Mitochondrial Morphogenesis,Dendrite Development,and Synapse Formation in Cerebellum Require both Bcl-w and the Glutamate Receptor δ2

Bcl-w belongs to the prosurvival group of the Bcl-2 family, while the glutamate receptor δ2 (Grid2) is an excitatory receptor that is specifically expressed in Purkinje cells, and required for Purkinje cell synapse formation. A recently published result as well as our own findings have shown that Bcl-w can physically interact with an autophagy protein, Beclin1, which in turn has been shown previously to form a protein complex with the intracellular domain of Grid2 and an adaptor protein, nPIST. This suggests that Bcl-w and Grid2 might interact genetically to regulate mitochondria, autophagy, and neuronal function. In this study, we investigated this genetic interaction of Bcl-w and Grid2 through analysis of single and double mutant mice of these two proteins using a combination of histological and behavior tests. It was found that Bcl-w does not control the cell number in mouse brain, but promotes what is likely to be the mitochondrial fission in Purkinje cell dendrites, and is required for synapse formation and motor learning in cerebellum, and that Grid2 has similar phenotypes. Mice carrying the double mutations of these two genes had synergistic effects including extremely long mitochondria in Purkinje cell dendrites, and strongly aberrant Purkinje cell dendrites, spines, and synapses, and severely ataxic behavior. Bcl-w and Grid2 mutations were not found to influence the basal autophagy that is required for Purkinje cell survival, thus resulting in these phenotypes. Our results demonstrate that Bcl-w and Grid2 are two critical proteins acting in distinct pathways to regulate mitochondrial morphogenesis and control Purkinje cell dendrite development and synapse formation. We propose that the mitochondrial fission occurring during neuronal growth might be critically important for dendrite development and synapse formation, and that it can be regulated coordinately by multiple pathways including Bcl-2 and glutamate receptor family members.     

The Postnatal Development of the Cerebellum— A fMRI and Silver Study

Summary The aim of this study was to evaluate the postnatal development of the cerebella of the pig and to compare this with the activation of the fMRI. The cells in the cerebella were studied by silver technique and the activation of the fMRI in the cerebella was initiated by flexion and extension of the hind paw. Our results showed an increase of the branching of the cells of the cerebellar cortex postnatally, coordinated with registration of fMRI active sites in the cerebella at 6-month postnatal. We concluded that the full maturation of the cerebella was around 6-month postnatal in the pig.     

Use of M1–M5 Muscarinic Receptor Knockout Mice as Novel Tools to Delineate the Physiological Roles of the Muscarinic Cholinergic System

In this review we report recent findings on the physiological role of the five known muscarinic acetylcholine receptors (mAChRs) as shown by gene targeting technology. Using knockout mice for each mAChRs subtype, the role of mAChRs subtypes in a number of physiological functions was confirmed and new activities were discovered. The M₁ mAChRs modulate neurotransmitter signaling in cortex and hippocampus. The M₃ mAChRs are involved in exocrine gland secretion, smooth muscle contractility, pupil dilation, food intake, and weight gain. The role of the M₅ mAChRs involves modulation of central dopamine function and the tone of cerebral blood vessels. mAChRs of the M₂ subtype mediate muscarinic agonist-induced bradycardia, tremor, hypothermia, and autoinhibition of release in several brain regions. M₄ mAChRs modulate dopamine activity in motor tracts and act as inhibitory autoreceptors in striatum. Thus, as elucidated by gene targeting technology, mAChRs have widespread and manifold functions in the periphery and brain.     

Can Clues from Evolution Unlock the Molecular Development of the Cerebellum?

The cerebellum sits at the rostral end of the vertebrate hindbrain and is responsible for sensory and motor integration. Owing to its relatively simple architecture, it is one of the most powerful model systems for studying brain evolution and development. Over the last decade, the combination of molecular fate mapping techniques in the mouse and experimental studies, both in vitro and in vivo, in mouse and chick have significantly advanced our understanding of cerebellar neurogenesis in space and time. In amniotes, the most numerous cell type in the cerebellum, and indeed the brain, is the cerebellar granule neurons, and these are born from a transient secondary proliferative zone, the external granule layer (EGL), where proliferation is driven by sonic hedgehog signalling and causes cerebellar foliation. Recent studies in zebrafish and sharks have shown that while the molecular mechanisms of neurogenesis appear conserved across vertebrates, the EGL as a site of shh-driven transit amplification is not, and is therefore implicated as a key amniote innovation that facilitated the evolution of the elaborate foliated cerebella found in birds and mammals. Ellucidating the molecular mechanisms underlying the origin of the EGL in evolution could have significant impacts on our understanding of the molecular details of cerebellar development.     

Alterations in the Cell Cycle in the Cerebellum of Hyperbilirubinemic Gunn Rat: A Possible Link with Apoptosis?

Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.     

Changes in Cationic Selectivity of the Nicotinic Channel at the Rat Ganglionic Synapse: A Role for Chloride Ions?

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.     

Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection: Evaluation in Murine Skin

Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended.     

Muscarinic Stimulation Promotes Cultured Purkinje Cell Survival: A Role for Acetylcholine in Cerebellar Development?

Abstract: The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6–7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 µ M ) and pirenzepine (10 n M ), but not by methoctramine (25 n M ). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high-affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine-sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.     

Does Lighting Manipulation During Incubation Affect Hatching Rhythms and Early Development of Sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LD_W), blue light (LD_B; λ_peak?=?463?nm), or red light (LD_R; λ_peak?=?685?nm). Eggs exposed to LD_B had the highest hatching rate (94.5%?±?1.9%), whereas LD_R and DD showed the lowest hatching rate (54.4%?±?3.9% and 48.4%?±?4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LD_B (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LD_W and LD_R (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LD_B showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LD_R and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish. (Author correspondence: borja@um.es)     

Origin of glutaminyl-tRNA synthetase: An example of palimpsest?

Sequence data and evolutionary arguments suggest that a similarity may exist between the C-terminal end of glutaminyl-tRNA synthetase (GlnRS) and the catalytic domain of glutamine amidotransferases (GATs). If true, this would seem to imply that the amidation reaction of the Glu-tRNA^GIn complex was the evolutionary precursor of the direct tRNA^GIn aminoacylation pathway. Since the C-terminal end of GlnRS does not now have an important functional role, it can be concluded that this sequence contains vestiges that lead us to believe that it represents a palimpsest. This sequence still conserves the remains of the evolutionary transition: amidation reaction aminoacylation reaction. This may be important in deciding which mechanism gave origin to the genetic code organization. These observations, together with results obtained by Gatti and Tzagoloff [J. Mol. Biol. (1991) 218: 557–568], lead to the hypothesis that the class I aminoacyl-tRNA synthetases (ARSs) may be homologous to the GATs of the trpG subfamily, while the class Il ARSs may be homologous to the GATs of the purF subfamily. Overall, this seems to point to the existence of an intimate evolutionary link between the proteins involved in the primitive metabolism and aminoacyl-tRNA synthetases.     

Giant Cells: Contradiction to Two-Hit Model of Tuber Formation?

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by the formation of hamartomatous lesions in many organs, including brain, heart or kidneys. It has been found that TSC is caused by the mutation in one of the two tumor suppressor genes: TSC1 or TSC2, encoding hamartin and tuberin, respectively. According to Knudson’s two-hit model of tumorigenesis, second-hit mutation and resulting loss of heterozygosity (LOH) of a tumor suppressor gene is necessary for tumor formation. In fact, LOH is commonly found in several types of hamartomas formed in the process of tuberous sclerosis, but, interestingly, not in brain lesions, containing characteristic giant cells. In this paper, we review literature covering origination of giant cells and present several hypotheses explaining why in spite of the presence of hamartin and tuberin, brain lesions form in TSC patients.     

Giant Cells: Contradiction to Two-Hit Model of Tuber Formation?

1. Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by the formation of hamartomatous lesions in many organs, including brain, heart or kidneys. It has been found that TSC is caused by the mutation in one of two tumor suppressor genes: TSC1 or TSC2, encoding hamartin and tuberin, respectively. 2. According to Knudson's two-hit model of tumorigenesis, second-hit mutation and resulting loss of heterozygosity (LOH) of a tumor suppressor gene is necessary for tumor formation. In fact, LOH is commonly found in several types of hamartomas formed in the process of tuberous sclerosis, but, interestingly, not in brain lesions, containing characteristic giant cells. 3. In the present paper we review literature covering origination of giant cells and present several hypotheses explaining why in spite of the presence of hamartin and tuberin, brain lesions form in TSC patients.     

Animal Cognition: An Insect's Sense of Time?

For Immanuel Kant, time was the very form of the inner sense, the bedrock of our consciousness and also the origin of arithmetic ability. New research on bumblebees has shown that even an invertebrate with a brain the size of a pinhead can actively sense the passage of elapsed time, allowing it to predict when certain salient events will occur in the future.     

Paranthropus boisei: An example of evolutionary stasis?

Of the presently recognised early hominid species, Paranthropus boisei is one of the better known from the fossil record and arguably the most distinctive; the latter interpretation rests on the numbers of apparently derived characters it incorporates. The species as traditionally diagnosed is distributed across approximately one million years and is presently confined to samples from East African sites. The hypodigm has been examined for evidence of intraspecific phyletic evolution, with particular emphasis on the areas best represented in the fossil record, namely the teeth and mandible. The results of this examination of 55 mandibular and dental variables, which uses the Γ test statistic for the detection of trend, and nonparametric spline regression (Loess regression) for investigating pattern and rate of temporal change, show that within Paranthropus boisei sensu stricto most evidence of temporally related morphological trends relates to the morphology of the P₄ crown. There is little or no evidence of any tendency to increase in overall size through time. Fossils from the Omo Shungura Formation and West Turkana which have been recovered from a time period earlier than the P. boisei sensu stricto hypodigm resemble the latter taxon in some features, but differ from it in aspects of cranial morphology. There is insufficient fossil evidence of the earlier taxon to tell whether it changes through time, but when trends of 47 mandibular and dental variables are assessed across the combined East African “robust” australopithecine sample, there is evidence for a relatively abrupt change around 2.2–2.3 Myr in approximately 25% of the dental and mandibular remains. Some of these changes correspond with the temporal trends within P. boisei sensu stricto, but others, such as mandible height, do not. If the earlier material is ancestral to P. boisei sensu stricto, evidence from the teeth and jaws is consistent with a punctuated origin for the latter taxon. © 1994 Wiley-Liss, Inc.     

Kinetic and Thermodynamic Analysis During Dissimilatory γ-FeOOH Reduction: Formation of Green Rust 1 and Magnetite

The oldest sedimentary rocks on Earth, the 3.8‐Ga Isua Iron‐Formation in southwestern Greenland, are metamorphosed past the point where organic‐walled fossils would remain. Acid residues and thin sections of these rocks reveal ferric microstructures that have filamentous, hollow rod, and spherical shapes not characteristic of crystalline minerals. Instead, they resemble ferric‐coated remains of bacteria. Modern so‐called iron bacteria were therefore studied to enhance a search image for oxide minerals precipitated by early bacteria. Iron bacteria become coated with ferrihydrite, a metastable mineral that converts to hematite, which is stable under high temperatures. If these unusual morphotypes are mineral remains of microfossils, then life must have evolved somewhat earlier than 3.8 Ga, and may have involved the interaction of sediments and molecular oxygen in water, with iron as a catalyst. Timing is constrained by the early in fall of planetary materials that would have heated the planet's surface.

Because there are no earlier sedimentary rocks to study on Earth, it may be necessary to expand the search elsewhere in the solar system for clues to any biotic precursors or other types of early life. Evidence from Mars shows geophysical and geochemical differentiation at a very early stage, which makes it an important candidate for such a search if sedimentation is an important process in life's origins. Not only does Mars have iron oxide‐rich soils, but its oldest regions have river channels where surface water and sediment may have been carried, and seepage areas where groundwater may have discharged. Mars may have had an atmosphere and liquid water in the crucial time frame of 3.9–4.0 Ga. A study of morphologies of iron oxide minerals collected in the southern highlands during a Mars sample return mission may therefore help to fill in important gaps in the history of Earth's earliest biosphere.