Correlations between brainstem NMDA receptor changes and active neuronal cell death after intermittent hypercapnic hypoxia in the developing piglet

The role of the N-methyl-D-aspartate (NMDA) receptor in cell death was evaluated in the piglet brainstem after exposure to intermittent hypercapnic hypoxia (IHH). Study groups comprised controls (n=6) and piglets exposed to IHH on 2 (n=6) or 4 (n=5) successive days prior to euthanasia. All piglets had the caudal medulla evaluated at 13-14 days of age using double immunohistochemistry for TUNEL and the NMDA receptor 1 (NR1) subunit. The percent of TUNEL positive neurons amongst NR1 (% TUN in NR1) and non-NR1 neurons (% TUN in non-NR1) was determined in eight nuclei. After 2 days of IHH, %TUN in NR1 was increased in the dorsal motor nucleus of the vagus (DMNV, P=0.007) and the inferior olivary nucleus (ION, P=0.05). After 2 days IHH, %TUN in non-NR1 neurons was increased in the lateral reticular nucleus (LRt, P=0.05), nucleus of the solitary tract (NTS, P=0.004) and gracile nucleus (P=0.05). After 4days IHH, the increase of %TUN in NR1 was sustained in the ION (P=0.05), while %TUN in non-NR1 neurons was sustained in NTS (P=0.04) and LRt (P=0.006). Daily IHH exposure induces neuronal death within NR1 and non-NR1 neurons, but the neuronal phenotype is consistent within affected brainstem nuclei. Involvement of the NMDA receptor tended to occur in nuclei with higher basal NR1 expression, and thus occurred in nuclei relevant to cardiorespiratory function. We speculate that IHH exposures, such as occurs during obstructive apnea or facial entrapment in prone sleeping during infancy, can induce abnormalities of cardiorespiratory control.     

Brain-derived neurotrophic factor mRNA and protein in the piglet brainstem and effects of Intermittent Hypercapnic Hypoxia

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for the development of normal respiratory rhythm and ventilatory control. Chronic exposure to Intermittent Hypercapnic Hypoxia (IHH) has been shown to alter ventilatory responses of piglets. This study investigated changes in BDNF distribution and expression in seven nuclei of the caudal medulla, from piglets exposed to IHH for 1, 2, 3, or 4 days before death, using non-radioactive in situ hybridisation (for mRNA) and immunohistochemistry (for protein). Compared to controls, BDNF mRNA was markedly increased across the entire medulla of the brainstem, after all durations of IHH (1-4 days). In contrast, BDNF protein expression increased after 1 day of exposure to IHH (p=0.003), but, thereafter, was not different to controls. Amongst individual nuclei, neurons of the dorsal motor nucleus of the vagus (DMNV) showed increased BDNF mRNA (p<0.01), but decreased protein expression (p=0.05) after all durations of IHH. In the ION, both mRNA and protein for BDNF were significantly increased after 1 day IHH (p<0.01 and p=0.001, respectively), but these increases were not sustained. This study is the first to investigate changes in BDNF expression in response to environmental challenges during postnatal development in the brainstem. Implications of the wide distribution of BDNF in the piglet caudal medulla and increased expression after IHH exposure are discussed, with particular reference to roles for BDNF-dependent neurons at this stage of development.     

Tracing of projection neurons from the cervical dorsal horn to the medulla with the anterograde tracer biotinylated dextran amine

In addition to the well-defined role of dorsal horn neurons in pain transmission, neurons in the superficial laminae also provide a rich source of synaptic input to cardiovascular and respiratory centers in the medullary reticular formation. In this study, ascending projection neurons from the superficial laminae of the cervical enlargement were studied in the rat using the anterograde tracer biotinylated dextran amine (BDA). Ipsilateral microinjection of BDA into the cervical spinal cord (C6-C8) resulted in extensive labeling of dorsal horn neurons in laminae I-V. Axons and terminal processes of cervical dorsal horn cells projecting to the medulla were present in the cuneate nucleus (Cu), the nucleus of the solitary tract (NTS), the lateral reticular nucleus, (LRt) as well as the caudal and rostral ventrolateral medulla (VLM). The highest density of BDA labeling was found ipsilaterally in the Cu, LRt, caudal and rostral VLM, while a moderate density of labeling was present in the NTS caudal to the area postrema (AP). Moderate-to-weak labeling was also found in the LRt, the caudal and rostral VLM contralateral to the BDA injection. These results support the existence of a spinomedullary pathway that transmits noxious and innocuous Adelta and C fiber-mediated sensory signals to the medulla. Neurons in this ascending spinal pathway likely participate in the patterning of autonomic responses evoked by pain or during exercise.     

Active caspase-3 in the sudden infant death syndrome (SIDS) brainstem

In a retrospective postmortem study, we examined the neuronal expression of active caspase-3, a specific apoptotic marker, in the brainstem of 67 infants dying from sudden infant death syndrome (SIDS), and 25 age-matched control infants (non-SIDS). Neuronal immunostaining for active caspase-3 was semi-quantitatively scored in nuclei from five brainstem levels: rostral, mid and caudal pons, and rostral and caudal medulla. Regardless of the cause of death (SIDS vs. non-SIDS), age-related differences in active caspase-3 expression were identified, predominantly in the medulla. No gender-related differences were identified. Comparing SIDS to non-SIDS cases, increased active caspase-3 expression was restricted to four nuclei in the caudal pons (abducens, facial, superior olivary, and pontine nuclei) and two nuclei in the rostral medulla (hypoglossal and dorsal motor nucleus of the vagus). We conclude that neuronal apoptosis is increased in the brainstem of SIDS compared to non-SIDS infants.     

Intermittent hypercapnic hypoxia effects on the nicotinic acetylcholine receptors in the developing piglet hippocampus and brainstem

This study investigated the effects of acute (1 day) vs repeated (4 days) exposure to intermittent hypercapnic hypoxia (IHH) on the immunohistochemical expression of α2, α3, α5, α7, α9 and β2 nicotinic acetylcholine receptor (nAChR) subunits in the developing piglet hippocampus and brainstem medulla, and how prior nicotine exposure alters the response to acute IHH. Five piglet groups included: 1 day IHH (1D IHH, n = 9), 4 days IHH (4D IHH, n = 8), controls exposed only to air cycles for 1 day (1D Air, n = 6) or 4 days (4D Air, n = 5), and pre-exposed to nicotine for 13 days prior to 1 day IHH (Nic + 1D IHH, n = 7). The exposure period alternated 6 min of HH (8%O₂, 7%CO₂, balance N₂) and 6 min of air over 48 min, while controls were switched from air-to-air. Results showed that: 1. repeated IHH induces more changes in nAChR subunit expression than acute IHH in both the hippocampus and brainstem medulla, 2. In the hippocampus, α2 and β2 changed the most (increased) following IHH and the CA3, CA2 and DG were mostly affected. In the brainstem medulla, α2, α5, α9 and β2 were changed (decreased) in most nuclei with the hypoglossal and nucleus of the solitary tract being mostly affected. 3. Pre-exposure to nicotine enhanced the changes in the hippocampus but dampened those in the brainstem medulla. These findings indicate that the nAChRs (predominantly with the α2/β2 complex) are affected by IHH in critical hippocampal and brainstem nuclei during early brain development, and that pre-exposure to nicotine alters the pattern of susceptibility to IHH.     

The effects of nicotine on the alpha-7 and beta-2 nicotinic acetycholine receptor subunits in the developing piglet brainstem

Exposure to cigarette smoke is a major risk factor for sudden infant death syndrome (SIDS). We tested the hypothesis that nicotine increases expression of the nicotinic acetylcholine receptor (nAChR) subunits α7 and β2 in a piglet model. Piglets exposed to 2 mg/kg/day nicotine for 14 days postnatally (n = 14) were compared to non-exposed controls (n = 14), (equal gender proportions). Immunohistochemistry was performed to identify and quantify changes in, α7 and β2 nAChR subunits in 8 nuclei of the medulla at both the rostral and caudal levels. Compared to controls, nicotine exposed piglets had decreased α7 in the rostral dorsal motor nucleus of the vagus (rDMNV) (p = 0.01), and increased β2 in the caudal DMNV (cDMNV) (p = 0.05), caudal nucleus of the spinal trigeminal tract (cNSTT) (p = 0.03) and caudal nucleus of the solitary tract (cNTS) (p = 0.04). Analysis by gender showed that in the control group, compared to males, females had higher β2 in the caudal hypoglossal (cXII) (p < 0.01) and caudal inferior olivary (p = 0.04) nuclei, while in the nicotine group females had higher β2 in the cDMNV (p = 0.02). Compared to control males, nicotine exposed males had lower β2 in the cXII (p < 0.01). Overall, changes in α7 were specific to nicotine exposure with no gender differentiation. Changes in β2 were more widespread but showed gender-specific effects. These findings provide evidence that early postnatal exposure to nicotine significantly affects nAChR subunit expressions in the developing brainstem.     

Projections of cervicothoracic dorsal roots to the cuneate nucleus of the rat, with observations on cellular "bricks"

The distribution of cervicothoracic (C1–T2) dorsal roots to the cuneate and accessory cuneate nuclei was studied in the rat with the Fink- Heimer I technique following single extradural rhizotomies. Cytoarchitectural analysis of the cuneate nucleus revealed an anatomically distinct caudal and rostral region. The caudal region was characterized by discrete aggregates of cells arranged as “slabs” or “bricks” which continue vertically almost to the ventral limit of the cuneate nucleus. In contrast, the cells of the rostral region were organized in a non-focal manner. The projection of the cervicothoracic dorsal roots to the cuneate nucleus reflected the cytoarchitectural pattern observed; non-focal terminal fields of degeneration in the rostral region and discrete “terminal field bands” of degeneration isolated from one another by degeneration-free zones in the caudal region. However, the projections of dorsal roots C5 and T2 to the caudal region is non-focal. In both regions of the cuneate nucleus, the distribution of dorsal root degeneration was topographically organized with cranial roots terminating ventrolaterally and more caudal roots, dorsomedially. The amount of intersegmental overlap of dorsal root terminal fields was greater in the rostral than in the caudal region. Individual dorsal roots projected differentially to the two regions of the cuneate nucleus. Roots C3 and C4 distributed primarily to the rostral region whereas C5 to T1 distributed to both regions. T2 root projected primarily to the caudal region. Dorsal roots C1 and C2 did not terminate in either region of the cuneate nucleus. All roots studied projected heavily and topographically to the accessory cuneate nucleus. Extensive overlap of the very dense terminal fields characterized the dorsal root projections to the accessory cuneate nucleus. On the basis of cytoarchitectonics and dorsal root projections, a dual organization of the cuneate nucleus was revealed. This organization reflected previous anatomical and electrophysiological studies in the rat and paralleled the organization described in the dorsal column nuclei of the cat. The significance of the dual organization with respect to dorsal column function was discussed, as was the finding of the lack of C1 and C2 dorsal root projections to the cuneate nucleus.     

Projections of the paratrigeminal nucleus to the ambiguus, rostroventrolateral and lateral reticular nuclei, and the solitary tract

The paratrigerminal nucleus (Pa5), a constituent of the spinal interstitial system, was linked to the pressor effect caused by bradykinin injected in the dorsal lateral medulla of the rat. The nucleus receives primary afferent sensory fibers contained in branches of the trigeminal, glossopharyngeal and vagus nerves. In this investigation connections of the paratrigeminal nucleus to other medullary structures were studied with the use of retrograde and anterograde neuronal tracers. Fluorescent light microscopy analyses of medullary sections of rats injected with the retrograde transport tracer Fluoro-gold in the nucleus of the solitary tract (NTS) or in the pressor area of the rostral ventrolateral medulla (RVLM) revealed labeled neuronal cell bodies in the ipsi- and contralateral Pa5. FluoroGold microinjections in the caudal ventrolateral medulla (CVLM) did not produce fluorescent labeling of Pa5 neurons. Microinjection of the anterograde transport neuronal tracer biocytin in the Pa5 produced bilateral labeling of the solitary tract (sol). rostroventrolateral reticular nucleus (RVL), ambiguus nucleus (Amb), lateral reticular nucleus (LRt) and ipsilateral parabrachial nuclei, but not the contralateral Pa5. Confocal laser microscopy showed fluorescence labeling of fibers and presumptive terminal varicosities in the NTS, RVL, Amb and LRt. The present findings showing the paratrigeminal nucleus interposed between sensory afferent and stuctures associated to cardiovascular and respiratory functions, suggest that the structure may act as a medullary relay nucleus for sensory stimuli directly connecting primary afferents to structures mediating cardiovascular and respiratory reflexes.     

Central projections of cervical primary afferent fibers in the guinea pig: an HRP and WGA/HRP tracer study

The central course and the projections of the first and the second cervical dorsal root ganglia and of suboccipital muscle primary afferent fibers in the guinea pig were studied by means of anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA/HRP) or aqueous solution of horseradish peroxidase (HRP). Injections of WGA/HRP into the second cervical dorsal root ganglion produced labeling in the dorsal and ventral horns. Within the spinal cord, the largest amount of HRP reaction product was found within the lateral third of the substantia gelatinosa and within the central cervical nucleus. The main area of termination in the medulla was the external cuneate nucleus. However, HRP reaction product was also found within the medial and inferior vestibular nuclei, cell group x, the perihypoglossal nuclei, the nucleus of the solitary tract, and the nucleus of the spinal trigeminal tract. Descending fibers could be detected as caudal as spinal segment T5. Injections of WGA/HRP into the first cervical dorsal root ganglion produced heavy terminal label within the central cervical nucleus but not within the substantia gelatinosa. Again, the external cuneate nucleus was the main area of termination within the medulla. Label could not be observed within the vestibular nuclear complex or within the spinal trigeminal nucleus. Injections of aqueous HRP into the suboccipital muscles produced heavy transganglionic label within the central cervical nucleus, whereas the substantia gelatinosa totally lacked terminal label. Ascending proprioceptive fibers reached the external cuneate nucleus and group x. Scanty projections could be detected within the vestibular nuclei as well as within the perihypoglossal nuclei except for the nucleus prepositus hypoglossi. Label was absent in the spinal trigeminal nucleus.     

Serotoninergic receptor 1A in the sudden infant death syndrome brainstem medulla and associations with clinical risk factors

The immunoreactivity of the serotoninergic receptor subtype 1A (5HT_1AR) was quantitatively analyzed in the human infant brainstem medulla (caudal and rostral levels). We hypothesized that immunoreactivity of 5HT_1AR would be reduced in infants diagnosed with sudden infant death syndrome (SIDS). In particular that those infants with known clinical risk factors (including cigarette smoke exposure, bed sharing and sleep position) would have greater changes than those without clinical risks. Comparing SIDS (n = 67) to infants who died suddenly with another diagnosis (non-SIDS, n = 25), we found decreased 5HT_1AR immunoreactivity in the majority of the nuclei studied at the rostral medulla level including dorsal motor nucleus of the vagus (DMNV), nucleus of the solitary tract, vestibular, and inferior olivary nucleus (ION). There was a significant relationship with all risk factors for 5HT_1AR, especially for DMNV, suggesting that 5HT_1ARs are highly vulnerable to various insults within the SIDS DMNV. This study not only provides further evidence of abnormalities within the brainstem serotoninergic system of SIDS infants, but also shows that these changes may be associated with exposure to clinical risk factors.     

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.     

Brainstem projections to the rat cuneate nucleus

Neurons in the pontomedullary tegmentum have been proposed as a final common pathway subserving descending inhibition in the dorsal column nuclei. To investigate the anatomical substrate for these descending effects, brainstem projections to the cuneate nucleus of rats were studied with injections of lectin-conjugated horseradish peroxidase. In rats with iontophoretic tracer injections in this nucleus, many labeled neurons were detected near the injection site, especially ventral and caudal to it. Intrinsic reciprocal projections were observed after injections in caudal, middle, or rostral levels of the cuneate nucleus. Neurons were labeled in the red nucleus, in agreement with previous anatomical studies, and also in the trigeminal, vestibular, and cochlear nuclei. An ipsilateral dorsomedial group of neurons was labeled in the upper cervical segments and scattered neurons were also labeled bilaterally near the central canal. Sparse retrograde labeling in the tegmentum was focused in the lateral paragigantocellular nucleus and caudal raphe. Consistent with the retrograde experiments, anterograde labeling after pressure injections of lectin-conjugated horseradish peroxidase in the pontomedullary tegmentum was very sparse within the dorsal column nuclei; labeling was dense, however, in the region immediately ventral to these nuclei. These results confirm previous work indicating that the activity of cuneate neurons is modulated by brainstem sensory nuclei. However, it appears that direct projections to the cuneate nucleus from pontine and rostral medullary regions are sparser than previously suggested. The last link of a polysynaptic descending inhibitory pathway may include GABAergic neurons immediately adjacent to the dorsal column nuclei and/or intrinsic to these nuclei.     

Prenatal cigarette smoke exposure effects on apoptotic and nicotinic acetylcholine receptor expression in the infant mouse brainstem

Infants exposed to cigarette smoked during pregnancy into infancy have increased respiratory and cardiac abnormalities. Nicotine, the major neurotoxic component of cigarette smoke, induces its actions by binding to nicotinic acetylcholine receptors (nAChR), with one downstream effect being increased apoptosis. Using a pre- into post- natal cigarette smoke exposure mouse model (SE), we studied the immunohistochemical expression of nAChR subunits α2, α3, α4, α5, α7, α9, β1 and β2 and two markers of apoptosis, active caspase-3 and TUNEL, in seven nuclei of the medulla and facial nucleus of the pons in male mice. Pups of dams exposed to two cigarettes (nicotine ≤1.2 mg, CO ≤15 mg) twice daily for six weeks prior to mating, during gestation and lactation (n = 5; SE), were compared to pups exposed to air under the same condition (n = 5; SHAM) at P20. Results showed that the hypoglossal nucleus had increased α3, α4, α7, α9, Casp-3 and TUNEL, dorsal motor nucleus of the vagus had increased α3, α5, α7, β1 and Casp-3, nucleus of the solitary tract had increased α3 but decreased α4, α5, β1 and apoptosis, cuneate nucleus had increased α3, β2 and Casp- 3, but decreased α5, nucleus of the spinal trigeminal tract had increased α3, α7, β1, lateral reticular nucleus had decreased β1, inferior olivary nucleus had increased β1 but decreased apoptosis, and the facial had increased α2, α3 and α7. This is the first study to demonstrate that nAChR subunits are affected following pre- into post-natal SE and that they simultaneously coincided with changes in apoptotic expression.     

Organization of projections from the gracile, medial cuneate and lateral nuclei in the North American opossum. Horseradish peroxidase study of the cells projecting to the cerebellum, thalamus and spinal cord

Using the horseradish peroxidase technique on the North American opossum, we were able to locate the neurons within the dorsal column and lateral cuneate nuclei which innervate the cerebellum and thalamus as well as those within the dorsal column nuclei which project spinalward. The medial and lateral cuneate nuclei supply axons to the anterior lobe, the paramedian lobule and the pyramis of the cerebellum and the lateral nucleus provides an additional projection to the uvula. The cerebellar projections from these nuclei arise from neurons located rostral to the obex. The thalamic projections from the gracile and medial cuneate nuclei originate from neurons throughout their rostral to caudal extent, although most of them are located just rostral to the obex. Neurons within the lateral cuneate nucleus which innervate the thalamus are found at intermediate rostrocaudal levels where most of them approximate the medial cuneate nucleus. The medial cuneate also projects to at least lumbar levels of the spinal cord in the opossum and neurons giving rise to such connections are found at the level of the obex and caudal to it. Neurons within the dorsal part of the dorsal column nuclei were labelled only after thalamic injections. Our results in the opossum are compared with those obtained in several placental mammals.     

Fos and FRA protein expression in rat precerebellar structures during the Neurolab Space Mission

Changes in gene expression were examined in precerebellar structures during and after space flight. These structures included the inferior olive (IO), the source of climbing fibers, and the lateral reticular nucleus (LRt) and basilar pontine nuclei (PN), sources of mossy fibers. We examined two immediate early gene products with two different time courses of expression: Fos, which persists only for a few (6-8)h after activation and FRA expression, which lasts for longer periods of time, i.e. hours and/or days after activation.Gravity effects on Fos and FRA gene expression were evident in vestibular and visual areas of the IO, including the dorsomedial cell column, the beta subnucleus and the dorsal cap of Kooy of the medial nucleus (which projects to the flocculonodular lobe, i.e. to the vestibular area of the IO involved in the olivary control of the vestibulo-ocular reflex (VOR)). Gene expression also affected the subnuclei A, B, and C and the caudal part of the medial IO. These olivary regions do not receive vestibular afferents, but rather spinal afferents, and are particularly involved in the olivary control of the vestibulospinal reflex (VSR). Changes in Fos expression were also observed in the LRt and the PN. We suggest that sensory substitution, in which signals produced by a subject's own activity replace activity normally provided by macular stimulation, contributes to the recovery of microgravity-related postural and motor deficits.While no consistent increases in FRA expression occurred in vestibular IO regions 24h after launch, consistent increases in FRA expression occurred 24h after landing. We hypothesize that this asymmetrical pattern of gene expression resulted from (i). tonic microgravity experienced after launch counteracting the effects of increased phasic gravitational forces experienced during launch, and (ii). the tonic gravitational field experienced after landing potentiating the effects of increased phasic gravitational forces experienced during landing.The specificity of these results is demonstrated by an absence of direct gravity-related changes in Fos expression in other precerebellar structures such as the external cuneate nucleus, group X, and the dorsal column nuclei that transmit exteroceptive and proprioceptive signals to thalamic nuclei and somatosensory areas of the cerebral cortex. The gravity-related Fos and FRA expression changes in the IO and the LRt seen here are of interest in view of the important role their projections play in adaptive gain changes of the VOR and VSR during sustained visuo-vestibular and neck-vestibular stimulation.     

Physiopathology of the cerebellum in the monkey. I. Origin of cerebellar afferent nervous fibers from the spinal cord and brain stem

Complete cerebellectomy is associated with almost total cell loss in the reticulotegmental, lateral reticular and pontine nuclei and in the principal and accessory olivary nuclei but not in the perihypoglossal and reticular paramedian nuclei in the monkey. The latter structure which is a prominent structure in the cat is underdeveloped or absent in this species. It also results in important retrograde degenerating changes of the neurons of the lateral cuneate nucleus, the dorsal nucleus of Clarke and the border cells of Cooper and Sherrington as disclosed in 1 monkey with a short-term cerebellectomy. A few neurons of the principal cuneate nucleus also undergo retrograde degeneration in the immediate postoperative period. The present findings suggest that the caudal part of the medial accessory olive and its “dorsal cap” are anatomically related to the contralateral nodulus and flocculus, respectively, whereas the rostral part of the medial accessory olive is more directly related to the neovermis. The dorsal accessory olive appears to be related to the contralateral cerebellar nuclei and more specifically, the fastigial nucleus. These results also favour the existence of cerebellopetal fibers from the principal cuneate nucleus and of a few non-cerebellopetal fibers from the lateral cuneate nucleus. The present findings support the suggestion of Cooper and Sherrington pointing to the existence of spinocerebellar fibers originating in the border cells of the ventral horn at the level of the low thoracic and lumbar segments of the cord. However the possibility that such “spinocerebellar” fibers may distribute collateral endings to the dorsolateral area of the medulla or even terminate in this area cannot be entirely ruled out on the basis of the present material. A similar feature possibly explains the fact that most (if not all) cells of the dorsal nucleus of Clarke resist the interruption of their axons at cerebellar level as suggested by the findings in monkeys with long standing lesions.     

Spino-bulbar, spino-thalamic and medical lemniscal connections in the American opossum, Didelphis marsupialis virginiana

Projections from the spinal cord and dorsal column nuclei to more rostral levels of the neuraxis were investigated in seventeen adult opossums by the Nauta-Gygax and Fink-Heimer techniques. In all cases with spinal cord lesions a greater number of degenerating fibers distributed to the medulla and pons than to the midbrain and diencephalon. Numerous degenerating fibers ended within the medial reticular formation of the medulla and caudal pons, and within the lateral reticular formation of the rostral pons and midbrain. Degenerating fibers were numerous in the reticular formation following cervical and thoracic lesions, but sparse in specimens with damage restricted to either the lumbar or sacral spinal cord. The dorsal column nuclei received afferent connections from the well known dorsal funicular pathway and, although to a much lesser extent, from the main ventrolateral spinal bundle. Although most of the latter fibers ended in the subnucleus dorsalis and spinal vestibular nucleus, some penetrated into the gracile and cuneate nuclei. Conspicuous terminal degeneration was present within the inferior olivary nucleus following cervical and thoracic lesions, but was lacking in cases of either caudal lumbar or sacral cord lesions. The location of terminal degeneration within the lateral reticular nucleus is dependent upon the level of the lesion in the spinal cord. Degenerating fibers ended within the lateral vestibular nucleus in all cases of spinal cord hemisection, and within the medial portion of the facial nucleus in cases with a lesion rostral to C-4. After cervical and thoracic hemisections terminal fiber degeneration was present within the midbrain tegmentum, the periaqueductal gray, the intercollicular nucleus (Mehler,'69), the posterior thalamic nucleus, the ventrobasal nucleus, the parafascicular nuclei and the caudal nucleus ventralis lateralis. All thalamic nomenclature was taken from Oswaldo-Cruz and Rocha-Miranda, '68. In animals with more caudal lesions, no fiber degeneration was evident within the nucleus ventralis lateralis and so little within the ventrobasal nucleus that it was impossible to ascertain a somatotopic pattern of spinothalamic projections. Lesions of the dorsal column nuclei caused terminal degeneration within the inferior olivary nucleus, the pars lateralis of the nucleus of the inferior colliculus, the zona incerta, the posterior thalamic nucleus, the caudal part of the ventral lateral thalamic nucleus and the ventrobasal nucleus of the thalamus. Diffuse connections with the reticular formation, periaqueductal gray, midbrain tegmentum and the parafascicular complex were also observed. The results from small lesions indicate that the input to the ventrobasal nucleus in the opossum is organized in the typical mammalian fashion.     

Expression of active caspase-3 in mitotic and postmitotic cells of the rat forebrain

Active caspase-3 immunoreactivity was detected in the rat forebrain proliferative regions at birth and remained high in these areas for about 2 weeks, during which period labeled cells were present centroperipherally across the olfactory bulb. By the end of the third postnatal week, only a small number of immunolabeled cells remained in these forebrain structures. Active caspase-3 immunolabeling was localized mostly to cell nuclei and co-localized partially with TuJ1 and NeuN immunoreactivity, but not with glial fibrially acidic protein, OX-42, gamma-aminobutyric acid, or terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive labeling. Active caspase-3 and 5-bromo-2'-deoxyuridine (BrdU) double-labeled nuclei were seen in the proliferative regions after 2 hours and in the periglomerular region of the bulb after 7 days following BrdU injections. Examination of the cells with electron microscopy confirmed that the active caspase-3-containing nuclei in the proliferative regions often had infoldings and appeared to be undergoing division. Some of the cells with active caspase-3-labeled nuclei in the bulb had synapses on their somata or dendrites. Labeled dendritic spines and a few axon terminals were also observed in the olfactory bulb. Taken together, it appears that a wave of active caspase-3-positive cells are dividing in the proliferative zones and then migrating to the bulb as they differentiate into neurons. Therefore, active caspase-3 may play a role in cellular processes such as neuronal differentiation, migration, and plasticity, in addition to its role in cell death.     

Dorsal column nuclei projections to the cerebellar cortex in cats as revealed by the use of the retrograde transport of horseradish peroxidase.

The existence of a cerebellar projection from the dorsal column nuclei (gracile and cuneate nuclei, DCN) has been proposed on electrophysiological grounds but questioned when studied with neuroanatomical techniques. The retrograde transport of horseradish peroxidase (HRP) has been used for the present study and provides anatomical evidence of a DCN-cerebellar pathway. In adult cats, 1 to 6 mul of 30% HRP were injected in pars intermedia of the anterior lobe (lobules IV-V), in paramedial lobule and in vermis of the anterior (lobules IV-V) and of the posterior lobe (lobule VII). After survival of 24 to 48 hours, all animals were perfused with a double aldehyde mixture and serial 40 mu sections through the medulla oblongata were incubated for visualization of HRP. In all cases, medullary nuclei known to project to the injected cortical regions of the cerebellum contained HRP-positive neurons mainly ipsilateral to the injection (e.g., external cuneate nucleus) or mainly contralateral to it (e.g., inferior olivary complex). Following ipsilateral injections in either the paramedian lobule or the pars intermedia, HRP-positive neurons in the cuneate nucleus were concentrated in its rostral portion where multipolar cells with radiating dendrites predominate. In contrast, none of the clusters region, in the caudal part of the cuneate nucleus, displayed HRP-positive granules. In cases in which the anterior vermis was injected a few labelled cells were present in the rostral part of the gracile nucleus but not in the clusters region of this nucleus. No labelling of DCN neurons was evident after posterior vermis injection. To compare the distribution of cells contributing to the DCN-cerebellar pathway with that of thalamic relay cells in the DCN, 0.5 to 3 mul of 30% HRP were injected in the nucleus ventralis posterolateralis of the thalamus in another series of cats. Contralateral to the thalamic injection, labelled cells were concentrated in the clusters region of the gracile and cuneate but rostrally in these nuclei they were scattered among unlabelled neurons. The preferential location in the DCN of cells which project to the cerebellum and of cells which project to the thalamus stresses the heterogeneous organization of these nuclei along the rostrocaudal axis. Further, the results indicate that regions of the DCN which have been distinguished on the basis of cytoarchitectonics (Kuypers and Tuerk, '64) and of afferents (Rustioni, '73, '74) differ also in their efferent projections.     

Research on alteration of neurons in vagal nuclei in medulla oblongata in newborns with respiratory distress

Neuronal and axonal degenerative changes in motor vagal neurons (DMNV) and sensory vagal neurons (nTS) in the medulla oblongata in newborns were studied. Material was taken from the autopsies of newborns, live and dead newborns, in different gestational weeks (aborted, immature, premature and mature). 46 cases were studied. Material for research was taken from the medulla oblongata and lung tissue. Serial horizontal incisions were made in the medulla oblongata (± 4 mm), commencing from the obex, where the DMNV and nTS vagal nuclei were explored. Fixed cuttings in buffered formalin (10%) were used for histochemical staining. Serial cuttings were done with a microtome (7 μm). Pulmonary infections, being significant (p < 0.05), have an important place when studying respiratory distress (RD) in newborns. Morphological changes of nerve cells in DMNV and nTS nuclei in the medulla oblongata in newborns in different gestational weeks are more emphasized in matures in comparison to aborted and immature (p < 0.05). Depending on the lifetime of dead newborns, neuronal morphological changes in vagus nerve nuclei are significant (p < 0.05). Therefore, it can be concluded that pulmonary infections are often caused due to dramatic respiratory distress in newborns, while hypoxaemic changes in the population of vagus nerve neurons in respiratory distress are more emphasized in matures.