How do we get from hyperexcitability to excitotoxicity in amyotrophic lateral sclerosis?

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease that, at present, has no effective cure. Evidence of increased circulating glutamate and hyperexcitability of the motor cortex in patients with amyotrophic lateral sclerosis have provided an empirical support base for the 'dying forward' excitotoxicity hypothesis. The hypothesis postulates that increased activation of upper motor neurons spreads pathology to lower motor neurons in the spinal cord in the form of excessive glutamate release, which triggers excitotoxic processes. Many clinical trials have focused on therapies that target excitotoxicity via dampening neuronal activation, but not all are effective. As such, there is a growing tension between the rising tide of evidence for the 'dying forward' excitotoxicity hypothesis and the failure of therapies that target neuronal activation. One possible solution to these contradictory outcomes is that our interpretation of the current evidence requires revision in the context of appreciating the complexity of the nervous system and the limitations of the neurobiological assays we use to study it. In this review we provide an evaluation of evidence relevant to the 'dying forward' excitotoxicity hypothesis and by doing so, identify key gaps in our knowledge that need to be addressed. We hope to provide a road map from hyperexcitability to excitotoxicity so that we can better develop therapies for patients suffering from amyotrophic lateral sclerosis. We conclude that studies of upper motor neuron activity and their synaptic output will play a decisive role in the future of amyotrophic lateral sclerosis therapy.

Activity-Dependent Global Downscaling of Evoked Neurotransmitter Release across Glutamatergic Inputs in Drosophila.

Within mammalian brain circuits, activity-dependent synaptic adaptations, such as synaptic scaling, stabilize neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. We tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. We used motoneuron-specific genetic manipulations to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. We discovered an adaptation which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This "presynaptic downscaling" maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. Our results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side.SIGNIFICANCE STATEMENT To date, compensatory adaptations which stabilise target cell activity through activity-dependent global scaling have been observed only within central circuits, and on the postsynaptic side. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, we tested whether activity-dependent global scaling could also manifest within peripheral circuits. We uncovered a compensatory adaptation which causes global scaling within a peripheral circuit and on the presynaptic side through uniform downscaling of evoked neurotransmitter release. Unlike in central circuits, uniform scaling maintains functionality over a wide, rather than a narrow, operational range, affording robust and stable activity. Activity-dependent global scaling therefore operates on both the presynaptic and postsynaptic sides to maintain target cell activity.

Drosophila larval motor patterning relies on regulated alternative splicing of Dscam2.

Recent studies capitalizing on the newly complete nanometer-resolution Drosophila larval connectome have made significant advances in identifying the structural basis of motor patterning. However, the molecular mechanisms utilized by neurons to wire these circuits remain poorly understood. In this study we explore how cell-specific expression of two Dscam2 isoforms, which mediate isoform-specific homophilic binding, contributes to motor patterning and output of Drosophila larvae. Ablating Dscam2 isoform diversity resulted in impaired locomotion. Electrophysiological assessment at the neuromuscular junction during fictive locomotion indicated that this behavioral defect was largely caused by weaker bouts of motor neuron activity. Morphological analyses of single motor neurons using MultiColour FlpOut revealed severe errors in dendrite arborization and assessment of cholinergic and GABAergic projections to the motor domain revealed altered morphology of interneuron processes. Loss of Dscam2 did not affect locomotor output, motor neuron activation or dendrite targeting. Our findings thus suggest that locomotor circuit phenotypes arise specifically from inappropriate Dscam2 interactions between premotor interneurons and motor neurons when they express the same isoform. Indeed, we report here that first-order premotor interneurons express Dscam2A. Since motor neurons express Dscam2B, our results provide evidence that Dscam2 isoform expression alternates between synaptic partners in the nerve cord. Our study demonstrates the importance of cell-specific alternative splicing in establishing the circuitry that underlies neuromotor patterning without inducing unwanted intercellular interactions.

Synaptic remodeling follows upper motor neuron hyperexcitability in a rodent model of TDP-43.

Amyotrophic Lateral Sclerosis (ALS) is an incurable disease characterized by relentlessly progressive degeneration of the corticomotor system. Cortical hyperexcitability has been identified as an early pre-symptomatic biomarker of ALS. This suggests that hyperexcitability occurs upstream in the ALS pathological cascade and may even be part of the mechanism that drives development of symptoms or loss of motor neurons in the spinal cord. However, many studies also indicate a loss to the synaptic machinery that mediates synaptic input which raises the question of which is the driver of disease, and which is a homeostatic response. Herein, we used an inducible mouse model of TDP-43 mediated ALS that permits for the construction of detailed phenotypic timelines. Our work comprehensively describes the relationship between intrinsic hyperexcitability and altered synaptic input onto motor cortical layer 5 pyramidal neurons over time. As a result, we have constructed the most complete timeline of electrophysiological changes following induction of TDP-43 dysfunction in the motor cortex. We report that intrinsic hyperexcitability of layer 5 pyramidal neurons precedes changes to excitatory synaptic connections, which manifest as an overall loss of inputs onto layer 5 pyramidal neurons. This finding highlights the importance of hyperexcitability as a primary mechanism of ALS and re-contextualizes synaptic changes as possibly representing secondary adaptive responses. Recognition of the relationship between intrinsic hyperexcitability and reduced excitatory synaptic input has important implications for the development of useful therapies against ALS. Novel strategies will need to be developed that target neuronal output by managing excitability against synapses separately.

Influence of cannabinoids upon nerve-evoked skeletal muscle contraction.

Endocannabinoids play important roles in regulating CNS synaptic function and peripheral metabolism, but cannabinoids can also act acutely to modulate contraction strength in skeletal muscle. Nerve terminals and the skeletal muscle sarcolemma express components of the cannabinoid signaling system. Endocannabinoids, N-arachidonylethanolamine (anandamide, AEA) and 2-arachidonoyl-glycerol (2-AG), are produced by skeletal muscle. They may be involved in the acute regulation of neuromuscular transmission, by adjusting the parameters for quantal acetylcholine release from the motor nerve terminal. Downstream of neuromuscular transmission, cannabinoids may also act to limit the efficiency of excitation-contraction coupling. Improved understanding of the distinct signaling actions of particular cannabinoid compounds and their receptor/transduction systems will help advance our understanding of the role of endocannabinoids in skeletal muscle physiology. Cannabinoids might also offer the potential to develop new pharmacotherapeutics to treat neuromuscular disorders that affect muscle strength.

Dscam2 suppresses synaptic strength through a PI3K-dependent endosomal pathway.

Dscam2 is a cell surface protein required for neuronal development in Drosophila; it can promote neural wiring through homophilic recognition that leads to either adhesion or repulsion between neurites. Here, we report that Dscam2 also plays a post-developmental role in suppressing synaptic strength. This function is dependent on one of two distinct extracellular isoforms of the protein and is autonomous to motor neurons. We link the PI3K enhancer, Centaurin gamma 1A, to the Dscam2-dependent regulation of synaptic strength and show that changes in phosphoinositide levels correlate with changes in endosomal compartments that have previously been associated with synaptic strength. Using transmission electron microscopy, we find an increase in synaptic vesicles at Dscam2 mutant active zones, providing a rationale for the increase in synaptic strength. Our study provides the first evidence that Dscam2 can regulate synaptic physiology and highlights how diverse roles of alternative protein isoforms can contribute to unique aspects of brain development and function.

Unusual chromosomal distribution of a major satellite DNA from Discoglossus pictus (Amphibia, Anura).

A new highly abundant satellite DNA from Discoglossus pictus (Dp-sat1) was isolated and characterized. The repetitive unit (0.51 kb) has 2 HindIII sites and only one SpeI site: digestion of genomic DNA with HindIII produces 3 fragments: HA (0.17 kb), HB (0.34 kb), and HC = HA + HB (0.51 kb), while digestion with SpeI produces the whole repetitive unit (0.51 kb) that contains both HindIII sites. Sequence analysis of cloned repeats indicates an average A + T content of 71%, with many A- and T-runs. Southern blot analysis shows an arrangement of multiple bands of the 0.51 kb monomer in SpeI-digested DNA, while HindIII-digested DNA shows a ladder composed of all the possible combinations of the 3 digested fragments. Quantitative dot-blot indicates that Dp-sat1 accounts for about 6% of the D. pictus genome: this value represents about 1.5 x 10(6) copies of repetitive units per nucleus. This satellite DNA is also a major repetitive DNA in 4 other Discoglossus species, in which the repetitive unit presents the same size and restriction sites except in D. montalentii where it contains a unique HindIII site. This satellite DNA was absent in all the other tested archaeo- and neo-bratrachian species, as well as non-amphibian species. Fluorescent in situ hybridization (FISH) analysis shows that Dp-sat1 is localized only in peri- and/or para-centromeric areas of the 7 small chromosome pairs, while no labeling was observed in the 7 large chromosome pairs. Remarkably, Dp-sat1 heterochromatin is found only at one pole of the nucleus, suggesting that during interphase all 7 small chromosome pairs are located in the same nuclear region.

Cytological evidence for population-specific sex chromosome heteromorphism in Palaearctic green toads (Amphibia, Anura).

A chromosome study was carried out on a number of European and Central Asiatic diploid green toad populations by means of standard and various other chromosome banding and staining methods (Ag-NOR-, Q-, CMA3-, late replicating [LR] banding pattern, C-and sequential C-banding + CMA3 + DAPI). This study revealed the remarkable karyological uniformity of specimens from all populations, with the only exception being specimens from a Moldavian population, where one chromosome pair was heteromorphic. Though similar in shape, size and with an identical heterochromatin distribution,the difference in the heteromorphic pair was due to a large inverted segment on its long arms. This heteromorphism was restricted to females, suggesting a female heterogametic sex chromosome system of ZZ/ZW type at a very early step of differentiation.

Morphology of the pancreas of some species belonging to the genera Phelsuma and Gecko (family Gekkonidae): evidence of apoptotic process during the seasonal cycle.

In this study we investigated comparative morphology of the endocrine pancreas of several species belonging to the family Gekkonidae and apoptotic processes of the pancreas which may be correlated to the seasonal cycle. The following species of the family Gekkonidae were studied: Phelsuma lineata, P. madagascariensis, P. dubia, P. abotti, Gekko gecko, G. vittatus, and Geckonia chazaliae. In all these species the pancreas consisted of large and medium islets as well as endocrine cells which were scattered throughout the acinar cells. Exocrine parenchyma consisted of tubuli-acini. Four mayor cell types were identified in the endocrine pancreas, using immunocytochemistry: glucagon-immunoreactive (A) cells, insulin-immunoreactive (B) cells, somatostatin-immunoreactive (D) cells, and pancreatic polypeptide immunoreactive (PP) cells. In the endocrine pancreas the amount of A cells and B cells was either equal or a prevalence of A cells was observed. In the wet season the pancreatic morphology presented normal features with very rare apoptotic cells. The animals belonging to the genus Phelsuma taken in the dry season (July) showed numerous vacuolated, Caspase 3, 9 and 11-immunoreactive acinar and some endocrine cells containing picnotic nuclei which were positive to tunel reaction. The animals belonging to the genus Gekko taken at the end of the dry season (October) exhibited strongly vacuolated, Caspase 3, 9 and 11-immunoreactive endocrine and some acinar cells containing nuclei which were positive to tunel reaction. These apoptosis events could be a reaction in response to stress mechanisms, such as a starvation period during the dry season.

Different genomic evolutionary rates in the various reptile lineages.

Although Reptiles occupy a strategic position among terrestrial vertebrates, studies of the composition and evolution of their genome are scarce. The cytogenetic analysis of nearly 1400 species evidenced different karyotypical evolutionary rates and different G-banding structures in turtles and crocodiles on the one hand and squamates on the other. A similar dichotomy was also identified through the study of the quantitative and compositional characteristics of the genome. The different evolutionary rates of chromosome morphology and genome size and composition and the diversification of coding and non-coding sequences bear an interesting relationship to the number of extant species and the extinction rates of the reptilian orders and suborders studied, suggesting a large role for such different evolutionary rates in the phylogenesis of this class. The different molecular and structural organisation of chromosomes could be an important, though by no means the sole, factor affecting the genome's evolutionary rate.

Detection of DNA in ancient bones using histochemical methods.

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian "Casti Amanti" house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4'-'6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.

Karyology of the Antarctic scallop Adamussium colbecki, with some comments on the karyological evolution of pectinids.

Karyotype, location of the nucleolar organiser region (NOR) and heterochromatin presence and composition were studied in the Antarctic scallop Adamussium colbecki Smith, 1902. The karyotype exhibits 2n = 38 chromosomes with 11 pairs of metacentrics, 5 of submetacentrics, one subtelocentric and two telocentrics. Ag-NOR, CMA(3), DA/MM and NOR-FISH evidenced paracentromeric NORs on the short arm of 2nd pair chromosomes. Digestion with three restriction endonucleases followed by sequential staining with Giemsa, CMA(3) and DAPI evidenced on all chromosomes centromeric heterochromatin positive for both DAPI and CMA(3). In situ hybridisation analysis showed the presence of an AT-rich satellite DNA in the centromeric heterochromatin of several chromosomes. A mosaicism was detected in the germinal cell lines of one specimen, as in six of the 20 plates examined the set had 37 chromosomes with a missing pair of telocentrics and an unpaired metacentric. Comparison of the chromosome sets of all the pectinids studied to date and comparison with a phyletic tree obtained from molecular mitochondrial genes studies yielded good agreement between karyotype morphology and taxonomic classification.

Base composition of DNA from some reptiles.

The DNA base composition was studied in 9 reptilian species (4 chelonians, 3 saurians, and 2 ophidians) by the thermal denaturation method. This preliminary investigation has brought to light differences in GC percentages and in the shape of melting curves between chelonian DNA and that from saurians and ophidians.

Relationships between DNA content and cell morphometric parameters in reptiles.

The relationships between the genome size and various cellular morphometric parameters have been, studied in 38 reptilian species. The nuclear volume, the cell volume and the cell surface area show a direct, linear and statistically significant correlation with the nuclear DNA content; the cell surface/cell volume ratio tends to decrease as DNA increases. The results are similar to those previously observed in Amphibia, which suggests, that at least in poikilothermic vertebrates, the correlations between DNA amounts and cell sizes are controlled by substantially similar mechanisms. A comparison of the average variations of the various morphometric parameters among each other and with the average variation of the genome size suggests that these parameters are not regulated all in the same way.

Karyology of the primitive salamanders, family Hynobiidae.

Karyotypes have been studied in 3 species of Hynobius and in 1 species each of the remaining genera of Hynobiids (Ranodon, Batrachuperus, Salamandrella and Onychodactylus). All species have large diploid numbers, between 56 and 66, and asymmetrical and bimodal karyotypes. DNA contents (2C) were found to vary between 33 and 51 pg. Determination was not possible in Onychodactylus where higher values may be suspected. Some of the karyotypes investigated are similar to those of Cryptobranchids. Phylogenetic implications are discussed.

An immunocytochemical study of intrapancreatic ganglia, nerve fibres and neuroglandular junctions in Brockmann bodies of the tompot blenny (Blennius gattoruggine), a marine teleost.

The innervation of the Brockmann bodies in the teleost fish, Blennius gattoruggine, was studied using immunocytochemical techniques at both the light and electron microscopy levels. Islet innervation consisted of intrapancreatic ganglia, generally localized inside the rim of the exocrine tissue of the Brockmann bodies, in proximity to the islet, nerve fibres and nerve terminals with synaptic complexes. The intrapancreatic ganglia were of variable size, with different numbers of ganglionic cells, that appeared unipolar in section. The cell bodies showed immunoreactivity to galanin, oxytocin, peptide tyrosine tyrosine and glucagon. The extrinsic and intrinsic nerve fibres passed through the exocrine parenchyma and crossed the connectival septa and islet connectival sheath, penetrating into the islets, where they became increasingly thinner. They terminated on the endocrine cells with dilated nerve terminals. At least three types of terminals were detected, depending on the different vesicle content: peptidergic, cholinergic or adrenergic. They presented specialized synaptic structures, the neuroglandular junctions, some of which contained neurosecretory granules immunogold labelled by galanin antiserum. This new finding confirms the role of galanin as a neurotransmitter. This rich supply of innervation may be important in the regulation and integration of islet secretion.

Genome size evolution in vertebrates: trends and constraints.

1. Studies on the genomic evolution in vertebrates have highlighted the differences existing between anamniotes and amniotes, both in quantitative and compositional terms. 2. These differences do not seem to depend on a different tendency to genic amplification, but rather on the existence of more strict and efficient constraints in amniotes. 3. Some constraints, that may be defined as "intrinsic", would act directly on the genome; among these particularly important is the chiasma frequency during meiosis. 4. Other, "extrinsic", constraints, would act indirectly through genic products or through cell morphometric parameters. 5. The genome size increase seems to depend on various mechanisms. The most wide-spread one seems to be the amplification of interspersed repetitive and non-repetitive sequences.

Occurrence of G-banding in metaphase chromosomes of Encarsia berlesei (Hymenoptera: Aphelinidae).

Well-defined G-bands were obtained on somatic metaphase chromosomes of Encarsia berlesei using trypsin and warm 2x SCC in sequence. The G-banded pattern allowed rapid identification of all five metacentric chromosomes, which appeared uniformly lighted when stained with DAPI fluorochrome dye. It is stressed that ageing affects G-banding in this insect species; in fact, good banded chromosomes were obtained on 1-month air-stored chromosomes. Evidence for asynchronous condensation on the chromosomes of this species is also provided.