Restoration of mitochondria axonal transport by adaptor Disc1 supplementation prevents neurodegeneration and rescues visual function.

Deficits in mitochondrial transport are a common feature of neurodegenerative diseases. We investigated whether loss of components of the mitochondrial transport machinery impinge directly on metabolic stress, neuronal death, and circuit dysfunction. Using multiphoton microscope live imaging, we showed that ocular hypertension, a major risk factor in glaucoma, disrupts mitochondria anterograde axonal transport leading to energy decline in vulnerable neurons. Gene- and protein-expression analysis revealed loss of the adaptor disrupted in schizophrenia 1 (Disc1) in retinal neurons subjected to high intraocular pressure. Disc1 gene delivery was sufficient to rescue anterograde transport and replenish axonal mitochondria. A genetically encoded ATP sensor combined with longitudinal live imaging showed that Disc1 supplementation increased ATP production in stressed neurons. Disc1 gene therapy promotes neuronal survival, reverses abnormal single-cell calcium dynamics, and restores visual responses. Our study demonstrates that enhancing anterograde mitochondrial transport is an effective strategy to alleviate metabolic stress and neurodegeneration.

Differential patterns of inputs create functional zones in central nucleus of inferior colliculus.

Distinct pathways carry monaural and binaural information from the lower auditory brainstem to the central nucleus of the inferior colliculus (ICC). Previous anatomical and physiological studies suggest that differential ascending inputs to regions of the ICC create functionally distinct zones. Here, we provide direct evidence of this relationship by combining recordings of single unit responses to sound in the ICC with focal, iontophoretic injections of the retrograde tracer Fluoro-Gold at the physiologically characterized sites. Three main patterns of anatomical inputs were observed. One pattern was identified by inputs from the cochlear nucleus and ventral nucleus of the lateral lemniscus in isolation, and these injection sites were correlated with monaural responses. The second pattern had inputs only from the ipsilateral medial and lateral superior olive, and these sites were correlated with interaural time difference (ITD)-sensitive responses to low frequency (<500 Hz). A third pattern had inputs from a variety of olivary and lemniscal sources, notably the contralateral lateral superior olive and dorsal nucleus of the lateral lemniscus. These were correlated with high-frequency ITD sensitivity to complex acoustic stimuli. These data support the notion of anatomical regions formed by specific patterns of anatomical inputs to the ICC. Such synaptic domains may represent functional zones in ICC.

Functional recovery of stepping in rats after a complete neonatal spinal cord transection is not due to regrowth across the lesion site.

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage.

Contralesional axonal remodeling of the corticospinal system in adult rats after stroke and bone marrow stromal cell treatment.

BACKGROUND AND PURPOSE: Motor recovery after stroke is associated with neuronal reorganization in bilateral hemispheres. We investigated contralesional corticospinal tract remodeling in the brain and spinal cord in rats after stroke and treatment of bone marrow stromal cells. METHODS: Adult male Wistar rats were subjected to permanent right middle cerebral artery occlusion. Phosphate-buffered saline or bone marrow stromal cells were injected into a tail vein 1 day postischemia. An adhesive removal test was performed weekly to monitor functional recovery. Threshold currents of intracortical microstimulation on the left motor cortex for evoking bilateral forelimb movements were measured 6 weeks after stroke. When intracortical microstimulation was completed, biotinylated dextran amine was injected into the left motor cortex to anterogradely label the corticospinal tract. At 4 days before euthanization, pseudorabies virus-152-EGFP and 614-mRFP were injected into left or right forelimb extensor muscles, respectively. All animals were euthanized 8 weeks after stroke. RESULTS: In normal rats (n=5), the corticospinal tract showed a unilateral innervation pattern. In middle cerebral artery occlusion rats (n=8), our data demonstrated that: 1) stroke reduced the stimulation threshold evoking ipsilateral forelimb movement; 2) EGFP-positive pyramidal neurons were increased in the left intact cortex, which were labeled from the left stroke-impaired forelimb; and 3) biotinylated dextran amine-labeled contralesional axons sprouted into the denervated spinal cord. Bone marrow stromal cells significantly enhanced all 3 responses (n=8, P<0.05). CONCLUSIONS: Our data demonstrated that corticospinal tract fibers originating from the contralesional motor cortex sprout into the denervated spinal cord after stroke and bone marrow stromal cells treatment, which may contribute to functional recovery.

Sensory sciatic nerve afferent inputs to the dorsal lateral medulla in the rat.

Investigations show the paratrigeminal nucleus (Pa5) as an input site for sensory information from the sciatic nerve field. Functional or physical disruption of the Pa5 alters behavioral and somatosensory responses to nociceptive hindpaw stimulation or sciatic nerve electrostimulation (SNS), both contralateral to the affected structure. The nucleus, an input site for cranial and spinal nerves, known for orofacial nociceptive sensory processing, has efferent connections to structures associated with nociception and cardiorespiratory functions. This study aimed at determining the afferent sciatic pathway to dorsal lateral medulla by means of a neuronal tract-tracer (biocytin) injected in the iliac segment of the sciatic nerve. Spinal cord samples revealed bilateral labeling in the gracile and pyramidal or cuneate tracts from survival day 2 (lumbar L1/L2) to day 8 (cervical C2/C3 segments) following biocytin application. From day 10 to day 20 medulla samples showed labeling of the contralateral Pa5 to the injection site. The ipsilateral paratrigeminal nucleus showed labeling on day 10 only. The lateral reticular nucleus (LRt) showed fluorescent labeled terminal fibers on day 12 and 14, after tracer injection to contralateral sciatic nerve. Neurotracer injection into the LRt of sciatic nerve-biocytin-treated rats produced retrograde labeled neurons soma in the Pa5 in the vicinity of biocytin labeled nerve terminals. Therefore, Pa5 may be considered one of the first sites in the brain for sensory/nociceptive inputs from the sciatic nerve. Also, the findings include Pa5 and LRt in the neural pathway of the somatosympathetic pressor response to SNS and nocifensive responses to hindpaw stimulation.

Role of the dorsal paragigantocellular reticular nucleus in paradoxical (rapid eye movement) sleep generation: a combined electrophysiological and anatomical study in the rat.

It is well known that noradrenergic locus coeruleus neurons decrease their activity during slow wave sleep and are quiescent during paradoxical sleep. It was recently proposed that their inactivation during paradoxical sleep is due to a tonic GABAergic inhibition arising from neurons located into the dorsal paragigantocellular reticular nucleus (DPGi). However, the discharge profile of DPGi neurons across the sleep-waking cycle as well as their connections with brain areas involved in paradoxical sleep regulation remain to be described. Here we show, for the first time in the unanesthetized rat that the DPGi contained a subtype of neurons with a tonic and sustained firing activation specifically during paradoxical sleep (PS-on neurons). Noteworthy, their firing rate increase anticipated for few seconds the beginning of the paradoxical sleep bout. By using anterograde tract-tracing, we further showed that the DPGi, in addition to locus coeruleus, directly projected to other areas containing wake-promoting neurons such as the serotonergic neurons of the dorsal raphe nucleus and hypocretinergic neurons of the posterior hypothalamus. Finally, the DPGi sent efferents to the ventrolateral part of the periaqueductal gray matter known to contain paradoxical sleep-suppressing neurons. Taken together, our original results suggest that the PS-on neurons of the DPGi may have their major role in simultaneous inhibitory control over the wake-promoting neurons and the permissive ventrolateral part of the periaqueductal gray matter as a means of influencing vigilance states and especially PS generation.

Multisensory processing via early cortical stages: Connections of the primary auditory cortical field with other sensory systems.

It is still a popular view that primary sensory cortices are unimodal, but recent physiological studies have shown that under certain behavioral conditions primary sensory cortices can also be activated by multiple other modalities. Here, we investigate the anatomical substrate, which may underlie multisensory processes at the level of the primary auditory cortex (field AI), and which may, in turn, enable AI to influence other sensory systems. We approached this issue by means of the axonal transport of the sensitive bidirectional neuronal tracer fluorescein-labeled dextran which was injected into AI of Mongolian gerbils (Meriones unguiculatus). Of the total number of retrogradely labeled cell bodies (i.e. cells of origin of direct projections to AI) found in non-auditory sensory and multisensory brain areas, approximately 40% were in cortical areas and 60% in subcortical structures. Of the cell bodies in the cortical areas about 82% were located in multisensory cortex, viz., the dorsoposterior and ventroposterior, posterior parietal cortex, the claustrum, and the endopiriform nucleus, 10% were located in the primary somatosensory cortex (hindlimb and trunk region), and 8% in secondary visual cortex. The cortical regions with retrogradely labeled cells also contained anterogradely labeled axons and their terminations, i.e. they are also target areas of direct projections from AI. In addition, the primary olfactory cortex was identified as a target area of projections from AI. The laminar pattern of corticocortical connections suggests that AI receives primarily cortical feedback-type inputs and projects in a feedforward manner to its target areas. Of the labeled cell bodies in the subcortical structures, approximately 90% were located in multisensory thalamic, 4% in visual thalamic, and 6% in multisensory lower brainstem structures. At subcortical levels, we observed a similar correspondence of retrogradely labeled cells and anterogradely labeled axons and terminals in visual (posterior limitans thalamic nucleus) and multisensory thalamic nuclei (dorsal and medial division of the medial geniculate body, suprageniculate nucleus, posterior thalamic cell group, zona incerta), and in the multisensory nucleus of the brachium of the inferior colliculus. Retrograde, but not anterograde, labeling was found in the multisensory pontine reticular formation, particularly in the reticulotegmental nucleus of the pons. Conversely, anterograde, but no retrograde, labeling was found in the visual laterodorsal and lateroposterior thalamic nuclei, in the multisensory peripeduncular, posterior intralaminar, and reticular thalamic nuclei, as well as in the multisensory superior and pericentral inferior colliculi (including cuneiform and sagulum nucleus), pontine nuclei, and periaqueductal gray. Our study supports the notion that AI is not merely involved in the analysis of auditory stimulus properties but also in processing of other sensory and multisensory information. Since AI is directly connected to other primary sensory cortices (viz. the somatosensory and olfactory ones) multisensory information is probably also processed in these cortices. This suggests more generally, that primary sensory cortices may not be unimodal.

Distribution of hypothalamic, hippocampal and other limbic peptidergic neuronal cell bodies giving rise to retinopetal fibers: anterograde and retrograde tracing and neuropeptide immunohistochemical studies.

In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the vitreous body resulted in nerve cell body labeling in several structures: the supraoptic and paraventricular nuclei, the hippocampus (CA1, CA3), the dentate gyrus, the indusium griseum, the olfactory tubercle, and the medial habenula, all of them belong to the limbic system. We estimated that the total number of retrogradely labeled cells is 1495+/-516. We have seen fiber labeling in the retinorecipient suprachiasmatic nucleus and in the primary visual center, the lateral geniculate body, but labeled nerve cell bodies in these structures were never seen. Iontophoretic application of Fluorogold into the hippocampal formation, where the major part of the biotinylated dextran amine-labeled cell bodies was observed, resulted in labeled fibers in the optic nerve and in the retina indicating that the retrogradely labeled cells in the hippocampus and the dentate gyrus among others are the cells of origin of the centrifugal visual fibers. Sections showing biotinylated dextran amine labeling were stained for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity using immunohistochemistry. Some biotinylated dextran amine-labeled cells also showed vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity. We conclude that the limboretinal pathway exists and that the cells of origin are partially vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactive.

Brain projections from the medullary dorsal reticular nucleus: an anterograde and retrograde tracing study in the rat.

In the last 15 years a role has been ascribed for the medullary dorsal reticular nucleus as a supraspinal pain modulating area. The medullary dorsal reticular nucleus is reciprocally connected with the spinal dorsal horn, is populated mainly by nociceptive neurons and regulates spinal nociceptive processing. Here we analyze the distribution of brain projections from the medullary dorsal reticular nucleus using the iontophoretic administration of the anterograde tracer biotinylated-dextran amine and the retrograde tracer cholera toxin subunit B. Fibers and terminal boutons labeled from the medullary dorsal reticular nucleus were located predominately in the brainstem, although extending also to the forebrain. In the medulla oblongata, anterograde labeling was observed in the orofacial motor nuclei, inferior olive, caudal ventrolateral medulla, rostral ventromedial medulla, nucleus tractus solitarius and most of the reticular formation. Labeling at the pons-cerebellum level was present in the locus coeruleus, A5 and A7 noradrenergic cell groups, parabrachial and deep cerebellar nuclei, whereas in the mesencephalon it was located in the periaqueductal gray matter, deep mesencephalic, oculomotor and anterior pretectal nuclei, and substantia nigra. In the diencephalon, fibers and terminal boutons were found mainly in the parafascicular, ventromedial, and posterior thalamic nuclei and in the arcuate, lateral, posterior, peri- and paraventricular hypothalamic areas. Telencephalic labeling was consistent but less intense and concentrated in the septal nuclei, globus pallidus and amygdala. The well-known role of the medullary dorsal reticular nucleus in nociception and its pattern of brain projections in rats suggests that the nucleus is possibly implicated in the modulation of: (i) the ascending nociceptive transmission involved in the motivational-affective dimension of pain; (ii) the endogenous supraspinal pain control system centered in the periaqueductal gray matter-rostral ventromedial medulla-spinal cord circuitry; (iii) the motor reactions associated with pain.

Traumatic axonal injury in the perisomatic domain triggers ultrarapid secondary axotomy and Wallerian degeneration.

Traumatic axonal injury (TAI) arising from diffuse brain injury (DBI) results in focally impaired axonal transport with progressive swelling and delayed disconnection over several hours within brainstem axons. Neocortical DBI-mediated perisomatic axotomy does not result in neuronal death, suggesting that a comparably delayed axotomy progression was responsible for this unanticipated response. To evaluate delayed perisomatic axotomy, the current study was initiated. Rats received intracerebroventricular 10-kDa dextran followed by moderate midline/central fluid percussion injury (FPI) or FPI alone. At 15, 30, 60, and 180 min post-injury, light and transmission electron microscopy identified impaired axonal transport via antibodies targeting amyloid precursor protein (APP), while double-label fluorescent microscopy explored concomitant focal axolemmal alterations via dextran-APP co-localization. At 15 min post-injury, perisomatic TAI was identified with LM within dorsolateral and ventral posterior thalamic nuclei. Using TEM, many sustaining somata and related proximal/distal axonal segments revealed normal ultrastructural detail that was continuous with focal axonal swellings characterized by cytoskeletal and organelle pathology. In other cases, axotomy was confirmed by loss of axonal continuity distal to the swelling. By 30 min post-injury, perisomatic axotomy predominated. By 60-180 min, somatic, proximal axonal segment, and swelling ultrastructure were comparable to earlier time points although swelling diameter increased. Distal axonal segment ultrastructure now revealed the initial stages of Wallerian degeneration. The site of perisomatic axotomy did not internalize dextran, suggesting that its pathogenesis occurred independent of altered axolemmal permeability. Collectively, this DBI-mediated ultrarapid perisomatic axotomy and its sequelae further illustrate the varied axonal responses to trauma.

Reconfiguration of multiple motor networks by short- and long-term actions of an identified modulatory neuron.

The pyloric and gastric motor pattern-generating networks in the stomatogastric ganglion of the lobster Homarus gammarus are reconfigured into a new functional circuit by burst discharge in an identified pair of modulatory projection interneurons, originally named the pyloric suppressor (PS) neurons because of their inhibitory effects on pyloric network activity. Here we elucidate the actions of the PS neurons on individual members of the neighbouring gastric circuit, as well as describing their ability to alter synaptic coupling between the two networks. PS neuron firing has two distinct effects on gastric network activity: an initial short-lasting action mediated by transient inhibition of most gastric motoneurons, followed by a long-lasting circuit activation associated with a prolonged PS-evoked depolarization of the medial gastric (MG) motoneuron and the single network interneuron, Int1. These long-lasting effects are voltage-dependent, and experiments with hyperpolarizing current injection and photoablation suggest that excitation of both the MG neuron and Int1 is critical for PS-elicited gastric network rhythmicity. In parallel, PS neuron discharge persistently (lasting several minutes) enhances the strength of an inhibitory synaptic influence of the MG neuron on the pyloric dilator (PD)-anterior burster (AB) pacemaker neurons, thereby facilitating operational fusion of the two networks. Therefore, a single modulatory neuron may influence disparate populations of neurons via a range of very different and highly target-specific mechanisms: conventional transient synaptic drive and up- or down-modulation of membrane properties and synaptic efficacy. Moreover, distinctly different time courses of these actions allow different circuit configurations to be specified sequentially by a given modulatory input.

Visual thalamocortical projections in the flying fox: parallel pathways to striate and extrastriate areas.

We studied thalamic projections to the visual cortex in flying foxes, animals that share neural features believed to resemble those present in the brains of early primates. Neurones labeled by injections of fluorescent tracers in striate and extrastriate cortices were charted relative to the architectural boundaries of thalamic nuclei. Three main findings are reported: First, there are parallel lateral geniculate nucleus (LGN) projections to striate and extrastriate cortices. Second, the pulvinar complex is expansive, and contains multiple subdivisions. Third, across the visual thalamus, the location of cells labeled after visual cortex injections changes systematically, with caudal visual areas receiving their strongest projections from the most lateral thalamic nuclei, and rostral areas receiving strong projections from medial nuclei. We identified three architectural layers in the LGN, and three subdivisions of the pulvinar complex. The outer LGN layer contained the largest cells, and had strong projections to the areas V1, V2 and V3. Neurones in the intermediate LGN layer were intermediate in size, and projected to V1 and, less densely, to V2. The layer nearest to the origin of the optic radiation contained the smallest cells, and projected not only to V1, V2 and V3, but also, weakly, to the occipitotemporal area (OT, which is similar to primate middle temporal area) and the occipitoparietal area (OP, a "third tier" area located near the dorsal midline). V1, V2 and V3 received strong projections from the lateral and intermediate subdivisions of the pulvinar complex, while OP and OT received their main thalamic input from the intermediate and medial subdivisions of the pulvinar complex. These results suggest parallels with the carnivore visual system, and indicate that the restriction of the projections of the large- and intermediate-sized LGN layers to V1, observed in present-day primates, evolved from a more generalized mammalian condition.

In vivo dynamic ME-MRI reveals differential functional responses of RA- and area X-projecting neurons in the HVC of canaries exposed to conspecific song.

HVC (nidopallial area, formerly known as hyperstriatum ventrale pars caudalis), a key centre for song control in oscines, responds in a selective manner to conspecific songs as indicated by electrophysiology. However, immediate-early gene induction cannot be detected in this nucleus following song stimulation. HVC contains neurons projecting either towards the nucleus robustus archistriatalis (RA; motor pathway) or area X (anterior forebrain pathway). Both RA- and area X-projecting cells show auditory responses. The present study analysed these responses separately in the two types of HVC projection neurons of canaries by a new in vivo approach using manganese as a calcium analogue which can be transported anterogradely and used as a paramagnetic contrast agent for magnetic resonance imaging (MRI). Manganese was stereotaxically injected into HVC and taken up by HVC neurons. The anterograde axonal transport of manganese from HVC to RA and area X was then followed by MRI during approximately 8 h and changes in signal intensity in these targets were fitted to sigmoid functions. Data comparing birds exposed or not to conspecific songs revealed that song stimulation specifically affected the activity of the two types of HVC projection neurons (increase in the sigmoid slope in RA and in its maximum signal intensity in area X). Dynamic manganese-enhanced MRI thus allows assessment of the functional state of specific neuronal populations in the song system of living canaries in a manner reminiscent of functional MRI (but with higher resolution) or of 2-deoxyglucose autoradiography (but in living subjects).

Distribution of androgen-binding protein in the rat hypothalamo-neurohypophyseal system, co-localization with oxytocin.

Androgen-binding protein (ABP) is known to be expressed in the male and female rat hypothalamus. In the present study, we observed immunocytochemically ABP in neurons of the magnocellular hypothalamic nuclei, in the preoptic region and in the lateral hypothalamus. Dense fiber networks with varicosities, containing ABP immunofluorescence, were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed a partial coexistence of ABP-and oxytocin immunoreactivity in a portion of the magnocellular perikarya. ABP was isolated by affinity chromatography from hypothalamus homogenates. Western blots resulted in immunoreactive (IR) bands with an approximate molecular weight of 35 and 50 kDa. Mass spectrometry of these preparations confirmed the presence of ABP, which was almost identical to ABP isolated from rat testis. It is likely that ABP, expressed in magnocellular oxytocinergic neurons, is subject to axonal transport and release in the hypothalamo-neurohypophyseal system.

Postnatal development of the somatosensory thalamocortical projection in rat and rabbit--a combined retrograde transport and stereological comparative study.

A comparative quantitative study of the somatosensory thalamocortical connections in the rat and rabbit, labeled with the fluorescent retrograde tracer Fluoro-Gold (FG), was conducted by means of unbiased stereology. FG was injected into the primary somatosensory cortex of the rat and rabbit in different age groups from P0 to P180 (P-postnatal day). The numerical density of retrogradely labeled the ventroposterolateral (VPL) projection neurons was analyzed. A significant decrease in this parameter was observed during the first two weeks of postnatal life in both studied species. Changes of the neuropil volume and selective elimination of early cortical connections stemming from the VPL may possibly cause this process. A withdrawal of axon collaterals from the expanded cortical sites as well as apoptosis (existing both in the VPL and parietal cortex) contribute to a decrease in the numerical density. Our observations allow us to conclude that the thalamocortical somatosensory connections established before the birth undergo significant quantitative changes in both studied species during the first two weeks of postnatal life and this period seems to be crucial for maturation of the thalamocortical loop.

Differentiation of lamina I spinomedullary and spinothalamic neurons in the cat.

We characterized spinomedullary neurons that project to the ventrolateral portion of the medulla that receives lamina I terminations in two sets of experiments in the cat. First, their distribution was examined using single unilateral iontophoretic injections of cholera toxin subunit B. The injection sites were characterized by microelectrode recordings from nociceptive- and thermoreceptive-specific units, indicative of lamina I input. The spinomedullary neurons were symmetrically distributed bilaterally, predominantly (63-69%) in lamina I but also in laminae V-VIII and the thoracic lateral horn (intermediolateral cell column). In horizontal sections, spinomedullary lamina I neurons included all three main morphological types described earlier. Second, spinomedullary and spinothalamic neurons were compared in retrograde double-labeling experiments. Different combinations of tracers were injected in the right thalamus and the left or right ventrolateral medulla (guided by recordings). The numbers of spinomedullary and spinothalamic neurons on the left side were comparable, and the segmental and laminar distributions were similar, except that a greater proportion of spinomedullary neurons originated from thoracic segments. However, the proportion of double-labeled neurons was consistently approximately 1%, indicating that spinomedullary and spinothalamic pathways arise from separate subpopulations. Spinomedullary neurons were more ventrally located within lamina I than spinothalamic neurons. A significantly greater proportion of spinomedullary neurons had fusiform somata (49% vs. 36%). These observations indicate that lamina I is the major source of spinal input to this portion of the ventrolateral medulla, that the projection includes several morphological types of inputs, and that this projection is distinct from the spinothalamic projection. These findings are consistent with the concept that lamina I projections constitute an ascending homeostatic afferent pathway relating the physiological condition of the body.

Tracing developing pathways in the brain: a comparison of carbocyanine dyes and cholera toxin b subunit.

The present study examined the efficiency of fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylinodocarbocyanine perchlorate and cholera toxin B subunit in tracing the crossed tectal projection to the nucleus rotundus of the thalamus (tectorotundal pathways) of paraformaldehyde-fixed and living chick embryos. The tracers were injected into the optic tectum under three experimental conditions (carbocyanine postfix, carbocyanine in vivo, and cholera toxin B subunit in vivo) and the anterograde transport of the nucleus rotundus was monitored and compared. In the carbocyanine postfix method, small crystals of carbocyanine dye were inserted into the tectum of paraformaldehyde-fixed embryos. A 6-month post-insertion period was required to label the crossed tectorotundal pathway. Results showed that tectal neurons did not begin to innervate the ipsilateral nucleus rotundus until embryonic day 9 and the contralateral nucleus rotundus until embryonic day 17. This slow progression of labeling through the crossed tectal projection resulted in significant contrast of the labeling between the ipsilateral and contralateral nuclei rotundus. In the carbocyanine in vivo method, a small volume of carbocyanine dye solution was injected into the tectum of living embryos. A 8- to 12-h survival period was sufficient enough to label the tectorotundal pathway. By embryonic day 8, the labeled axons terminated in the ipsilateral nucleus rotundus and the crossed tectorotundal projection was first detected by embryonic day 10. Similarly, in the cholera toxin B subunit in vivo method, a small volume of cholera toxin B subunit solution was injected into the tectum of living embryos. After a 6- to 10-h survival period, heavily labeled axons were found to innervate bilaterally the nucleus rotundus by embryonic day 8. This appeared to be the earliest schedule for detecting the crossed tectorotundal projection, compared with that of both the postfix and in vivo methods of carbocyanine dye. Based on the differences in the detectability of the crossed tectorotundal projection between the postfix and in vivo methods, the present data suggest that the former method is of limited purpose for labeling tectal collaterals during embryogenesis. Moreover, given the rapid transport rate and absence of photobleaching, which is often seen when using carbocyanine dye, the cholera toxin B subunit in vivo method appears to be the tracer of choice for investigating embryonic pathways.