Target selection of proprioceptive and motor axon synapses on neonatal V1-derived Ia inhibitory interneurons and Renshaw cells

The diversity of premotor interneurons in the mammalian spinal cord is generated from a few phylogenetically conserved embryonic classes of interneurons (V0, V1, V2, V3). Their mechanisms of diversification remain unresolved, although these are clearly important to understand motor circuit assembly in the spinal cord. Some Ia inhibitory interneurons (IaINs) and all Renshaw cells (RCs) derive from embryonic V1 interneurons; however, in adult they display distinct functional properties and synaptic inputs, for example proprioceptive inputs preferentially target IaINs, while motor axons target RCs. Previously, we found that both inputs converge on RCs in neonates, raising the possibility that proprioceptive (VGLUT1-positive) and motor axon synapses (VAChT-positive) initially target several different V1 interneurons populations and then become selected or deselected postnatally. Alternatively, specific inputs might precisely connect only with predefined groups of V1 interneurons. To test these hypotheses we analyzed synaptic development on V1-derived IaINs and compared them to RCs of the same age and spinal cord levels. V1-interneurons were labeled using genetically encoded lineage markers in mice. The results show that although neonatal V1-derived IaINs and RCs are competent to receive proprioceptive synapses, these synapses preferentially target the proximal somato-dendritic regions of IaINs and postnatally proliferate on IaINs, but not on RCs. In contrast, cholinergic synapses on RCs are specifically derived from motor axons, while on IaINs they originate from Pitx2 V0c interneurons. Thus, motor, proprioceptive, and even some interneuron inputs are biased toward specific subtypes of V1-interneurons. Postnatal strengthening of these inputs is later superimposed on this initial preferential targeting.     

Responses of cells in the rat supraoptic nucleus in vivo to stimulation of afferent pathways are different at different times of the light/dark cycle

To determine whether the daily rhythms of spike activity in the supraoptic nucleus (SON) were accompanied by changes in the behaviour of its inputs, we used conventional extracellular single cell recordings from cells in the SON of anaesthetized rats while stimulating the contralateral optic nerve and the ipsilateral suprachiasmatic nucleus (SCN). Neurones in the SON region were identified by antidromic activation and classified as oxytocin or vasopressin cells, on the basis of their spontaneous firing patterns. Approximately 27% of both oxytocin (29/108) and vasopressin (39/147) neurones were excited by stimulation of the optic nerve, and the majority of responses had a long latency (>20 ms). Very few oxytocin (3/108) and vasopressin cells (2/147) were inhibited by stimulation of the optic nerve. The pattern of the responses (excitatory, inhibitory or nonresponsive) of oxytocin and vasopressin cells to stimulation of the optic nerve was significantly related to the time of day (chi-square test; P = 0.012, oxytocin cells; P = 0.006, vasopressin cells). The proportion of oxytocin cells excited by stimulation of the optic nerve was highest at ZT 4-8 and lowest at ZT 20-24. For vasopressin cells, it was highest at ZT 12-16 and lowest at ZT 20-24. The proportion of excitatory, inhibitory and complex responses seen in oxytocin and vasopressin cells following stimulation of the SCN also changed and was significantly different at different times of day (oxytocin cells: highest proportion of excitatory responses at ZT 12-16, P = 0.029; chi-square test; vasopressin cells: highest proportion of excitatory responses at ZT 0-4, P = 0.005; chi-square test). Thus, inputs to oxytocin and vasopressin neurones from the optic nerve and some outputs from the SCN changed during the light/dark cycle. Such changes may contribute to the generation of 24-h rhythms in activity of oxytocin and vasopressin neurones and release of the peptides.     

Responses of cells in the rat suprachiasmatic nucleus in vivo to stimulation of afferent pathways are different at different times of the light/dark cycle

Conventional extracellular recordings were made from single cells in the suprachiasmatic nucleus (SCN) region of the anaesthetized rat. Each cell was tested for its response to stimulation at three sites; the contralateral optic nerve, the ipsilateral supraoptic nucleus (SON) or the ipsilateral arcuate nucleus (ARC) to determine whether the behaviour of the synapses in the SCN was different at different times. Responses to stimulation were tested once each hour and assessed by creating peristimulus time histograms. Excitatory, inhibitory or complex (consisting of more than one component) responses were seen. The responses of some cells that were recorded for several hours changed with time. Changes were seen in the responses of SCN cells to stimulation of the ARC (31/91 cells) and the SON (26/90 cells) regions, but only rarely to stimulation of the optic nerve (2/72 cells). Such differences in proportion are unlikely to have occurred by chance (P < 0.001; chi-square test). Changes seen included the appearance of both excitatory and inhibitory responses in cells that were initially unresponsive. In some cells, one component of a complex response remained constant while another component changed with time. When the cells in the SCN were treated as a group, the proportion of excitatory, inhibitory or complex responses to ARC stimulation did not remain constant throughout the light/dark cycle (P = 0.014; chi-square test). The proportion of excitatory, inhibitory or complex responses to SON and optic nerve stimulation showed no significant variation with the light/dark cycle. If a change in response can be interpreted as a change in the behaviour of a neural connection, the results imply that some of the projections to the SCN from within the hypothalamus change at different times of the light/dark cycle, whereas no change could be seen in the input from the optic nerve. Thus, some of the connections of the SCN appear not to be hard wired, but change rapidly with time.     

Cell cycle inhibitors p21 and p16 are required for the regulation of Schwann cell proliferation

Regulated cell proliferation is a crucial prerequisite for Schwann cells to achieve myelination in development and regeneration. In the present study, we have investigated the function of the cell cycle inhibitors p21 and p16 as potential regulators of Schwann cell proliferation, using p21- or p16-deficient mice. We report that both inhibitors are required for proper withdrawal of Schwann cells from the cell cycle during development and following injury. Postnatal Schwann cells express p21 exclusively in the cytoplasm, first detectable at postnatal day 7. This cytoplasmic p21 expression is necessary for proper Schwann cell proliferation control in the late development of peripheral nerves. After axonal damage, p21 is found in Schwann cell nuclei during the initiation of the proliferation period. This stage is critically regulated by p21, since loss of p21 leads to a strong increase in Schwann cell proliferation. Unexpectedly, p21 levels are upregulated in this phase suggesting that the role of p21 may be more complex than purely inhibitory for the Schwann cell cycle. However, inhibition of Schwann cell proliferation is the overriding crucial function of p21 and p16 in peripheral nerves as revealed by the consequences of loss-of-function in development and after injury. Different mechanisms appear to underlie the inhibitory function, depending on whether p21 is cytoplasmic or nuclear.     

"Dormant basket cell" hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat

The "dormant basket cell" hypothesis suggests that postinjury hippocampal network hyperexcitability results from the loss of vulnerable neurons that normally excite insult-resistant inhibitory basket cells. We have reexamined the experimental basis of this hypothesis in light of reports that excitatory hilar mossy cells are not consistently vulnerable and inhibitory basket cells are not consistently seizure resistant. Prolonged afferent stimulation that reliably evoked granule cell discharges always produced extensive hilar neuron degeneration and immediate granule cell disinhibition. Conversely, kainic acid-induced status epilepticus in chronically implanted animals produced similarly extensive hilar cell loss and immediate granule cell disinhibition, but only when granule cells discharged continuously during status epilepticus. In both preparations, electron microscopy revealed degeneration of presynaptic terminals forming asymmetrical synapses in the mossy cell target zone, including some terminating on gamma-aminobutyric acid-immunoreactive elements, but no evidence of axosomatic or axoaxonic degeneration in the adjacent granule cell layer. Although parvalbumin immunocytochemistry and in situ hybridization revealed decreased staining, this apparently was due to altered parvalbumin expression rather than basket cell death, because substance P receptor-positive interneurons, some of which contained residual parvalbumin immunoreactivity, survived. These results confirm the inherent vulnerability of dendritically projecting hilar mossy cells and interneurons and the relative resistance of dentate inhibitory basket and chandelier cells that target granule cell somata. The variability of hippocampal cell loss after status epilepticus suggests that altered hippocampal structure and function cannot be assumed to cause the spontaneous seizures that develop in these animals and highlights the importance of confirming hippocampal pathology and pathophysiology in vivo in each case.     

Number of oligodendrocyte progenitors recruited to the lesioned spinal cord is modulated by the levels of the cell cycle regulatory protein p27Kip-1

Remyelination is a critical step for recovery of function after demyelination and defines the ability to generate new myelin. This repair process is dependent on the presence of resident oligodendrocyte progenitors (OLPs) that have been shown to remyelinate axons after demyelination. We have previously shown that the levels of the cell cycle inhibitor p27Kip-1 modulate the number of neonatal cortical OLPs. We now asked whether this cell cycle molecule plays also a role in regulating the number of adult OLP in the spinal cord after demyelination induced by lysolecithin injection. The proliferative response of OLP in the spinal cord of injected wild-type (wt) and p27Kip-1 null mice was evaluated 3 days after lesion. In vivo labeling with bromodeoxyuridine (BrdU) was used to identify cells in S phase. Double immunofluorescence for the OLP marker NG2, and for BrdU was used to count the number of proliferating progenitors. Consistent with a role of p27Kip-1 in regulating the number of adult OLP in the injured spinal cord, a larger number of proliferating OLPs was observed in p27Kip-1null mice compared with wild-type controls. These cells were able to differentiate as assessed by the presence of MBP+ cells in the spinal cord 14 days after injury. We conclude that the cellular levels of the cell cycle inhibitor p27Kip-1 modulate the repair response of OLPs to injury in the adult spinal cord.     

Insulin-like growth factor 1 inhibits hair cell apoptosis and promotes the cell cycle of supporting cells by activating different downstream cascades after pharmacological hair cell injury in neonatal mice

Sensorineural hearing loss, which is mainly caused by cochlear hair cell damage, is an intractable disease, as cochlear hair cells and supporting cells are unable to proliferate in postnatal mammals. As a novel and potent treatment for sensorineural hearing loss, we have studied IGF-1 and found that it protects cochlear hair cells from the damage caused by noise and ischemic trauma. Through a clinical trial, we have also confirmed that IGF-1 is an effective treatment for idiopathic sudden sensorineural hearing loss. In the current study, we attempted to identify the downstream pathways of the IGF-1 signal and the mechanisms by which IGF-1 protects the neonatal mouse cochlear hair cells that have been damaged by neomycin. IGF-1 activated both the PI3K/Akt and MEK/ERK pathways to maintain the hair cell numbers in the injured cochlea. The PI3K/Akt pathway specifically protected the cochlear inner hair cells through the inhibition of apoptosis. In contrast, the MEK/ERK pathway induced the cell cycle promotion of Hensen's and Claudius' cells, the supporting cells that are located lateral to the outer hair cells of the cochlea. This cell cycle promotion of the supporting cells resulted in the maintenance of the outer hair cell numbers. These results indicate that IGF-1 is a growth factor that efficiently regulates different mechanisms through different downstream cascades, thereby protecting cochlear hair cells.     

The cyclin-dependent kinase inhibitors p19(Ink4d) and p27(Kip1) are coexpressed in select retinal cells and act cooperatively to control cell cycle exit

Cyclin-dependent kinase inhibitors (cdki's), including p19(Ink4d) and p27(Kip1), mediate exit from the cell cycle. To determine the function of these cdki's in regulating neurogenesis, we examined retina from wild-type, Ink4d-null, and Ink4d/Kip1-double null animals. Ink4d was expressed in progenitors and select neurons in the mature retina. Ink4d-null retina showed an extended period of proliferation, followed by apoptosis. Colabeling for p19(Ink4d) and p27(Kip1) revealed that a subpopulation of cells expressed both inhibitors. Deletion of Ink4d and Kip1 resulted in continued proliferation that was synergistic. This hyperproliferation led to an increase in number of horizontal cells and differentiated neurons reentering the cell cycle. Deletion of Ink4d and Kip1 also exacerbated the retinal dysplasia observed in Kip1-null mice, which was shown to be partly dependent on p53. These data indicate that select retinal cells express both p19(Ink4d) and p27(Kip1) and that they act cooperatively to ensure cell cycle exit.     

The inhibitory effect of tumor necrosis factor and interleukin-1 on Ia induction by interferon-gamma on endothelial cells from murine central nervous system microvessels

The effect of both tumor necrosis factor-alpha (TNF) and interleukin-1 (IL-1) on interferon-gamma (IFN)-induced Ia expression was studied using cultured endothelial cells (EC) isolated from cerebral microvessels of SJL mice. TNF inhibited Ia induction by IFN in a dose-dependent manner. The degree of inhibition by TNF was related to the IFN concentration: 200 U/ml TNF inhibited Ia expression induced by 20 U/ml IFN by 80% and Ia induced by 100 U/ml IFN by 45%. FACS analysis revealed the induction of Ia antigen on 30-40% of EC after 3 days' culture with IFN; TNF significantly reduced the percent of EC expressing Ia antigens. Identical treatment of SJL astrocytes showed TNF augmented Ia expression induced by IFN. IL-1 also inhibited Ia induction by IFN in a manner similar to that observed with TNF. The percent reduction of Ia-positive EC by Il-1 (2.0 U/ml) was 30% and 50% during incubations with 100 and 20 U/ml IFN, respectively. When combined at suboptimal concentrations IL-1 and TNF synergistically inhibited Ia expression induced by IFN. These results demonstrate that TNF acts on EC and astrocytes in a disparate manner and indicate that TNF and IL-1 can synergistically down-regulate immune responses involving central nervous system EC.     

Proliferation and death of conditionally immortalized neural cells from murine neocortex: p53 alters the ability of neuron-like cells to re-enter the cell cycle

Neurons are distinctive in that they are generally considered to be permanently post-mitotic cells. The oncoprotein p53 is a key regulator in neuronal development, notably in cell proliferation and neuronal death. We hypothesize that p53 maintains the post-mitotic characteristic of differentiated neurons. New lines of conditionally immortalized cortical cells were generated to test this hypothesis. Populations of cells were obtained from the neocortices of dual transgenic mice that were null for p53 and expressed a temperature-sensitive SV40 large T antigen. At a permissive temperature (32 degrees C), the cells continued to proliferate and most expressed nestin and proteins associated with glia. At a non-permissive temperature (39 degrees C), the cells expressed cytoskeletal proteins associated with differentiated neurons such as microtubule associated protein 2 and neurofilament 200. Under permissive conditions, both p53(+/-) and p53(-/-) cells exhibited similar cycling behaviors; the length of the cell cycle was 13-15 h and >85% of the cells were actively cycling. In non-permissive conditions, most p53(+/-) cells stopped dividing, whereas the p53(-/-) cells continued to proliferate. The survival of the cells also differed. In the non-permissive conditions, many p53(+/-) cells died following treatment with a neurotoxin (ethanol, 400 mg/dl), whereas the p53(-/-) cells did not. After re-introduction to the permissive conditions, both cell lines expressed neuron-like characteristics, but only the p53(-/-) cells retained their ability to cycle. Therefore, p53-mediated activities appear to be involved in the proliferation, survival, and post-mitotic nature of neuron-like cells.     

Reduction of the cell cycle length by decreasing G1 phase and cell cycle reentry expand neuronal progenitor cells in the subventricular zone of adult rat after stroke.

A critical determinant of proliferation of progenitor cells is the duration of the cell division cycle. Stroke increases proliferation of progenitor cells in the subventricular zone (SVZ). Using cumulative and single S-phase labeling with 5-bromo-2'-deoxyuridine, we examined cell cycle kinetics of neural progenitor cells in the SVZ after stroke. In nonstroke rats, 20% of the SVZ cell population was proliferating. However, stroke significantly increased dividing cells up to 31% and these cells had a cell cycle length (T(C)) of 15.3 h, significantly (P < 0.05) shorter than the 19 h Tc in nonstroke SVZ cells. Few terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells were detected in the SVZ cells of nonstroke and stroke groups, suggesting that the majority of dividing cells in the SVZ do not undergo apoptosis. Cell cycle phase analysis revealed that stroke substantially shortened the length of the G1 phase (9.6 h) compared with the G1 phase of 12.6 h in nonstroke SVZ cells (P < 0.03). This reduction in G1 contributes to stroke-induced reduction of T(C) because no significant changes were detected on the length of S, G2 and M phases between two groups. Furthermore, compared with progenitor cells in nonstroke SVZ (10%), a greater proportion (14%) of progenitor cells in stroke SVZ reentered the cell cycle after mitosis (P < 0.05). These results show that an increase in proliferating progenitor cells in the SVZ contributes to stroke-induced neurogenesis and this increase is regulated by shortening the length of the cell cycle, decreasing the G1 phase and increasing cell cycle reentry.     

Ultrastructural features and synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from those of granule cells in the granule cell layer

Several investigators have shown the existence of dentate granule cells in ectopic locations within the hilus and molecular layer using both Golgi and retrograde tracing studies but the ultrastructural features and synaptic connections of ectopic granule cells were not previously examined. In the present study, the biocytin retrograde tracing technique was used to label ectopic granule cells following injections into stratum lucidum of CA3b of hippocampal slices obtained from epileptic rats. Electron microscopy was used to study hilar ectopic granule cells that were located 20–40 μm from the granule cell layer (GCL). They had ultrastructural features similar to those of granule cells in the GCL but showed differences, including nuclei that often displayed infoldings and thicker apical dendrites. At their origin, these dendrites were 6 μm in diameter and they tapered down to 2 μm at the border with the GCL. Both biocytin-labeled and unlabeled axon terminals formed exclusively asymmetric synapses with the somata and proximal dendrites of hilar ectopic granule cells. The mean number of axosomatic synapses for these cells was three times that for granule cells in the GCL. Together, these data indicate that hilar ectopic granule cells are postsynaptic to mossy fibers and have less inhibitory input on their somata and proximal dendrites than granule cells in the GCL. This finding is consistent with recent physiological results showing that hilar ectopic granule cells from epileptic rats are more hyperexcitable than granule cells in the GCL.     

Evidences that beta1 integrin and Rac1 are involved in the overriding effect of laminin on myelin-associated glycoprotein inhibitory activity on neuronal cells

During neurite elongation, migrating growth cones encounter both permissive and inhibitory substrates, such as laminin and MAG (myelin-associated glycoprotein), respectively. Here, we demonstrated on two neuronal cell lines (PC12 and N1E-115), that laminin and collagen hampered, in a dose-dependent manner, MAG inhibitory activity on several integrin functions, i.e., neurite growth, cell adhesion and cell spreading. Using a function blocking antibody, in PC12 cells, we showed that alpha1beta1 integrin is required in these phenomena. In parallel, we observed that MAG perturbs actin dynamics and lamellipodia formation during early steps of cell spreading. This seemed to be independent of RhoA activation, but dependent of Rac-1 inhibition by MAG. Laminin overrode MAG activity on actin and prevented MAG inhibition NGF-induced Rac1 activation. In conclusion, we evidenced antagonistic signaling between MAG receptors and beta1 integrins, in which Rac-1 may have a central function.     

Ca2+/calmodulin-dependent modulation of cell cycle elements pRb and p27kip1 involved in the enhanced proliferation of lymphoblasts from patients with Alzheimer dementia

Failure of cell cycle regulation in neurons might be critically involved in the process of neurodegeneration in Alzheimer's disease (AD). We present here evidence to support the hypothesis that cell cycle alterations occur in cells other than neurons in AD sufferers. Lymphocytes from AD patients immortalized with Epstein-Barr virus showed an enhanced rate of proliferation and increased phosphorylation of the retinoblastoma protein (pRb) and other members of the family of pocket proteins compared with cell lines derived from normal age-matched controls. The calmodulin antagonist calmidazolium, as well as W-7 and W-13, abrogated the enhanced activity of AD cells without altering the normal basal rate of proliferation. The effect of calmidazolium was accompanied by partially dephosphorylation of pRb. No changes were found in the expression levels of the G1 cyclin/Cdks complexes. However, lymphoblasts derived from AD patients showed reduced levels of the Cdk inhibitor p27(kip1), which were restored after anti-calmodulin treatment of the cultures. These observations suggest that in AD cells the enhanced rates of cell proliferation and phosphorylation of pRb and the intracellular content of p27(kip1) may be interrelated events controlled by a mechanism dependent on the Ca(2+)/calmodulin signaling pathway. The distinct functional features of lymphoblastoid cells from AD patients offer an invaluable, noninvasive tool to investigate the etiopathogenesis, and eventually, for the early diagnosis and prognosis of this devastating disease.     

Absence of the cell cycle inhibitor p21Cip1 reduces LPS-induced NO release and activation of the transcription factor NF-kappaB in mixed glial cultures

We have studied possible differences in glial activation between cells from wild-type and p21Cip1-/- mice. We compared the effect of serum mitogenic stimulation on proliferation rate and on the total number of glial cells after 7 days of culture. No differences between wild-type and p21Cip1-/- glial cells were observed. We also compared the effect of lipopolysaccharide (LPS) from Escherichia coli, an agent widely used to induce glial activation. Nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) release, and nuclear factor kappa-B (NF-kappaB) activation were evaluated as indicators of glial activation. We observed an attenuation of NO release and NF-kappaB activation in p21Cip1-/- glial cells when compared with glial cells from wild-type mice. In contrast, TNF-alpha release was enhanced in p21Cip1-/- glia. These results suggest that the cell cycle inhibitor p21Cip1 plays a role in the inflammatory response induced by LPS.     

Developmental expression of the cell cycle and apoptosis controlling gene,Lot1, in the rat cerebellum and in cultures of cerebellar granule cells

The Lot1 gene encodes a zinc finger protein that, in vitro, concurrently regulates apoptosis and cell cycle arrest and belongs to a recently identified family of proteins with oncogenic and tumor-supressor functions. The present study, based on the development of the first antibody reportedly produced against rat Lot1, examines protein expression during normal development of the rat cerebellum and following methylazoxymethanol (MAM) administration, which results in hypoplasia of the cerebellar granule cell population. Using light microscopic immunocytochemistry, specific immunostaining for the Lot1 protein was observed at postnatal days 2 to 7 in the superficial external granule layer composed primarily of proliferating neuronal precursor cells. Purkinje cells showed distinct nuclear labeling at P7. In the adult cerebellum, the overall low Lot1 level was essentially associated with Purkinje cells. Experimentally altered developmental conditions, such as those obtained through MAM-induced microencephaly, did not drastically affect the pattern of Lot1 expression. In particular, Purkinje cells continued to show normal levels of immunoreactivity notwithstanding the altered cerebellar architecture. Primary cultures of cerebellar granule cells showed a temporal pattern of Lot1 expression resembling that of in vivo development, with mRNA and protein levels progressively decreasing with differentiation. When cerebellar granule cells were exposed to different neurotoxic challenges, Lot1 appeared not affected by purely apoptotic cell death, while transitorily induced by mixed necrotic-apoptotic cell death.