Interleukin-1beta immunoreactivity in identified neurons of the rat magnocellular neurosecretory system: evidence for activity-dependent release

Interleukin-1beta has been demonstrated in neurons of the rat hypothalamus, including cells of the magnocellular neurosecretory system and tuberoinfundibular system (Lechan et al., [1990] Brain Res. 514:135-140). Despite its potential importance to regulation of neuroendocrine function, however, neither the specific cell types that express interleukin-1beta or the conditions that may result in its release have yet been described. Therefore, we utilized a combination of immunocytochemical and immunoelectron microscopic localization, in conjunction with Western blot analysis, on normonatremic, hypernatremic, and lactating rats to assess the site of synthesis and potential secretion characteristics of interleukin-1beta in the rat magnocellular neurosecretory system. Interleukin-1beta immunoreactivity was localized within both oxytocin and vasopressin neurons in the paraventricular, supraoptic, accessory and periventricular hypothalamic nuclei. Additionally, interleukin-1beta immunoreactive fibers were localized in the zona interna and zona externa of the median eminence and in the neurohypophysis. Immunoelectron microscopic analysis revealed that interleukin-1beta immunoreactivity is associated with small spherical structures, distinct from neurosecretory granules, in neurosecretory axons within the neurohypophysis. Furthermore, stimulation of heightened neurosecretory activity via chronic osmotic challenge and lactation resulted in a marked diminution in levels of interleukin-1beta immunoreactivity in the neurohypophysis with a subsequent return to normal levels after cessation of the stimuli. Western blot analysis confirmed the existence of interleukin-1beta protein in the neurohypophysis and provided further evidence for reduction in levels of IL-1beta immunoreactivity after stimulation of secretory activity. These results suggest an endogenous neuronal source of interleukin-1beta exists within the rat magnocellular neurosecretory system under normal physiological conditions. The potential for activity-dependent release of IL-1beta and implications for the involvement of interleukin-1beta in regulation of neurosecretory activity are discussed.     

Target dependence of hypoglossal motor neurons during development in maturity

We have investigated the target dependence of hypoglossal motor neurons in postnatal rats by transecting the hypoglossal nerve and preventing reinnervation of the tongue. After transection in early postnatal life, approximately 60% of hypoglossal motor neurons die and surviving neurons are markedly atrophic compared to contralateral controls. In maturity, there is also substantial neuronal atrophy and about 30% of motor neurons appear to die after the procedure. However, most hypoglossal neurons in adults survive transection for periods up to 1 year. The adult response is present by 3 weeks of age. The time course of neuronal atrophy and death after permanent target deprivation was investigated in adult animals. One month after the hypoglossal nerve was deflected, there was marked axonal atrophy, although somatic atrophy was minimal. By 3 months after the procedure substantial neuronal atrophy and apparent cell loss (about 30%) had occurred. There was little change between 3 and 6 months. We conclude that hypoglossal motor neurons are influenced by connections with their targets in postnatal life. Even in maturity, neurons require target connections for maintenance of axonal and somatic morphology. However, the majority of motor neurons in adult animals can survive target deprivation for prolonged periods.     

Early stages in the development of spinal motor neurons.

In order to identify early events in the differentiation of motor neurons, the expression of several developmentally regulated, neuronal molecules was investigated by immunohistochemistry on consecutive sections of cervical spinal cord. Motor neurons are among the first neurons to be born and to differentiate within the embryonic rat spinal cord. They undergo their terminal mitosis on embryonic days 10 and 11 (E10-11) and acquire detectable levels of the transmitter synthesizing enzyme, choline acetyltransferase, by E11.5. Staining with antibodies to the 68 kD neurofilament protein revealed motor neurons extending processes out the ventral root as early as E10.5. Monoclonal antibodies to two different epitopes on the cell adhesive molecule, NCAM, bound to myotomes on E10.5, and began to recognize ventral horn neurons by E11. Two other markers of developing neurons, the growth-associated protein, GAP-43, and the surface glycoprotein, TAG-1, were clearly detected on young motor neurons by E11.5. Thus, during the 36 hours following the final mitosis of their precursors, motor neurons acquire cytoskeletal, enzymatic, and cell surface components that distinguish them from other developing cells within the spinal cord. Not all of the newly acquired molecules continue to be expressed by motor neurons. Immunoreactivity for TAG-1 was lost by E12.5, followed by a gradual reduction of immunoreactivity for GAP-43 and the highly polysialylated form of NCAM. By E15, only antibodies to choline acetyltransferase (Phelps et al., J. Comp. Neurol. 307:1-10, 1990), and to neurofilaments, selectively stained motor neurons within the embryonic spinal cord. The transient presence of GAP-43, TAG-1, and the embryonic form of NCAM coincides with a period of vigorous axonal growth and declines when motor neurons reach their targets. This report describes the temporal sequence of early stages in the differentiation of the rodent motor neuronal phenotype. Some of these changes may be related to interactions with their synaptic partners.     

Long-term enhancement produced by activity-dependent modulation of Aplysia sensory neurons

We have investigated long-lasting enhancement of signaling effectiveness in the tail sensory neurons of Aplysia using both intracellular and extracellular stimulation. The pairing of high frequency homosynaptic activation with heterosynaptic modulation produced significantly greater enhancement of monosynaptic connections to identified motor neurons than did homosynaptic activity, heterosynaptic modulation, or test stimuli alone. Enhancement of the monosynaptic excitatory postsynaptic potential produced by pairing persisted for at least 4 hr, and the kinetics of decay of this potentiation indicated a time constant of about 5 hr. Although unpaired stimulation produced much weaker enhancement, both homosynaptic activity and heterosynaptic modulation alone produced enhancement lasting more than 90 min. The results are consistent with the possibility that intrinsic electrical activity can amplify the modulatory effects of a paired extrinsic chemical signal to produce long-term changes in synaptic strength. Paired stimulation also produced a relative enhancement of the excitability of the sensory neuron soma as judged by changes in action potential threshold. The lack of generalized changes in the postsynaptic cell and the observation of pairing-induced long-term changes in action potential threshold in the presynaptic cell soma suggest that long-term enhancement produced by pairing has a presynaptic locus in this system. Since pairing-specific enhancement can encode associations between sensory and motivational events in these cells, this form of plasticity may function as a form of associative memory. Similarities between long-term paired enhancement in this system and associative long-term potentiation in other systems suggest that activity-dependent neuromodulation might be involved in cellular memory in other systems as well.     

Spatiotemporal distribution of dying neurons during early mouse development

Apoptosis is a critical cellular event during several stages of neuronal development. Recently, we have shown that biotinylated annexin V detects apoptosis in vivo in various cell lineages of a wide range of species by binding to phosphatidylserines that are exposed at the outer leaflet of the plasma membrane. In the present study, we tested the specificity by which annexin V binds apoptotic neurons, and subsequently investigated developmental cell death in the central and peripheral nervous system of early mouse embryos at both the cellular and histological level, and compared the phagocytic clearance of apoptotic neurons with that of apoptotic mesodermal cells. Our data indicate: (i) that biotinylated annexin V can be used as a sensitive marker that detects apoptotic neurons, including their extensions at an early stage during development; (ii) that apoptosis plays an important part during early morphogenesis of the central nervous system, and during early quantitative matching of brain-derived neurotrophic factor and neurotrophic factor 3 responsive postmitotic large clear neurons in the peripheral ganglia with their projection areas; and (iii) that apoptotic neurons are removed by a process that differs from classical phagocytosis of non-neuronal tissues.     

Immunocytochemical and ultrastructural evidence of dendritic degeneration in motor neurons of aluminum-intoxicated rabbits

Summary Using immunocytochemical and ultrastructural methods, we observed extensive and characteristic dendritic changes in motor neurons of rabbits inoculated intracisternally with aluminum phosphate. Anti-microtubule-associated protein 2 immunostaining revealed markedly reduced immunoreactivity in motor neuron dendrites and a reduced number of dendritic trees in aluminum phosphate-intoxicated rabbits. These dendritic changes were confirmed at the ultrastructural level; neurofilamentous accumulations, membranous inclusions and disrupted microtubules were common features of motor neuron dendrites, but less prominent in motor neuron axons. These observations suggest that dendrites are characteristically involved in aluminum intoxication in addition to the widely reported accumulation of phosphorylated neurofilament in perikarya and axons.     

A semaphorin code defines subpopulations of spinal motor neurons during mouse development

Abstract In the spinal cord, motor neurons (MNs) with similar muscle targets and sensory inputs are grouped together into motor pools. To date, relatively little is known about the molecular mechanisms that control the establishment of pool-specific circuitry. Semaphorins, a large family of secreted and cell surface proteins, are important mediators of developmental processes such as axon guidance and cell migration. Here, we used mRNA in situ hybridization to study the expression patterns of semaphorins and their receptors, neuropilins and plexins, in the embryonic mouse spinal cord. Our data show that semaphorins and their receptors are differentially expressed in MNs that lie in distinct locations within the spinal cord. Furthermore, we report a combinatorial expression of class 3 (secreted) semaphorins and their receptors that characterizes distinct motor pools within the brachial and lumbar spinal cord. Finally, we found that a secreted semaphorin, Sema3A, elicits differential collapse responses in topologically distinct subpopulations of spinal MNs. These findings lead us to propose that semaphorins and their receptors might play important roles in the sorting of motor pools and the patterning of their afferent and efferent projections.     

Sialyl cholesterol enhances the development of grafted neurons and motor recovery.

Trophic actions of alpha-sialyl cholesterol (SC) and its sialidase-tolerant derivative, alpha-(3 beta-hydroxysialyl) cholesterol (SCt), were carried out on the development of midbrain neurons both in vitro and in vivo transplantation studies. Low to moderate concentrations of SC (0.01 to 0.05 micrograms/ml) facilitated neurite extension but had no effects on cell survival of primary cultured midbrain neurons. However, high concentration of SC (0.1 micrograms/ml) disturbed both neurite genesis and cell survival. SCt had a similar effect on midbrain neurons. At higher concentrations, SC and SCt induced concentration-dependent morphological changes in astrocytes from flat to fibrous. The effect on astrocytes was stronger in SCt than SC. At highest concentration tested (20 micrograms/ml), the proliferation of astrocytes was completely blocked, cells became detached and finally died. This effect of SC and SCt was partially blocked by simultaneous application of aFGF. Following dopaminergic cell grafting in vivo, SC and SCt had biphasic effects: a low dose (0.2 mg/kg, SC) enhanced motor recovery at 4 and 6 weeks after transplantation, while the highest dose (20 mg/kg, SC) disturbed motor recovery at all periods tested. These effects on motor recovery were paralleled by an effect on neurite genesis as studied by tyrosine hydroxylase immunostaining. Thus, at low concentrations, SC and SCt are neurotrophic agents that stimulate the development and differentiation of dopaminergic neurons.     

Molecular factors underlying selective vulnerability of motor neurons to neurodegeneration in amyotrophic lateral sclerosis

Current research evidence suggests that genetic factors, oxidative stress and glutamatergic toxicity, with damage to critical target proteins and organelles, may be important contributory factors to motor neuron injury in amyotrophic lateral sclerosis (ALS). Various molecular and neurochemical features of human motor neurons may render this cell group differentially vulnerable to such insults. Motor neurons are large cells with long axonal processes which lead to requirements for a high level of mitochondrial activity and a high neurofilament content compared to other neuronal groups. The lack of calcium buffering proteins parvalbum in and calbindin D28k and the low expression of the GluR2 AMPA receptor subunit may render human motor neurons particularly vulnerable to calcium toxicity following glutamate receptor activation. Motor neurons also have a high perisomatic expression of the glutamate transporter protein EAAT2 and a very high expression of the cytosolic free radical scavenging enzyme Cu/Zn superoxide dismutase (SOD1) which may render this cell group vulnerable in the face of genetic or post-translational alterations interfering with the function of these proteins. More detailed characterisation of the molecular features of human motor neurons in the future may allow the strategic development of better neuroprotective therapies for the benefit of patients afflicted by ALS.

     

Postnatal development of nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the retina of the golden hamster

The histochemical method was used to investigate the postnatal development of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) -positive neurons in retinas of the golden hamster. NADPH-d-positive neurons were discernible in the retina at postnatal day (P)1. From P4 onward to adulthood, when the retina acquired its laminated characteristics, NADPH-d- positive neurons were observed in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Results showed that NADPH-d-positive neurons in INL and GCL followed different time courses and patterns in their development. NADPH-d-positive neurons in INL underwent a sharp increase from P4 to P8 (3.6-fold), followed by a decrease to 46% of the maximum at P12. This value was maintained relatively constant to the adult level. The mean diameters of NADPH-d-positive neurons in INL, which were smaller than those in the GCL for all ages, increased from P8 to P12 and from P20 to adulthood. As for neurons in the GCL, the increase in cell number was not so apparent for the earlier postnatal days until P20; thereafter, an obvious increase to the adult level was observed. The mean diameters of the NADPH-d-positive cell bodies in the GCL increased with age, except for P16-P20, during which time there was a slight and insignificant decrease. The tendency of changes in cell density was basically similar to that of the total number for both the INL and the GCL. Between P12 and P20, the density distribution map of the NADPH-d-positive neurons underwent dramatic changes: The highest density shifted from the upper central retina at the earlier postnatal days to the lower central retina in the adult. The two waves of increase in NADPH-d-positive neurons coincide with the process of axonal elongation and synaptogenesis and the acquisition of visual function and experience. It is suggested that these NADPH-d-positive neurons are related to these two developmental events.     

Synaptopodin maintains the neural activity-dependent enlargement of dendritic spines in hippocampal neurons

Synaptopodin (SYNPO) is an F-actin interacting protein expressed in dendritic spines and upregulated during the late-phase of long-term potentiation. Here, we investigated whether SYNPO regulates spine morphology through interactions with F-actin, the major cytoskeletal element of spines. In primary hippocampal neuron cultures, both endogenous and exogenous SYNPO localized preferentially in large spines under basal conditions. SYNPO overexpression did not affect the number or volume of spines in unstimulated neurons. Pharmacological activation of synaptic NMDA receptors transiently increased spine volume in control neurons, while the increase was persistent in neurons overexpressing SYNPO. In addition, exogenous SYNPO in PtK2 cells suppressed staurosporine-dependent disruption of F-actin stress fibers, suggesting that SYNPO protected F-actin from disruption. These results suggest that SYNPO stabilized activity-dependent increases in spine volume and imply that late-phase changes in spine morphology involve SYNPO.     

Prenatal development of the human spinal cord. I. Ventral motor neurons

Differentiation of the ventral motor neurons were followed in the developing human spinal cord from week 8 to week 26 of intrauterine life by thionin staining and the rapid Golgi method. Ventral roots were seen as translucent rootlets by week 8 and the ventral motor neurons were clearly identifiable by week 10, indicating that the ventral grey was earlier to differentiate than the dorsal grey, which showed a small, darkly stained, undifferentiated cell population. Lateral groupings of the motor neurons in the cervical and lumbar enlargements were obvious by week 10. Nissl substance and dendrites had reached an adult pattern by about week 18 of intrauterine life.     

Genetic disorders of motor neurons

Disorders of the motor neuron are etiologically and clinically heterogeneous and cause serious disability and death. Whereas mendelian inheritance can be demonstrated in a subset of these disorders, the genetic contribution to the sporadic forms of motor neuron degeneration are not well understood. In families with spinal muscular atrophy, Kennedy disease and amyotrophic lateral sclerosis, genetic linkage analysis and positional cloning have proven to be extremely productive. The genetics of these neurodegenerative disorders are reviewed.