Abnormality in the cerebellar folial pattern of C57BL/6J mice

The characteristic folial pattern of the mouse cerebellum is formed during postnatal development. We observed this process in C57BL/6J (B6) mice in detail, and found an abnormal folial pattern in a specific region (lobules VIII and IX of the vermis) in a substantial number of B6 mice. The frequency of this abnormality increased during postnatal development and reached 55% in the adult. Thus, the present study showed an abnormality in the cerebellar folial pattern of B6 mice, a mouse widely used in knockout studies, and called for caution in the phenotypic analysis of knockout mice of the B6 genetic background.     

Somatostatin expression in the cerebellar cortex during postnatal development

Summary The distribution of somatostatin-immunoreactive (SOM-IR) elements in the cerebellar cortex of the rat has been studied at different stages of postnatal development (from birth to day 30) and in adult animals using immunohistochemistry. The results showed that in vermis of new born animals there are three main groups of SOM-IR structures within the cortex which subsequently spread along the Purkinje cell layer. In addition, both in the vermis and in the lateral lobes, numerous more evenly distributed SOM-positive cells and fibers could be seen. SOM-IR Golgi cells, Purkinje cells and climbing fibers could then be recognized during the subsequent developmental stages. In the vermal zone, SOM-IR Purkinje cells formed patches, which seemed to be part of a sagittal columnar or band-like organization. This was most obvious between days 5 and 21 of postnatal development. Subsequently there was a reduction in the number of immunoreactive Purkinje cells but a patchy disposition remained. In addition high numbers of SOM-IR Purkinje and Golgi cells and also climbing fibers were identified in the flocculus and paraflocculus at all stages of development studied, and they were also seen in the adult rats in these regions. In the lateral lobes expression of SOM-like immunoreactivity (LI) decreased and almost completely disappeared in adult animals. The present results demonstrate that a SOM or a SOM-LI peptide can be transiently detected in many Purkinje and Golgi cells in the cerebellar cortex, suggesting a role in events related to developmental processes. However, in some regions and structures SOM-LI can be seen also in adult animals. Dedicated to: Prof. Alf Brodal     

Sequential accumulation of iron in glial cells during chicken cerebellar development

Iron is an essential, but potentially harmful, metal in the brain. In normal brain, iron has been reported to accumulate mainly in glial cells and occasionally in neurons in some particular nuclei. However, the majority of investigations have targeted the adult brain. Here, we investigated spatiotemporal localization of iron in developing and adult chicken cerebellum using iron histochemistry. Iron reactivity was not detected in the chick cerebellum until embryonic day 12. Iron accumulation was first found in mature myelinating oligodendrocytes located in the inner part of the cerebellar folium at embryonic day 14. From embryonic day 20, iron-positive mature myelinating oligodendrocytes were localized in the white matter and the granular layer. From post-hatching day 2, iron accumulation was observed in Bergmann glia in the Purkinje cell layer as well as in mature myelinating oligodendrocytes. Iron accumulation in microglia was observed in the granular and molecular layers at post-hatching month 12. Our data indicate that during cerebellar development iron is accumulated in a unique sequence according to individual requirements or microenvironmental demands.     

Development and migration of Purkinje cells in the mouse cerebellar primordium

Summary The mode of Purkinje cell migration in the mouse cerebellar primordium was examined immunohistochemically, by marking Purkinje cells with anti-spot 35 antibody and labeling them with 5-bromodeoxyuridine. The cells migrated radially from the neuroepithelium of the fourth ventricle towards the cortical surface between the 13th and 17th days (E13–E17) of gestation. Regional differences in the migratory process were evident: the final settlement of the Purkinje cells proceeded earlier in the lateral and posterior parts of the primordium, exhibiting latero-medial and posteroventral-anterodorsal diminishing sequences. To elucidate the factors involved in the migration, the arrangement of radial glial fibers, and expression of the cell adhesion molecule, tenascin, were examined immunohistochemically with the monoclonal antibody 1D11, a marker for both immature and mature astroglia, and an anti-tenascin antibody. At E14, 1D11-immunopositive fibers were seen to extend from the ventricle to the pial surface, and the cell bodies of immature glia migrated after E15 towards the cortex, shortening the radial processes whose end-feet were attached to the pia mater. Tenascin, which possesses a neuron-glial adhesiveness, was also expressed on the radial fibers during the migration of the Purkinje cells. The fibers were closely apposed to the migratory Purkinje cells, and their arrangement and orientation accorded with the migratory direction of the Purkinje cells. Further, changes in the molecular species of antigens detected by both the 1D11 and anti-tenascin antibodies were observed by immunoblotting analysis during the course of cerebellar development. These findings suggest that the arrangement of radial glia and expression of adhesion molecules may be involved in the control and guidance of Purkinje cell migration.This paper is dedicated to Professor Fred Walberg on the occasion of his 70th birthday     

Time of origin and distribution of a new cell type in the rat cerebellar cortex

Summary A new cell type was identified in the granular layer of the rat cerebellum. It has a lightly staining nucleus with a nucleolus, it is spherical in shape and is larger than granule cells and smaller than Golgi cells. These pale cells are preferentially concentrated in the nodulus, the ventral uvula, the lingula, the flocculus, and parts of the paraflocculus. According to autoradiographic (³H-thymidine) evidence, over 60% of these pale cells are formed on embryonic days 19 and 20, and their production comes to an end soon after birth, prior to the differentiation of granule cells. The possible relation of pale cells to vestibular afferents of the cerebellar cortex is discussed.     

Afferent volleys in limb nerves influencing impulse discharges in cerebellar cortex I. In mossy fibers and granule cells

Summary This paper is the first of a series in which the processing of information in the cerebellum has been studied by investigating the effects that known inputs from limb nerves produce on the unitary spike potentials in the cerebellar cortex. These spikes have been recorded extracellularly at all depths along microelectrode tracks in the 5th, 4th and 3rd lobules of the anterior lobe in the lateral vermis or in the pars intermedia. These units have a background frequency of discharge, often very irregular, and computer averaging techniques have been employed in order to derive reliable information on the time course and intensity of the excitatory and/or inhibitory actions produced by the input against this background.Most of the spike responses recorded from the granular layer fall into two classes, one characteristic of impulses in mossy fibers, and the other of impulse discharges from granule cells. Both in the spontaneous background and in the response to afferent volleys in limb nerves the mossy fibers exhibit a performance in close accord with that described for the discharges up the spino-cerebellar tracts. The short latency of 6–9 msec for hindlimb stimuli and the high frequency burst response of 2–4 impulses are characteristic. The mossy fibers displayed a wide variety of responses to the wide range of testing inputs, there being various combinations of excitatory and inhibitory responses and also delayed excitatory actions, all of which must be assumed to be reflections of synaptic influences on the cells of origin of the mossy fibers in the spinal cord.Granule cells have a longer latency by several milliseconds, 9–20 msec for the hindlimb, and a slower frequency in their burst response which tended to be longer and more irregular. The small unitary spike potentials are more difficult to isolate. Also with repetitive stimulation granule cells are more readily depressed than are mossy fibers.Usually a granule cell exhibits a wider range of response to the various cutaneous and muscular afferents of a limb. Both mossy fibers and granule cells may display reciprocal responses to volleys from muscle nerves to antagonistic muscles. This attempt to define properties of the mossy fiber and granule cell spike potentials should help in their identification in future investigations.Post-Doctoral Fellow NINDS (1F2NB40,544101 NSRB).Post-Doctoral Fellow UHF Grant No. FTF-3-UB-70.     

Effects of maternal alcohol consumption during breastfeeding on motor and cerebellar Purkinje cells behavior in mice

Purkinje cells (PCs) are the sole output from the cerebellar cortex. Their electrophysiological behavior may serve as indicator of chronic ethanol effects on the cerebellum. Here, we studied the effects of ethanol consumption through breastfeeding on motor behavior, histology and PCs electrophysiology. Mice with different maternal drinking regimen (ethanol, E or sucrose, S) during prenatal (E/and S/) and postnatal period (/E and/S) were compared. Motor performance in the runway and rotarod tests was significantly worse in mice exposed to ethanol prenatally (E/E and E/S) than in mice exposed to sucrose (S/S), with a limited influence, if any, of mother regimen during lactation (E/S vs E/E). A loss of 20–25% of PCs was found for both E/S and E/E compared to S/S mice but PC numbers were similar in S/E and S/S. Mean PC spontaneous simple spike firing rate and rhythmicity were higher in E/S and E/E than in S/S but there was no difference between S/E and S/S. Complex spike frequency was similar in all groups. In contrast, complex spike duration and the related pause induced on the simple spike firing were shorter in E/E and in E/S, but no difference was found between S/E and S/S. We conclude that cerebellar dysfunction induced by maternal ethanol consumption in mice depends upon the drinking regimen during pregnancy and not during lactation.     

Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra. Attempted replacement of these neurons by stem cells has proved inconclusive. Bone marrow mesenchymal stem cells (MSC) are multipotent, differentiating into a variety of cells, including neuron-like cells. We used the 6-hydroxydopamine (6-OHDA) animal model of Parkinson's disease to assess migration and differentiation of transplanted MSC. We found in rodents that transplanted MSC survive better in the 6-OHDA-induced damaged hemisphere compared to the unlesioned side. Moreover, contralaterally engrafted MSC migrated through the corpus callosum to populate the striatum, thalamic nuclei and substantia nigra of the 6-OHDA-lesioned hemisphere. In conclusion, we demonstrate that 6-OHDA-induced damage increases the viability of transplanted MSC and attracts these cells from the opposite hemisphere.     

Is motilin a cerebellar peptide in the rat?

Summary Motilin was demonstrated by the immunoperoxidase technique in endocrine cells of the gastrointestinal tract using several specific antisera. Motilin-like immunoreactivity could only be demonstrated with one of these antisera and was observed in Purkinje cells and dendrites of the cerebellum, in pyramidal cells and dendrites of the cerebral cortex and in dendrites of the CA3 field of the hippocampus of the rat.Very low motilin-like immunoreactivity was found in cerebellum as well as in cerebral cortex using radioimmunoassay. However, using reverse phase liquid chromatography combined with UV-detection and radioimmunoassay, no peak of a peptide corresponding to synthetic motilin was detectable in rat cerebellar extracts, in contrast to findings in rat duodenum. The results do not suggest that motilin is an intrinsic neuroactive substance of the cerebellum.     

Nuclear envelope invaginations in hamster pyramidal cells during development and aging

Nuclear envelope invaginations were observed in pyramidal cell nuclei of the hamster frontal cortex during development and aging. These invaginations which began to appear at 10 days did not recede at maturity as has been observed in certain other cell types, but persisted in the adult hamster and during subsequent aging. Morphometric data showed a significant increase in the number of nuclear envelope invaginations and in their length per unit area of the nucleus. This increase was positively correlated with age until 500 days and is suggestive of a continued high metabolic activity that did not subside following the rapid growth phase of the pyramidal neurons.     

Understanding the development and function of T follicular helper cells

A fundamental function of T helper (Th) cells is to regulate B-cell proliferation and immunoglobulin class switching, especially in the germinal centers. Th1 and Th2 lineages of CD4⁺ T cells have long been considered to play an essential role in helping B cells by promoting the production immunoglobulin G2a (IgG2a) and IgG1/IgE, respectively. Recently, it has become clear that a subset CD4⁺ T cells, named T follicular helper (Tfh) cells, is critical to B-cell response induction. In this review, we summarize the latest advances in our understanding of the regulation of Tfh cell differentiation, the relationship of Tfh cells to other CD4⁺ T-cell lineages, and the role of Tfh cells in health and disease.     

Effects of berberine on 6-hydroxydopamine-induced neurotoxicity in PC12 cells and a rat model of Parkinson's disease

Protoberberine isoquinoline alkaloids including berberine inhibit dopamine biosynthesis and aggravate l-DOPA-induced cytotoxicity in PC12 cells. In this study, the effects of berberine on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in PC12 cells and on unilateral 6-OHDA-lesioned rats were investigated. In PC12 cells, berberine at 10 and 30 μM associated with 6-OHDA (10, 20, and 50 μM) enhanced cytotoxicity at 48 h compared to 6-OHDA alone, indicated by an increase in apoptotic cell death. In addition, treatment with berberine (5 and 30 mg/kg, i.p.) for 21 days in 6-OHDA-lesioned rats markedly depleted tyrosine hydroxylase-immunopositive cells in the substantia nigra as compared to berberine-untreated rats. Further, the levels of dopamine and norepinephrine were also significantly decreased by berberine administration (5 and 30 mg/kg) in the striatal regions of 6-OHDA-lesioned rats. These results suggested that berberine aggravated 6-OHDA-induced cytotoxicity in PC12 cells, and led to the degeneration of dopaminergic neuronal cells in the substantia nigra of 6-OHDA-lesioned rats. It is, therefore, suggested that the use of long-term l-DOPA therapy with isoquinoline derivatives including berberine may need to be examined for the presence of adverse symptoms.     

Cutaneous mechanoreceptors influencing impulse discharges in cerebellar cortex. II. In Purkyně cells by mossy fiber input

Summary This paper gives an account of single Purkyn cell responses when three types of mechanical stimulation, as in the previous paper, are applied to the forefoot and hindfoot of the decerebrate unanesthetized cat. Attention was concentrated on the effects of brief mechanical pulses to the footpad. Recording was extracellular by glass microelectrodes and special precautions were taken in identifying the spike responses as being due to a single Purkyn cell and in securing its effective isolation for our computer averaging techniques, as described in the previous papers. All Purkyn cells were in the ipsilateral anterior lobe in the lateral vermis or pars intermedia of lobules III, IV, V, except for a few recordings in the extreme rostral zone of lobule VI.Mechanical pulses or taps evoked responses from many Purkyn cells which were pure excitatory, pure inhibitory or admixtures thereof. The latencies of onset were usually in the range of 12–20 msec from the onset of the tap, which tends to be a little longer than the observed latencies for mossy fiber responses described in the preceding paper. There was often a considerable difference in the sizes of the responses evoked from different pads of the same foot, and the usual threshold for response was below 0.2 mm amplitude. Durations of responses were usually 10–20 msec for excitation and 50–100 msec for inhibition.Pressure pulses to the central foot pads of 2 sec duration evoked a wide variety of responses: brief phasic at on and off that could be admixtures of excitation and inhibition; almost pure tonic excitations or inhibitions that were well maintained during the 2 sec; phasic-tonic responses in various relative degrees. Usually 500 g was maximally effective and the threshold was below 100 g.Hair receptors were stimulated preferentially by brief air jets, there being brief excitatory or inhibitory responses much as with taps, but with rather longer latency. The effective area was usually fairly extensive over the hairy skin of the foot.In general the effects on Purkyn cells by cutaneous mechanoreceptors acting via mossy fibers were in accord with the mossy fiber responses reported in the preceding paper and with the well-known excitatory and inhibitory effects that are exerted by mossy fiber inputs on Purkyn cells.     

Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat

The progressive renal disease model of chronic uninephrectomy-desoxycorticosterone-trimethylacetate (UNX-DOCA) hypertension is associated with mesangial proliferation as a major disease mechanism. A detailed structural analysis of the alterations in glomerular structure which accompany the development of sclerosis in this model has not been made. Male Munich-Wistar rats underwent UNX, received weekly injections of the aldosterone agonist DOCA and 1% sodium chloride as drinking solution and were compared with sham operated controls (CON). Thirty eight days after onset, UNX animals had an albuminuria of 183±180 mg/day versus 0.38±0.22 mg/day in CON. Kidneys were fixed by total body perfusion and renal tissue processed for light and electron-microscopy. Superficial and deep total glomerular volume increased from 2.18±0.15 (deep: 2.57±0.24) 10⁶ m³ in CON to 3.98±0.81 (deep: 3.95±0.63) 10⁶ m³ in UNX. In addition to overall tuft hypertrophy, structural analysis revealed severe destruction of tuft architecture with mesangial expansion and/or capillary ballooning, leading to local tuft enlargements. Podocytes overlying the expanded areas appeared unable to adapt to cover the increased tuft surfaces. They developed severe lesions in cell architecture leading to denudation of glomerular basement membrane (GBM)-areas. Naked GBM appears to represent a nidus for hyalinosis, thrombosis and synechia formation, which progresses to segmental sclerosis. In the UNX-DOCA model of chronic glomerular hypertension local mesangial expansion was frequently encountered but no evidence was found that mesangial proliferation and matrix production proceeded to sclerosis. The crucial damage to the glomerulus in this model would appear to be attributable to podocyte failure, with the resultant GBM denudation triggering synechia formation, hyalinosis and ultimately glomerulosclerosis.     

Cutaneous mechanoreceptors influencing impulse discharges in cerebellar cortex. III. In Purkyně cells by climbing fiber input

Summary An account is given of single Purkyn cell responses that are evoked by cutaneous mechanoreceptors acting via climbing fibers (CF). There was no complication by mossy fiber input, which was selectively depressed by light pentothal anesthesia.Brief mechanical pulses (taps) to the footpads and air jets on hairy skin were very effective in evoking CF responses, the usual range of latencies being 25–35 msec for the hindfoot and 19–30 msec for the forefoot. There was often a considerable difference in the effectiveness of the different pads of the same foot. For any particular Purkyn cell there was a close correspondence between the receptive fields for footpads and hairy skin.The threshold was often below 0.02 mm taps for the most sensitive pads, and was even lower for more prolonged mechanical displacements. Mechanical pulses of 80 to several hundred msec in duration were effective in evoking CF responses of Purkyn cells at off as well as at on. This finding and the very low threshold strongly suggest Pacinian corpuscles as the principal receptors concerned in activating the CF system.Repetitive taps to footpads were effective at slow frequencies, 5 or 10 Hz, but there was a response only to the first tap with a frequency of 65 Hz.     

The influence of a spatiotemporal 3D environment on endothelial cell differentiation of human induced pluripotent stem cells

Current EC differentiation protocols are inefficient, and the phenotypes of the differentiated ECs are only briefly stable, which significantly inhibits their utility for basic science research. Here, a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol, up to 45% of the differentiated hiPSCs assumed an EC phenotype, and after purification, greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. Gene and protein expression levels of CD31, CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively, these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and, furthermore, the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks in vitro.     

Serotonin and neuroendocrine peptides influence DNA synthesis in rat and human small intestinal cells in vitro

Animal studies suggest a mediator role for neuroendocrine peptides and amines in regulating cell proliferation in the gastrointestinal epithelium. Our aim was to examine the effect of serotonin and selected gastrointestinal peptides on DNA synthesis in a rat and human small intestinal cell line in vitro. IEC-6 and FHs-74 cells were incubated with epidermal growth factor (EGF), insulin-like growth factor II, glucagon, substance P, neurokinin A, calcitonin gene-related peptide (GRP, CCGRP), neurotensin and serotonin. The cells were labelled with [methyl-³H] thymidine and processed for autoradiography. DNA synthesis was evaluated by the labelling index. Epidermal growth factor, insulin-like growth factor II, glucagon, and substance P increased the labelling index in a dose-related manner (P < 0.003). In contrast, a significant dose-dependent reduction of the labelling index was observed after administration of serotonin and neurokinin A (P < 0.0001). Neurotensin and CGRP did not affect the labelling index. EGF, insulin-like growth factor II, glucagon, substance P, serotonin and neurokinin A may be important physiological regulators of proliferation, of gastrointestinal cells.     

Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells

Melatonin receptor activation has been linked to the regulation of neurotrophic factors, including the brain-derived neurotrophic factor (BDNF). To further characterize the effects of melatonin receptor stimulation on neuronal BDNF, we used a clinically available novel agonist for MT1 and MT2 melatonin receptors, ramelteon. Primary cultures of cerebellar granule cells (CGC) have been established as an in vitro model for studying neuronal BDNF. We took advantage of the availability of MT1- and MT2-deficient (knockout; KO) mice to employ primary CGC prepared from wild type (WT), MT1 KO, and MT2 KO mice. We investigated the effects of ramelteon on BDNF protein and mRNA content. Administered in a low nanomolar range, ramelteon increased BDNF protein content in all three types of mouse CGC. This ramelteon-triggered BDNF protein elevation was not preceded by a BDNF mRNA increase. However, it was prevented by treatment of cultures with a protein synthesis inhibitor cycloheximide. These results demonstrated that the MT1/MT2 melatonin receptor agonist ramelteon is capable of increasing BDNF protein in neurons expressing either of the two melatonin receptor types and that this action of ramelteon involves translational mechanisms. Further research is needed to explore the putative influence of ramelteon on BDNF-associated neuroplasticity.