 ipsilateral to the injections. They occurred as small clusters of cells along the longitudinal axis of C2. Labeled neurons were also observed contralaterally in the lateral cervical nucleus, dorsal horn (especially medial lamina VI), and loosely distributed in the ventral horn. The ipsilaterally projecting neurons were also labeled following small (0.2–0.5 μl) HRP injections restricted to individual spinothalamic terminal zones (intralaminar nuclei, ventrobasal complex-nucleus ventralis lateralis border zone, medial division of the posterior nuclei), indicating that as a group they project widely throughout the thalamus. Single unit recording methods were used to obtain complementary information on the functional properties of these neurons. The antidromic stimulation method was applied to identify units in C2 projecting to the ipsilateral thalamus in anesthetized, paralyzed cats. Three categories of ipsilaterally projecting C2 units were identified: (i) units not driven by any type of natural stimulation; (2) units having large cutaneous receptive fields (RFs) and wide dynamic response ranges (“widefield”), and (3) units with smaller RFs and varied properties (“other”). Widefield units with bilaterally symmetrical and asymmetrical RFs were observed. Co-stimulation of different portions of an excitatory RF produced summation of the unit response. Inhibitory RF components were identified in one third of the widefield units. Unit recordings after spinal tract lesions revealed that the afferent input passed via the ipsilateral lateral and/or ventral funiculi. Widefield unit responses to somatosensory stimuli could be inhibited by dorsal column conditioning stimulation. Several “other” units resembled widefield units, while a second group had small RFs restricted to the C2 dermatome. Possible functional roles of the projecting C2 neurons in somatosensory and non-specific systems are discussed.     

Circadian rhythms and energy metabolism with special reference to the suprachiasmatic nucleus

The role of the hypothalamic suprachiasmatic nucleus (SCN) was examined in rats and obtained following results: (a) The time-dependent (light > dark) hyperglycemic response to intracranial injection of 2-deoxy-d-glucose (2DG) disappeared in rats with bilateral lesions of the SCN, in rats on weeks 4–6 after surgical blinding, and in congenitally blind (hereditary microphthalmic) rats; (b) The hyperglycemia induced by electrical stimulation of the SCN was not observed in weeks 4–8 after surgical blinding; (c) Change in the blood glucose concentration after insulin injection into the SCN was eliminated by SCN lesions; (d) Alterations in activity of autonomic efferents to peripheral organs on light exposure disappeared after SCN lesions; (e) SCN lesions decreased the blood glucagon level and increased the blood insulin level; (f) SCN lesions decreased protein intake, and glucagon increased it; (g) Increases in the plasma renin activity and vasopressin concentration after water-deprivation were suppressed in hereditary microphthalmic rats with abnormal SCN. These findings suggest that the SCN is involved in the mechanism of blood glucose and body fluid intake as well as that of circadian rhythm.     

Circadian rhythms and energy metabolism with special reference to the suprachiasmatic nucleus

The role of the hypothalamic suprachiasmatic nucleus (SCN) was examined in rats and obtained following results: (a) The time-dependent (light > dark) hyperglycemic response to intracranial injection of 2-deoxy- -glucose (2DG) disappeared in rats with bilateral lesions of the SCN, in rats on weeks 4–6 after surgical blinding, and in congenitally blind (hereditary microphthalmic) rats; (b) The hyperglycemia induced by electrical stimulation of the SCN was not observed in weeks 4–8 after surgical blinding; (c) Change in the blood glucose concentration after insulin injection into the SCN was eliminated by SCN lesions; (d) Alterations in activity of autonomic efferents to peripheral organs on light exposure disappeared after SCN lesions; (e) SCN lesions decreased the blood glucagon level and increased the blood insulin level; (f) SCN lesions decreased protein intake, and glucagon increased it; (g) Increases in the plasma renin activity and vasopressin concentration after water-deprivation were suppressed in hereditary microphthalmic rats with abnormal SCN. These findings suggest that the SCN is involved in the mechanism of blood glucose and body fluid intake as well as that of circadian rhythm.     

Identification of prepositus neurons projecting to the oculomotor nucleus in the alert cat

A population of prepositus hypoglossi nucleus neurons with discharges correlated to gaze parameters and antidromically activated by stimulation of the ipsilateral oculomotor nucleus has been recorded in the alert cat. The latency for antidromic invasion was of 0.6–0.8 msec. Neuronal firing rate encoded eye position and eye velocity in the horizontal plane, showing in all cases ipsilateral ‘on’ direction.     

Morphological and functional properties of trigeminal nucleus oralis neurons projecting to the trigeminal motor nucleus of the cat

Horseradish peroxidase (HRP) was injected into the somata located in the rostrodorsomedial part (Vo.r) of the trigeminal nucleus oralis; an axonal projection to the trigeminal motor nucleus (Vmo) was demonstrated in two Vo.r neurons. The two neurons differed in their morphological and functional properties. The first Vo.r neuron responded to stimulation of low-threshold mechanoreceptors and its stem axon gave off massive axon collaterals that issued terminal branches to the dorsolateral subdivision of Vmo, Vo.r, and the medial and lateral parts of the lower brainstem reticular formation. The second Vo.r neuron was activated by stimulation of the tooth pulp or lingual nerve at twice longer latency than that of the first neuron. This stem axon was divided into two main ascending and one descending branches, and one of the main ascending branches was further bifurcated into two branches. The main non-bifurcated ascending branch gave off 4 collaterals, two of which sent terminal branches into the dorsolateral subdivision of Vmo and others into the Vo.r and juxta-trigeminal regions. The somato-dendroarchitectonic differences were also described in the two Vo.r neurons stained.     

Location of brain stem neurons projecting to the oculomotor nucleus in the cat

Horseradish peroxidase was injected into the oculomotor nucleus of 11 adult cats and the animals were killed after 48 h. In 7 cats satisfactory labeling occurred in neurons in the superior, medial, and lateral vestibular nuclei, the abducens nucleus, the infracerebellar nucleus, nucleus praepositus hypoglossi, and nucleus interpositus. These retrograde studies confirmed the major vestibulo-ocular pathways shown by anterograde degeneration methods and demonstrated additional projections to the third nerve nucleus which may be activated by peripheral vestibular input.     

An electrophysiological study of medullary neurons projecting to nucleus parabrachialis of the cat

Neuronal activity was recorded from a total of 868 cells in the medullas of cats in regions known to project to nucleus parabrachialis (NPB) of the pons and thought to be involved in respiration. Of 196 neurons antidromically (85) or synaptically (111) activated from NPB, 178 were silent in the decerebrate or barbiturate-anesthetized preparation, and could not be induced to activity by increasing CO₂, activating Breuer-Hering reflexes, stimulation of the vagus or vagotomy. We conclude that the direct pathway from the lateral tegmental field of the medulla to NPB is not significantly involved in respiratory function in these preparations.