AbstractUraeotyphlus oxyurus , Uraeotyphlus interruptus Pillai et Ravichandran, 1999, Uraeotyphlus narayani Seshachar, 1939 and Uraeotyphlus menoni Annandale, 1913 were cytogenetically analysed following conventional and differential staining techniques. These species show similar karyotypes with 2n=36 (FN=58). There were no traces of species-specific features in regard to C-banding and NOR staining. The comparative study of karyotypes shows chromosomal homologies among the four species. Chromosomal data seem to support the concept that two species groups exist in the genus Uraeotyphlus. Uraeotyphlus Peters, 1879is endemic to the Western Ghats region of peninsular India and constitutes one of the three genera within the family Ichthyophiidae Taylor, 1968 along with Caudacaecilia Taylor, 1968 and Ichthyophis Fitzinger, 1826, Uraeotyphlus interreptus Pillai & Ravichandran, 1999, Uraeotyphlus narayani Seshachar, 1939 and Uraeotyphlus menoni Annandale, 1913 were grouped as derived species, while of Uraeotyphlus gansi Gower, Rajendran, Nussbaum & Wilkinson, 2008, Uraeotyphlus oommeni Gower & Wilkinson, 2007 and Uraeotyphlus malabaricus  were considered as primitive ones  but which was subsequently found to be Uraeotyphlus prope interruptus of Ichthyophiidae. With this surge of interest to elucidate the phylogeny and evolution, Uraeotyphlus malabaricus species group, Uraeotyphlus gansi, bearing2n=42, FN=58, highest diploid number known thus far for a member of the genus Uraeotyphlus of the family Ichthyophiidae.Earlier, Uraeotyphlus oxyuryus and Uraeotyphlus interruptus and the results of reanalysis of chromosomes of Uraeotyphlus narayani and Uraeotyphlus menoni with a view to providing new insights into the intragenus relationships within the genus Uraeotyphlus.In this study, we present the karyotypes of in vivo colchicine treatment, chromosome preparations were obtained from the liver, the gut epithelium and the testis. Cell suspensions, hypotonic treatment and fixation of cells were performed as described earlier  was performed by applying the one-step silver nitrate method of PageBreakUraeotyphlus oxyurus  includes two pairs (1–2) of large metacentrics and one pair (3) of submetacentrics, while the B group consisted of three pairs (4–6) of medium sized metacentrics in which the pair four was slightly longer than other two pairs. The third group (C) included 5 pairs (7–11) of smaller submetacentrics, all in decreasing order of their total length. The fourth group (D) included mostly acrocentric (12–18) pairs.Uraeotyphlus narayani  are confined to chrormosomal pair 9 in the complement but were not consistently demonstrated in each and every cell. Whereas, interphase nuclei demonstrated 1 to 2 (or sometimes 3) numbers of silver nitrate (2–3) aggregates  chromosomes was found consistently identical in each of the four species belonging to Uraeotyphlus malabaricus group. Until now, none of these species karyotypes were known except a publication citing a variable karyotype for the species belonging to the genus, Uraeotyphlus, Uraeotyphlus gansi. Uraeotyphlus gansi bearing a different basic chromosomal number (2n=42) and morphology. A major attribute of Uraeotyphlus gansi karyotype is that it is similar to any of the known karyotypes of the species belonging to Ichthyophis and Caudacaecilia of the family Ichthyophiidae , which serves as a modal karyotype for the Uraeotyphlus malabaricus group species which could be considered a basal one among uraeotyphlids. Besides, based on the pronounced chromosomal homogeneity among the said species groups’ karyotypic specificities, this may be considered as of cytogenetic importance while placing emphasis on species differentiation within the genus. Uraeotyphlid chromosome complements belonging to family Ichthyophiidae seem to present bimodal diploid numbers: 2n=42 chromosomes in the basal type Uraeotyphlus malabaricus group and 2n=36 chromosomes in the case of derived species belonging to Uraeotyphlus oxyurus species complex.The four species karyotypes (2n=36) may be considered as derived ones from that of Uraeotyphlus oxyurus karyotypes and of Uraeotyphlus gansi karyotype are unique in possessing most chromosomal pairs as conserved. The centromeric position of the lowerset of chromosomes in the karyotype makes all the more significant for consideration of their karyotypic specificities.Presently, chromosomal data on ichthyophiid taxa indicate that they are all well-conserved among the species whose karyotypes are known, bearing identical chromosomal set in each case . The chaDuring the course of drawing karyological relationships prevailing between the two taxa  it is possible to infer that most submeta and metacentrics are conserved to a maximum extent and only acrocentrics mayhave paved the way for chromosome speciation events.PageBreak makes clear the occurrence of a succession of chromosomal rearrangements, mainly through pericentric inversion and / or fusion. This appears necessary in order to create the karyotype found in described karyotype from those of ancestral ones. This sequence of events could account for the appearance of reduction in basic chromosome numbers from 2n=42 chromosome to 2n=36 chromosomes  in which there is an enormous interspecific variation was evident in the distribution and amount of heterochromatin  which might form a monophyletic group. In the light of extensive chromosomal homology incurred, that does not preclude minor karyotypic differences necessitating in the use of other banding techniques for the improvement of karyological characterization.In conclusion, the cytogenetic study based on conventional Giemsa staining including C- and NOR bandings, upon four


Exposure to alcohol significantly alters the developmental trajectory of progenitor cells and fundamentally compromises tissue formation . Emerging research suggests that ethanol can impair mammalian development by interfering with the execution of molecular programs governing differentiation. For example, ethanol exposure disrupts cellular migration, changes cell–cell interactions, and alters growth factor signaling pathways. Additionally, ethanol can alter epigenetic mechanisms controlling gene expression. Normally, lineage-specific regulatory factors  establish the transcriptional networks of each new cell type; the cell’s identity then is maintained through epigenetic alterations in the way in which the DNA encoding each gene becomes packaged within the chromatin. Ethanol exposure can induce epigenetic changes that do not induce genetic mutations but nonetheless alter the course of fetal development and result in a large array of patterning defects. Two crucial enzyme complexes—the Polycomb and Trithorax proteins—are central to the epigenetic programs controlling the intricate balance between self-renewal and the execution of cellular differentiation, with diametrically opposed functions. Prenatal ethanol exposure may disrupt the functions of these two enzyme complexes, altering a crucial aspect of mammalian differentiation. Characterizing the involvement of Polycomb and Trithorax group complexes in the etiology of fetal alcohol spectrum disorders will undoubtedly enhance understanding of the role that epigenetic programming plays in this complex disorder. Exposure of the developing embryo and fetus to alcohol can have profound adverse effects on physical, behavioral, and cognitive development. The resulting deficits collectively have been termed fetal alcohol spectrum disorders (FASD). They range in severity from mild cognitive deficits to a well-defined syndrome , which is broadly characterized by low birth weight, distinctive craniofacial malformations, smaller-than-normal head size , and central nervous system dysfunction . The mecMammalian development consists of a series of carefully orchestrated changes in gene expression that occur as stem or progenitor cells differentiate to form the tissues and organs making up the growing fetus.Studies using a diverse range of model organisms have led to the conclusion that epigenetic modifications to the chromatin structure provide a plausible link between exposure to environmental substances that can harm the developing fetus  and lasting alterations in gene expression leading to disease phenotypes. Numerous studies have demonstrated that exposure to ethanol is associated with both genome-wide and gene-specific changes in DNA methylation , alteratAlthough the mechanisms by which alcohol impacts chromatin structure are not completely understood, recent work suggests that some epigenetic changes result from altered cellular metabolism. For example, HOX factors, which play crucial roles in directing organ patterning and morphogenesis , work together to produce a combinatorial “histone code” that serves to regulate cell-lineage– specific patterns of chromatin structure throughout development . Within figure 1ADrosophila melanogaster in the late 1970s to early 1980s revealed the existence of two large multiprotein complexes with diametrically opposite roles in the regulation of gene expression: the Polycomb group (PcG) and Trithorax group (TrxG)  . These tThe PcG proteins and the genes encoding them originally were discovered over 30 years ago as key regulators of the processes that specify which end of the embryo forms the head and which the rear during the development of Drosophila . Since tPRC2 has similar repressive properties to PRC1 and also is an essential regulator of cellular differentiation. It facilitates the silencing of developmentally crucial genes through mono-, di-, and trimethylation of the histone 3 lysine 27 and trimethylation of histone 3 lysine 9 , both ofAdding an additional layer of complexity, PRC2 associates with the mammalian enzymes responsible for DNA methylation ; this association aids in the ability of PRC2 complexes to repress their target loci . This phIn fruit flies, maternal transcription factors that were included in the egg cells and which are distributed unevenly throughout the developing embryo shape gene expression in the early embryo. The levels of these transcription factors diminish over time, and once they disappear from the developing embryo, the memory of which genes were active in a given cell is propagated through the action of the TrxG proteins . These pThe main core of TrxG complexes is composed of four proteins called WD40 repeat domain 5 (WDR5), retinoblastoma binding protein 5 (RbBP5), dosage compensation–related protein 30 (Dpy30), and absent, small, or homeotic-like protein (Ash2L) see . WDR5 reMLL1) has been eliminated, or knocked out, show alterations in H3K4 methylation. In contrast, knockout of the MLL2 gene in mouse embryonic stem cells leads to skewed differentiation but no concrete alterations to H3K4 methylation  proteins  and proteins called SET1A and SET1B . MLL1 inhylation . For thehylation . Thus, aPostmortem studies of children that succumbed to FAS revealed groups of poorly differentiated neuronal and glial cells at abnormal sites within the brain, suggesting large-scale problems with cellular proliferation and differentiation resulting from prenatal alcohol exposure . FurtherDlx2, Hand1, Msx2, Nestin, Nkx2.1, Nkx2.2, Olig2, Pax6, and Sox1 all are bivalently marked, whereas in multipotent, neural precursor cells (which only can develop further into different types of neurons) only Dlx2 and Pax6 maintain this conformation. Interestingly, two genes encoding marker proteins that are found only in a type of glial cell called astrocyte  establish novel bivalent domains so that these genes can be kept in an active or inactive state, depending on whether they will become nerve cells or astrocytes (During early mammalian development, approximately 2,000 genes are bivalently marked as described earlier, and these marks progressively resolve towards the lineage-specific patterns of chromatin organization characterizing each unique cell type . As devetrocytes . Proper trocytes . Similartrocytes . By propMsx2 and Pax6—leading to craniofacial abnormalities and excessive differentiation of glutamatergic neurons, respectively (Nkx2.1 and Olig2 are diminished by alcohol, potentially disrupting the balance between excitation and inhibition in the cerebral cortex after birth (Pax6 gene at a site other than where it usually is expressed could ameliorate the impact of ethanol on cell proliferation and neurogenesis. These results suggest that within a limited scope it may be possible to reverse alcohol’s effects on developmental programs.Importantly, the expression of many of these factors that are bivalently marked and regulated by PcG and TrxG is disrupted in various models of prenatal alcohol exposure; moreover, this disruption is associated with profound errors in neuronal patterning. For example, alcohol suppresses the activation of two neural precursor genes—ectively . Similarer birth . FinallyOne of the most difficult aspects in the study of FASDs has been trying to explain the wide range of severity and enormous variation in FASD-associated birth defects. The process of organ formation is initiated during the early stages of embryonic development, and different rudimentary organ systems are formed and grow during unique developmental windows . Each orSince their discovery, the PcG and TrxG protein complexes have been identified in numerous disease contexts, including cellular transformation of normal cells into tumor cells as well as structural defects and mental illness . These s


Learning is believed to depend on lasting changes in synaptic efficacy such as long-term potentiation and long-term depression. As a result, a profusion of studies has tried to elucidate the mechanisms underlying these forms of plasticity. Traditionally, experience-dependent changes at excitatory synapses were assumed to underlie learning and memory formation. However, with the relatively more recent investigation of inhibitory transmission, it had become evident that inhibitory synapses are not only plastic, but also provide an additional way to modulate excitatory transmission and the induction of plasticity at excitatory synapses. Thanks to recent technological advances, progress has been made in understanding synaptic transmission and plasticity from particular interneuron subtypes. In this review article, we will describe various forms of synaptic plasticity that have been ascribed to two fairly well characterized populations of interneurons in the hippocampus, those expressing cholecystokinin (CCK) and parvalbumin (PV). We will discuss the resulting changes in the strength and plasticity of excitatory transmission that occur in the local circuit as a result of the modulation of inhibitory transmission. We will focus on the hippocampus because this region has a relatively well-understood circuitry, numerous forms of activity-dependent plasticity and a multitude of identified interneuron subclasses. Our focus is restricted to studies performed in the hippocampus because the well-characterized circuitry has allowed for insight into how the numerous forms of plasticity expressed in inhibitory cells alters excitation and modulates network properties.Interneurons are classified according to several factors including axonal and dendritic connectivity, electrophysiological properties and expression of molecular markers. Based on these criteria, the hippocampus is one of the structures with the largest interneuron diversity  depression of GABA release, a phenomenon called depolarization-induced suppression of inhibition (DSI). This phenomenon was initially described more than 20 years ago in the cerebellum , eCBs can mediate a long-term depression of inhibitory transmission. Several minutes of CB1R activation are needed for a lasting depression to be induced. This more sustained CB1R activation probably allows for significant changes in second messenger and phosphorylation levels of downstream target molecules. Consistently, protein kinase A (PKA) activity and the active zone proteins RIM1α and Rab3b are needed for iLTD induction  -CA1 excitatory synapse. Not surprisingly, the time course of the facilitation follows the time course of the eCB-mediated plasticity. For instance, it was initially described that the dis-inhibition occurring during DSI provides a transient facilitation of LTP induction at excitatory synapses  to evoke an action potential (E-S coupling) and directly increase the size of the EPSP at the SC-CA1 synapse. The first effect was observed after inducing iLTD with synaptic activity of the SC inputs or with repetitive postsynaptic depolarization. Action potential firing was extracellularly monitored , which is a critical element in PV+ maturation during development, acts on adult PV+ cells to increase GABA transmission, resulting in a suppression of LTP induction at SC-CA1 synapses (Pitcher et al., in vivo in CA1, suggesting a complex interaction between different interneuron types in oscillatory activity (Wulff et al., in vivo that take into account the axonal arbors and cell-type specific markers of interneurons, have revealed that each cell type has a potential role in the network activity of the hippocampus during a specific behavior (for example, Klausberger et al., in vivo studies in which transmission from all PV+ cells has been removed reveal very interesting changes in behavior and hippocampal network activity (Korotkova et al., At the circuit level, a recent study reported that cholinergically-driven θ-oscillations in CA1 involves an inhibitory circuit consisting mainly of CCK+ interneurons (Nagode et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motoneuron death. Mutations in the gene for superoxide dismutase 1 (SOD1) cause a familial form of ALS and have been used to develop transgenic mice which overexpress human mutant SOD1 (mSOD) and these mice exhibit a motoneuron disease which is pathologically and phenotypically similar to ALS. Neuroinflammation is a pathological hallmark of many neurodegenerative diseases including ALS and is typified by the activation and proliferation of microglia and the infiltration of T cells into the brain and spinal cord. Although the neuroinflammatory response has been considered a consequence of neuronal dysfunction and death, evidence indicates that manipulation of this response can alter disease progression. Previously viewed as deleterious to neuronal survival, recent reports suggest a trophic role for activated microglia in the mSOD mouse during the early stages of disease that is dependent on instructive signals from infiltrating T cells. However, at advanced stages of disease, activated microglia acquire increased neurotoxic potential, warranting further investigation into factors capable of skewing microglial activation towards a neurotrophic phenotype as a means of therapeutic intervention in ALS. Neuroinflammation is a pathological hallmark of many neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). It is characterized by the activation and proliferation of microglia (microgliosis) and the accumulation of infiltrating T lymphocytes at sites of neurodegeneration. Although often considered a consequence to neuronal injury and degeneration, the neuroinflammatory response can have protective or deleterious effects on neuronal survival. These disparate effects are elicited by the heterogeneous activation programs of microglia, which in turn are dictated by their surrounding microenvironment and by infiltrating T cells.Typically diagnosed during the fifth decade of life, amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the degeneration of motoneurons in the brainstem and spinal cord and loss of descending motor tracts. Clinical manifestations of ALS include muscle weakness, spasticity, muscle atrophy, and advancing paralysis that culminates in respiratory failure, the usual cause of death in affected patients. ALS is a disease primarily of sporadic etiology with a plethora of aberrant physiological processes implicated in its pathogenesis including excitotoxicity, oxidative damage, the formation protein aggregates, and mitochondrial dysfunction . A pathoA small fraction of cases (~10%) termed familial ALS (fALS) are due to a variety of genetic mutations, with 20% of fALS cases due to dominantly inherited mutations in superoxide dismutase 1 (SOD1). SOD1 is a ubiquitously expressed, 32 kDa homodimeric cytosolic protein that catalyzes the dismutation of superoxide, a by-product of cellular respiration, to hydrogen peroxide. To date, over 125 different mutations that span the entire genomic sequence and protein structure of SOD1 have been identified as causing ALS  developeAn added complexity to mSOD pathogenicity is experimental evidence indicating that motoneuron death in the mSOD model is a noncell autonomous event. Although mSOD expression restricted to neurons is sufficient to cause motoneuron death if expressed at adequate levels , mSOD exWithin the CNS, populations of macrophages can be distinguished based on their anatomical location. Perivascular macrophages lie between the basal lamina of blood vessels and the glia limitans while meningeal macrophages lie within the leptomeninges that surround the CNS. Microglia are considered the CNS tissue-resident macrophage population and are found within the parenchyma of the CNS. These cells possess a characteristic stellate morphology, with long sinuous processes extending from a round cell body. In their quiescent state, microglia are highly dynamic cells, surveying their surrounding microenvironment through the constant extension and retraction of their processes; it is estimated that the entire extracellular space of the CNS is surveyed every few hours .The phenotype of resting microglia differs from that of other populations of tissue macrophages, being more similar to that of immature myeloid cells; microglia express only low levels of CD45, major histocompatibility complexes (MHCs), and are poor antigen presenting cells . The doβ and tumor necrosis factor α (TNF-α) and increased release of reactive oxygen species and nitric oxide through upregulated expression of NADPH oxidase and inducible nitric oxide synthase (iNOS), respectively , a potent stimulus for M1 activation of macrophages, result in increased microglial production of nitric oxide (NO) and reactive oxygen species (ROS), as well as increased levels of extracellular glutamate which culminates in the excitotoxic death of neurons [Often likened to a double-edged sword in the literature, activated microglia can produce substances that are either beneficial or toxic to surrounding neurons. M1-  activated microglia exhibit a proinflammatory phenotype characterized by the production of interleukin- (IL-) 1ectively . In vitr neurons . Treatme neurons ).−/− mice exhibit dysregulated microglial responses following peripheral injection of LPS, while CX3CR1 ablation in mSOD mice results in increased levels of neuronal loss [Neurons play an integral role in regulating microglial activation by expressing membrane bound and soluble mediators that enhance microglial production of anti-inflammatory cytokines and neurotrophins. For example, CD200 is a glycoprotein expressed by neurons and its cognate receptor (CD200R) is expressed by all myeloid cells including microglia. In the CNS of mice deficient for CD200R, microglia were observed to possess activated morphologies under steady-state conditions and exhibited an enhanced response following facial nerve axotomy compared to similarly treated wild-type mice  suggestinal loss . Neurona+ T cells interacts with Fas (CD95) expressed on host cells to induce the downstream activation of caspases, culminating in the apoptosis of host cells. Perforin induces the formation of pores on the target cell membrane, which can result in osmotic cell lysis and provides a means of entry for secreted granzymes [T cells are the central players in adaptive immunity and can be divided into different subsets based on the expression of cell surface molecules and function . Cytotoxranzymes .+ T cells have only limited ability to directly kill cells; they do not express Fas ligand or secrete granulysin and function mainly to activate and regulate the activity of other cells involved in the immune response, including macrophages and microglia [T lymphocytes expressing CD4 include helper T cells (Th) that are further classified according to cytokine production profiles and effector functions and T-regulatory cells Tregs; . Compareicroglia . For exaicroglia . Regulaticroglia . For eacicroglia .+ T cells, antigen is presented on MHC class II molecules on the membranes of APCs, typically dendritic cells, and activated macrophages. CD8+ T cells recognize antigen presented on MHC class I molecules which are expressed on the membranes of all nucleated cells with the exception of neurons and other cell populations within the CNS; however, under neuroinflammatory conditions, neurons upregulate their MHC class I expression, making them potential targets for CTLs [+ T cells, resulting in their activation and potential reactivity to neuronal cells [+ T cells must first be activated within secondary lymphoid organs before migration and extravasation into the CNS. This may be accomplished through antigen drainage of cerebrospinal fluid into the cervical lymphatics or through the migration of APCs residing in the perivascular space to lymph nodes [+ and CD8+ T cells, a secondary independent signal elicited through the binding of molecules present on the membranes of host cells is essential for activation and clonal expansion; this secondary signal from APCs may be stimulatory or inhibitory. The types of costimulatory or coinhibitory molecules expressed by APCs confer the nature of their functional activation states, while the density of costimulatory and coinhibitory molecules on T-cell membranes dictates the functional outcome of T-cell activation [Naive T cells are activated upon recognition and binding of antigen specific to their expressed T-cell receptor; differentiation to a specific effector subtype is determined by the local microenvironment. For CD4for CTLs . Notablyal cells . Neuronaph nodes . For bottivation . In the tivation .Activated T cells are capable of extravasating into the CNS where they perform immune surveillance, and in the steady state, variable numbers of T cells are present within the parenchyma of the CNS ; however+ T cells surveying the CNS for their cognate antigen, induces the CD8+ T-cell apoptosis [γ . B7-H1   monocytes in peripheral blood [κB (NF-κB), a transcription factor involved in the expression of proinflammatory mediators, are upregulated in the spinal cords of ALS patients [Notably, patients suffering from sporadic ALS have been reported to have increased levels of circulating inflammatory , a timetivation .+ Tregs into mSOD mice extended the stable phase of disease progression and survival times, suggesting that manipulation of the microglial response through the adoptive transfer of Treg cells or pharmaceutical agents that potentiate M2 activation in microglia may have therapeutic value. In fact, Neuraltus Pharmaceuticals  is currently conducting phase II clinical trials in patients suffering from ALS, PD, and AD using NP100, a pharmaceutical drug designed to skew macrophage activation towards an M2 phenotype to determine its efficacy in prolonging disease duration.Findings from these studies suggest that exploiting the neurotrophism of alternatively activated microglia, rather than dampening microglial activation generally, may have some therapeutic benefit in ALS; however, molecular targets enabling this manipulation remain elusive. Recently Beers et al.  demonstrPharmacological treatments for ALS have largely been ineffective at slowing the disease process, in part because the blood-brain barrier prevents the transmission of the majority of drugs from the blood into the CNS. This has spurred investigations into alternative therapeutic modalities for the treatment of ALS and other neurodegenerative diseases. Microglia are members of the mononuclear phagocyte system which also includes hematopoietic progenitors, blood monocytes, dendritic cells, and other populations of tissue macrophages . Under iInvestigations into the migration of bone-marrow-derived cells (BMDCs) into the CNS often employ bone marrow (BM) chimeric mice, typically created by exposing rodents to myeloablative levels of radiation followed by the adoptive transfer of labelled BM cells. The results of these studies suggest that while BMDCs contribute to the maintenance of meningeal and perivascular macrophage populations within the CNS, BMDCs make only limited contributions to the parenchymal microglial pool –57. HoweA caveat associated with the irradiation-BM reconstitution protocol employed to create BM-chimeric mice is that it introduces two confounding variables. First, irradiation elicits a widespread inflammatory response including increased levels of cytokines and chemokines within the CNS  and has Once considered a consequence of neuron death in chronic neurodegenerative disease, neuroinflammation is now recognized to influence disease progression in ALS and the mSOD mouse model. While microglial activation and T-cell infiltration have previously been implicated in exacerbating pathological processes and contributing neuron death in the mSOD mouse, experimental evidence has demonstrated that microglial activation together with the infiltration of instructive T cells has trophic effects on surrounding neurons until late stages of disease. Further investigations into phenomena that induce this phenotypical switch in activated microglia could potentially enable the exploitation of microglial neurotrophism and provide future therapeutic benefits.


Tissues in the anterior segment of the eye are particular vulnerable to oxidative stress. To minimise oxidative stress, ocular tissues utilise a range of antioxidant defence systems which include nonenzymatic and enzymatic antioxidants in combination with repair and chaperone systems. However, as we age our antioxidant defence systems are overwhelmed resulting in increased oxidative stress and damage to tissues of the eye and the onset of various ocular pathologies such as corneal opacities, lens cataracts, and glaucoma. While it is well established that nonenzymatic antioxidants such as ascorbic acid and glutathione are important in protecting ocular tissues from oxidative stress, less is known about the delivery mechanisms used to accumulate these endogenous antioxidants in the different tissues of the eye. This review aims to summarise what is currently known about the antioxidant transport pathways in the anterior eye and how a deeper understanding of these transport systems with respect to ocular physiology could be used to increase antioxidant levels and delay the onset of eye diseases. We are constantly exposed to oxidative stress through our environment, by products of metabolism and lifestyle factors. The positioning of tissues in the anterior segment of the eye makes them particularly susceptible to oxidative stress. Oxidative stress stimulates the production of unstable and highly reactive oxygen species (ROS) that are responsible for cellular damage. To combat the ubiquitous presence of ROS, ocular tissues have evolved diverse antioxidant defence systems to prevent permanent and lasting ROS-mediated tissue damage. However, as we age, prooxidants overwhelm the antioxidant defences resulting in oxidative stress and damage to tissues of the eye associated with the development of ocular pathologies such as corneal opacities, cataracts, and glaucoma. It is therefore no surprise that the therapeutic potential of natural and nutraceutical antioxidants in treating eye diseases is widely explored within vision research . HoweverThe cornea is the primary barrier between the environment and ocular tissues. It is responsible for ~75% of the total refractive power of the eye, a function owing to its transparency and unique curvature. The outermost layer of the cornea is the epithelium and is constantly exposed to mechanical stress, microbial invasion, external chemical insults, and UV radiation. The middle stromal layer accounts for nearly 90% of corneal thickness and plays a key role in maintaining corneal transparency, strength, and curvature –5. It co+, enter the lens predominantly via both poles along the extracellular spaces between fibre cells. The current then propagates across fibre cell membranes and flows towards the lens surface via an intracellular pathway mediated by gap junctions which connect all fibre cells. The influx of ions is accompanied by the convection of water, oxygen, and solutes to the deeper-lying fibre cells, while the efflux of ions facilitates the removal of fibre cell waste products. Consistent with this model, ion channels and transporters have been found in distinct fibre cell membrane domains that allow the uptake and export of solutes [The primary function of the lens is to focus light onto the retina. To achieve this, the lens is composed of an ordered arrangement of crystalline fibre cells which are derived from equatorial epithelial cells which exit the cell cycle and embark upon a differentiation process that produces extensive cellular elongation, the loss of cellular organelles and nuclei, and the expression of fibre-specific proteins . Since t solutes . Regiona solutes . It is t solutes .μm, offering negligible resistance to flow [μm, providing a greater resistance to aqueous outflow. The outermost portion of the TM is the cribriform or juxtacanalicular meshwork which lies immediately adjacent to the endothelial cells lining Schlemm's canal [The main function of the trabecular meshwork (TM) is to provide a site through which aqueous humour can leave the eye . The TM  to flow . Further to flow , 14. In 's canal . This la's canal , 14. Due's canal –15. The A common feature of these tissues is their avascular nature and their dependence on either tears or aqueous humour for their supply of glucose, amino acids, and antioxidants. The vast literature on exogenous antioxidant supplementation and the mixed findings from epidemiological studies on ocular health are not surprising given that (1) these compounds are unable to reach their target tissue via the circulatory system and (2) transporter proteins must be expressed in the cornea and lens to specifically accumulate these exogenous molecules into ocular cells. Current research is focused on designing more appropriate systems for drug delivery to the front of the eye with a great deal of attention paid towards improving ocular drug bioavailability and towards the development of noninvasive sustained drug release for anterior segment disorder. In comparison, less is known about the molecular identity of endogenous antioxidant transporter systems in the anterior eye and how these transport systems could be utilized to enhance the availability of antioxidants to specific regions within ocular tissues. μM [The two key antioxidants of the eye are ascorbic acid (vitamin C) and glutathione (GSH), which are present at 1 mM  and 2 μMμM , respectμM  and the  de novo; however humans and a few other species, including Wistar rats [α-tocopherol (vitamin E) by rescuing α-tocopheryl radicals in membranes [Many mammals have the ability to synthesise ascorbic acidtar rats , lack thtar rats  and, thetar rats . The higtar rats , preventtar rats . As a sctar rats . Ascorbitar rats  and thentar rats , 27. Ascembranes . As a reembranes . Aside fembranes , 31.de novo from the amino acids cysteine, glutamate, and glycine via the sequential actions of γ-glutamylsynthetase and GSH synthetase. GSH is a powerful antioxidant with multiple functions in the eye. It protects protein thiol groups by affording protection against ROS, is necessary for the functioning of several glutathione-dependent antioxidant enzymes that neutralize ROS, and is responsible for drug detoxification [Glutathione can also be obtained from the diet but is primarily synthesised fication . GSH alsfication , 34. GSH2O2 which in turn can be converted to water and oxygen by catalase [2O2 is an uncharged molecule that can easily cross membranes. GSH-related enzymes rely on the presence of glutathione to exert their antioxidant. Glutathione reductase (GR) is a NADPH-dependent enzyme that facilitates the conversion of oxidised glutathione (GSSG) to reduced glutathione (GSH), thus maintaining the pool of total glutathione in its reduced and active form. In concert with GR, glutathione peroxidase (GPX) uses GSH to detoxify organic hydroperoxides and hydrogen peroxide resulting in the formation of GSSG and water.In the anterior chamber of the eye, superoxide dismutase (SOD), catalase, and glutathione-related enzymes act in conjunction with each other to play a critical role in regulating the production of ROS and precluding any tissue damage. While ascorbic acid is the main antioxidant in the aqueous humour, it is not as efficient in detoxifying ROS as antioxidant enzymes such as SOD . SOD actcatalase . CatalasThe positioning of the cornea means that it is constantly exposed to high amounts of UV radiation. To scavenge ROS and protect itself from light-induced damage, the corneal epithelium contains high concentrations of ascorbic acid which far exceed concentrations found in other ocular tissues , 23. TheIn the lens, ascorbic acid has many roles including prevention of membrane lipid peroxidation and the protection of lens cation pumps , 43. AccIn cultured bovine trabecular meshwork cells, physiological ascorbic acid levels have been shown to promote cell growth, glycosaminoglycan production , and stide novo synthesis from its precursor amino acids, GSH regeneration from GSSG by glutathione reductase, and GSH efflux. Surprisingly, there is limited information regarding the molecular pathways involved in maintaining GSH homeostasis in the cornea. Previous work has investigated GSH uptake by measuring 35S-GSH in the different layers of the cornea and found that the highest concentrations of 35S-GSH were detected in the stroma [While overall GSH concentrations in the cornea are lower relative to ascorbic acid concentrations, GSH plays a major role in corneal defence. It is involved in maintaining the barrier function of the corneal endothelium, controlling normal hydration levels, protecting cell membrane integrity, and degrading xenobiotics agents. In other tissues, GSH levels are maintained via a combination of GSH uptake, e stroma . Howevere stroma . This sue stroma , 65, alt+-dependent glutamate transporters (EAATs) and the Na+-independent cystine/glutamate exchanger (Xc−) in the human corneal epithelium [c−, its substrate cysteine, and the rate limiting enzyme for GSH synthesis, γ-glutamylcysteine synthetase (γ-GCS), were also localised to the rat corneal epithelium [c− system mediates the Na+-independent exchange of extracellular cystine (the oxidised form of cysteine) for intracellular glutamate, while the EAATs maintain the outwardly directed glutamate concentration gradient required for glutamate exchange with cystine [c− system also operates in the cornea and that GSH synthesis is most likely restricted to the epithelial layer and not the endothelium. A recent study into potential pathways for uptake of precursor amino acids utilized for GSH synthesis colocalized the Naithelium   1, 2, 4, and 5 and the organic anion transporting polypeptide (Oatp) 1 and 2, although only the rat Oatps and not human OATP appear to contribute to GSH release [The liver and kidney are major sites of GSH uptake, synthesis, and export and can therefore offer potential clues as to the molecular pathways that may operate in the cornea to maintain GSH levels. Two members of the organic anion transporter (OAT1 and OAT3) and the sodium dicarboxylate 3 transporter (NaDC3) have been shown to be involved in GSH uptake across the renal basolateral membrane . NaDC3 i release –72. The  release . MRPs al release . Of the three putative GSH uptake transporters, only OAT3 and NaDC3 are expressed at the transcript and protein level in the rat cornea . Subsequc− and EAAT4/5 colocalize indicating that these transporters may work together to accumulate cysteine for GSH synthesis [c− appears to switch its glutamate recycling partner to alanine-serine-cysteine transporter (ASCT2) which transports glutamate at low pH, which coincides with the acidic environment in the lens core [c− and ASCT2 may work to accumulate cysteine where it can act as a low molecular mass antioxidant in this highly compact region. The glycine transporters, GLYT1 and GLYT2, were both shown to be localised to the lens cortex indicating a role in glycine uptake in cortical fibre cells  . In the ynthesis . On the ens core . Since tre cells . Interesre cells . Immunolμmol/g tissue wet weight [2O2, which is known to cause tissue damage that could compromise outflow [In the excised bovine trabecular meshwork, glutathione is reported at a concentration of 0.40 t weight . While t outflow , 80. In  outflow . Thus, tOcular tissues are continuously subjected to a variety of physical, chemical, and environmental insults that generate ROS and create an oxidative environment in the eye. The tissues in the anterior segment of the eye, particularly the cornea, lens, and trabecular meshwork, bear the brunt of this oxidative stress due in part to their positioning in the eye and the avascular nature of these tissues. Unsurprisingly these tissues utilise similar antioxidant defence systems  which ai2O2 [The ciliary body provides the aqueous humour with antioxidants, nutrients, and amino acids. However, in contrast to ion transport and fluid transport , little 2O2 . Recentl2O2 , 95. Bot2O2 . GSH canc−  system is likely to be more effective in accumulating cystine/cysteine directly into nuclear fibre cells. Moving ahead, identification of other transporters used to accumulate endogenous antioxidants in ocular tissues could highlight alternative pathways that could then be targeted to increase uptake and elevate antioxidant levels.With increasing age, a decrease in antioxidants and/or antioxidant enzyme activity occurs, leading to progressive tissue damage and ocular pathologies. A large number of studies have explored the use of antioxidants (both endogenous and exogenous) in treating eye diseases. In comparison, only a few studies have investigated the molecular processes by which antioxidants accumulate in ocular tissues. For example, the use of thiol antioxidants and their derivatives, such as cysteine, glutathione, N-acetylcysteine (NAC), and more recently N-acetylcysteine amide (NACA), has been examined in providing protection from age-related nuclear cataracts –102. TheWhile we have described the actions of antioxidant compounds and enzymes that directly minimize oxidative stress, we have omitted enzymatic repair systems (such as thioltransferases (TTase) and thioredoxins (Trx) ) found iDespite the presence of immense antioxidant capacity, with age, ocular tissues can readily succumb to ROS-mediated damage. Thus, it is important to understand the transport systems and repair pathways responsible for accumulating and maintaining these antioxidants in ocular tissues, so that more targeted therapies can be developed and made successful.


Huntington's disease (HD) is caused by a dominant mutation that results in an unstable expansion of a CAG repeat in the huntingtin gene leading to a toxic gain of function in huntingtin protein which causes massive neurodegeneration mainly in the striatum and clinical symptoms associated with the disease. Since the mutation has multiple effects in the cell and the precise mechanism of the disease remains to be elucidated, gene therapy approaches have been developed that intervene in different aspects of the condition. These approaches include increasing expression of growth factors, decreasing levels of mutant huntingtin, and restoring cell metabolism and transcriptional balance. The aim of this paper is to outline the nucleic acid-based therapeutic strategies that have been tested to date. Htt) that results in synthesis of an aberrant polyglutamine tract in huntingtin protein (HTT) and leads to neuronal dysfunction and neurodegeneration [Huntington's disease  causes HD is still poorly understood. Normal HTT has been shown to have multiple distinct functions in cells, including antiapoptotic activity [The mechanism by which the mutant huntingtin  [Htt gene) [To date, many animal models have been developed that closely mimic HD symptoms or pathology. Rodents  and nonhuman primates have been used most extensively to test HD gene therapy strategies. In general, HD animal models can be divided into chemically induced and genetic models. The chemically induced HD models include excitotoxic lesion models  induced) –19 and m genome) , 24 and tt gene) –27 animaHtt causes the disease. These can be broadly classed into strategies involving (i) increasing expression levels of growth factors, (ii) decreasing levels of mutant HTT, and (iii) restoring cell metabolism and transcriptional balance.The majority of therapeutics currently used to treat HD are designed to ameliorate the symptomatology of the condition, that is, psychiatric agents for the control of behavioural symptoms, motor sedatives, cognitive enhancers –30, and Since the behavioural phenotypes in HD arise from a progressive loss of mainly striatal and cortical neurons, neuroprotection and neurorestoration are one of the major gene therapy approaches being developed. Early gene therapy strategies for HD focused on the delivery of neurotrophic factor genes as a direct means for protecting vulnerable striatal neurons against mutant HTT-mediated toxicity. Other alternatives have included delivery of molecules aimed at directing neurogenesis—the production of new adult neurons to replace neurons lost in the disease .ex vivo modified cell grafts [≈43% in control groups), and attenuated functional disability associated with striatal lesions produced by 3-NP infusion. Locomotor coordination as assessed by performance on the accelerating rotarod of animals expressing the NTN-producing transgene was similar to those of unlesioned animals [Ex vivo delivery of GDNF also reduced neuronal death and maintained motor functions, although to a lesser extent. This was attributable to lower levels of transgene expression compared to those achieved by direct in vivo viral-mediated gene transfer, also, GDNF expression appeared limited to the area immediately surrounding the transplant core, and only 1% of total cells injected survived 3 months after transplantation [Neurotrophic factors prevent cell death in degenerative processes and enhance growth and function of neurons. Several neurotrophic factors have shown promise as therapeutic agents in cell lines and animal models. Nerve growth factor (NGF) was the first trophic factor evaluated in an excitotoxic rodent model of HD. However, the potent protection from degeneration with nearly two-thirds of the neurons in the QA-injured striatum rescued was found only when NGF was delivered into striatum by l grafts –42, but l grafts , 44. The animals . Similar animals  and mous animals  models oantation . Interesantation . This mi% reduction in lesion volumes and twice as big cell survival rate versus control groups. The experiments using the first technique showed either no difference or only a slight difference in neuron density between CNTF-treated and unlesioned brains, except for the lateral caudate where the decrease in density was 11% for NeuN-immunoreactive and 18% for calbindin-immunoreactive neurons. CNTF-expressing BHK cell grafts were also employed in a phase I clinical study involving a small number of HD patients. Whilst no side effects were observed over a period of 2 years, no clinical benefit was observed in these patients, most likely due to the low amounts of CNTF release produced by many of the capsules and low survival of the encapsulated cells [Encouraging results were obtained following ciliary neurotrophic factor (CNTF) infusion. Although lentiviral-mediated overexpression of CNTF in the striatum of YAC72 mice, a genetic mouse model of HD, reduced hyperactivity in 5 and 8 month old mice, no differences in rotarod performance or feet-clasping was found compared to controls . In excied cells .ex vivo BDNF gene delivery [Much research on HD has focused on brain-derived neurotrophic factor (BDNF), which is considered to be the main candidate for neuroprotective therapeutic strategies for HD. BDNF has been shown to modulate the onset and severity of motor and cognitive functions in HD mouse models –56. Mutadelivery , 62, nordelivery  has provper se and can cause tumour formation due to accidental insertional mutagenesis [Although increasing BDNF expression by means of viral vectors has led to encouraging results, a number of issues still remain to be resolved, as excess expression of the BDNF transgene can have a deleterious effect on neuronal circuits and learning and memory , and somagenesis . Furtheragenesis , 66.Htt expression. This is an attractive approach to therapy as it sidesteps the requirement to understand the mechanism by which mutant Htt causes the disease pathology. The therapeutic promise of this direct approach is underpinned by a pivotal study demonstrating that repression of mHtt expression in a conditional mouse model could reverse the pathological features of the disease including formation of neuronal inclusions and abnormal motor behaviour [Htt load using this approach during the disease course might facilitate better protein clearance by affected neurons and allow neurons to normalise changes induced by mHtt. Moreover, the ability to instigate treatments prior to onset of disease symptoms that is afforded by genetic screening for individuals that have inherited the HD mutation allows greater opportunity to make a significant impact on disease progression, when neuronal dysfunction might be prevalent but significant neurodegeneration and depletion of cortical and striatal neuron populations are yet to take place.More recently, the development of effective gene silencing approaches using RNA interference technology has led to evaluation of strategies aimed at selectively reducing mutant ehaviour . Thus deHtt expression in HD human cell lines. PNAs are DNA/RNA mimics with an uncharged amide backbone, that increases the affinity of PNA hybridization and facilitates recognition of RNA target, and LNA is an RNA analogue that contains a methylene bridge between the 2′-oxygen and 4′-carbon of the ribose, which reduces the conformational flexibility of the ribose and confers outstanding affinity to complementary hybridization. A comparison in the same model cell line revealed that inhibition by PNAs and LNAs that target CAG repeats is more effective than inhibition by an siRNA that targets a deletion polymorphism [Modified antisense short nucleotide technology provides fascinating opportunities for development of nucleic acid-based therapeutics. Peptide nucleic acid (PNA) peptide conjugates and locked nucleic acid (LNA) oligomers were found to be potent and allele-selective inhibitors of mutant morphism .Htt transcripts using species-specific short hairpin (shRNA) or short-interfering RNA (siRNA) in HD transgenic mouse models. Sequence differences between mouse host and human genes allowed allele-specific silencing of the pathogenic human Htt transcripts in the setting of preserved expression of endogenous Htt. Intraventricular infusions of lipid-encapsulated [≈70% leading to a 56–66% reduction in HD protein levels. As a consequence, numbers and the size of intranuclear inclusions were decreased, and R6/2 transgenic mice and mice with the AAV-mediated Htt expression, respectively, showed improved performance in motor tests including beam walking and an accelerating rotarod.Early RNA interference-based approaches involved targeted knockdown of mpsulated  or intrapsulated  siRNAs eHtt decreased mutant huntingtin mRNA and protein levels by 51–78% and 28–50%, respectively, reduced numbers of aberrant nuclear inclusions, and improved disease-associated behaviours such as the feet-clasping phenotype in HD mice and rotarod performance as well as spontaneous exploratory forepaw use in rats [Htt mRNA reduced Htt mRNA levels by more than 80% and almost completely prevented loss of dopamine and cAMP-regulated phosphoprotein-32 (DARPP-32) expression, and restored deficits in striatal glucose metabolism and mitochondrial complex II activity in a rat HD model. Expression of shRNA after appearance of HD pathology also produced a drastic reduction in the lesion size, associated with a partial clearance of HTT inclusions [One of the major hurdles for gene silencing is effective delivery of siRNA sequences to affected cells. The chronic nature of this disease suggests that continuous and long-term expression of siRNA will be required. This can be achieved by chronic infusion but whether lifelong infusion can be tolerated is unclear. An alternative approach is to use viral vectors to achieve long-term expression of shRNA molecules. AAV-mediated brain delivery of shRNA directed against human mutant  in rats –73. In tclusions .Htt might be tolerated to some extent in the adult brain. To determine whether silencing of the normal allele might exacerbate mHtt pathology, Drouet et al. [Htt alleles. Lentiviral vector-mediated expression of a pathogenic human htt171-82Q on a background of reduced wild-type rat Htt levels did not produce any differences in GABAergic neuron survival or HTT inclusion load compared to rats that received the htt171-82Q alone suggesting partial inactivation of the endogenous rat allele did not increase the vulnerability of striatal neurons to mHtt. Moreover, coincident nonallele-specific silencing of mHtt and wild-type Htt is well tolerated in mice, with minimal signs of toxicity for up to 9 months [Htt mRNAs by ≈60% showed significant improvements in rotarod performance as compared to control-treated HD171-82Q mice. Moreover, despite AAV1-mi2.4 failing to normalise the weight loss observed in these animals, survival rates of these mice were also increased [While the results of these proof-of-concept studies are encouraging, one challenge in extending this concept to human HD subjects is the potential requirement to develop approaches that selectively silence the mutant allele whilst leaving the normal allele intact. Normal huntingtin has roles in axonal guidance, cAMP signaling, long-term potentiation/depression, and calcium and glutamate signaling, raising concerns about potential toxicity resulting from loss of huntingtin, function . Moreovet et al.  and Boudt et al.  evaluate9 months . However9 months  which ma9 months . Moreove9 months . TransgeHtt transcript [An alternative approach might also involve targeting disease-allele-linked small nucleotide polymorphisms (SNPs)  or deletanscript , and it anscript . It is aHtt-related molecular pathways when expression of the wild-type huntingtin is inhibited [Htt in mouse striatum with a dataset obtained from early grade HD patient caudate-putamen relative to their respective controls. Ninety-two genes were found to be altered in both gene sets. Interestingly, 41 of these showed changes in expression level in the same direction and were enriched in pathways involving developmental regulation of gene transcription. Fifty-one other genes that were common to both gene sets changed in the opposite direction and were enriched for proteins involved in ion transport and synaptic transmission, many of which are downregulated in human HD. These authors propose that these transcriptional changes might result from toxic gain of function aspect and that knocking down both mutant and wild-type Htt might revert these changes. However, a recent and extensive analysis of gene expression changes altered by Htt deficiency compared with the effect of the gain of function conferred by increasing CAG repeat length suggests that it is important to understand the biological processes affected by the lowering of normal Htt expression [ Htt allele (Hdh null). Microarray comparison between a gene set whose expression was continuously altered with increasing CAG length with that of a huntingtin-null gene set showed that there was virtually no overlap in these gene sets suggesting the CAG expansion confers a simple gain of a novel function as opposed to a mixed gain of function/loss of function mechanism. Although the molecular responses are quite distinct, the gene expression changes result in varying degrees of interconnectedness at the network level. For example, pathways involved in the energy network such as carbohydrate metabolism/glycolysis are more prominently affected by CAG repeat length whereas oxidative respiration and tricarboxylic acid cycle pathways are more affected by huntingtin deficiency [While these results are encouraging and suggest stringent targeted knockdown of the mutant allele may not be necessary, these studies have also reported significant transcriptomic changes in nhibited , 82. Boupression . Panels ficiency . These aHtt mRNA expression involves boosting the capacity of the cell to lower amounts of mutant huntingtin protein. The neurodegenerative and motor, cognitive, and psychiatric symptoms of HD typically manifest in midlife suggesting that in the presymptomatic phase, neurons are able to cope to some extent with the expression of mutant huntingtin protein. In a conditional transgenic mouse model of HD, blockade of mHtt fragment expression in symptomatic mice leads to a disappearance of inclusions and an improvement in behavioural phenotypes, demonstrating that continuous production of the mutant protein is required to maintain the disease process and raising the possibility that HD may be reversible [in vitro and reducing striatal lesion by 35–65% in vivo [An alternative to knocking down versible . The hal in vivo . Another in vivo . An intr in vivo . It impr in vivo .An important aspect of HD pathogenesis is the impact of the mutant protein on mitochondrial function and cellular bioenergetics. Reduced glucose utilisation and activities of complexes of the electron transporter chain in the striatum of advanced-grade HD subjects suggests a general metabolic deficit in HD patients –98. Addiα (PGC-1α), a transcriptional coactivator that regulates expression of genes involved in mitochondrial biogenesis and oxidative phosphorylation [α expression levels are specifically reduced in the caudate-putamen, the first region affected in HD, in presymptomatic postmortem HD cases. Moreover, a differential cellular expression pattern of PGC-1α is found in the caudate-putamen, with decreased expression in the vulnerable medium spiny neurons but upregulation in nNOS-immunoreactive interneurons which are typically spared in the disease [α expression is due to mHtt-mediated repression of PGC-1α transcription via interference of CREB/TAF4-dependent regulation of PGC-1α gene expression. Inhibition of PGC1a expression leads to altered expression of genes involved in energy metabolism and diminished capacity of vulnerable neurons in response to energy demands in HD [α could protect against mHtt-induced mitochondrial dysfunction and striatal toxicity. Direct administration of lentiviral vector expressing PGC-1α into the striatum of R6/2 transgenic HD mice completely prevented neuronal atrophy, complementing in vitro data showing PGC-1α overexpression reversed mitochondrial dysfunction in mutant STHdhQ111 cells, and abrogated the toxicity of mHtt in transfected primary striatal neurons, potentially through inducing the expression of genes encoding reactive oxygen species defence enzymes including Cu/Zn superoxide dismutase (SOD1), manganese SOD (SOD2), catalase, and glutathione peroxidase [α overexpression results in improved survival and motor performance in the R6/2 mice remains to be confirmed. These results suggest that stimulation of pathways involved in energy metabolism controlled by PGC-1α by pharmacological or genetic means could provide potential clinical benefit at early stages of HD.Therapeutic agents that aim to ameliorate the cellular energy deficits by enhancing energy production and improvement of mitochondrial function in HD are neuroprotective. Lentiviral-mediated overexpression of two subunits of the succinate dehydrogenase (SDH) enzyme, the main component of mitochondrial complex II, restores the membrane potential and blocks neuronal death induced by mutant huntingtin in murine cell model of HD . Anotherrylation . PGC-1α  disease , 105. Deroxidase . This isroxidase . WhetherHuntingtin can interact with a number of transcription factors, for example, cAMP-response element binding (CREB) binding protein , 109, TAper se and/or cause insertional mutagenesis. The incorporation of a regulatory systems such as the tet system might increase the safety profile of such a treatment approach. Moreover, although studies using viral vectors have shown sustained gene expression for many years, maintaining long-term expression is still a current challenge.Despite significant achievements in nucleic acid-based gene therapy for HD, a range of technical problems is needed to be solved and questions to be answered. Each strategy described has specific disadvantages. Though many studies have proven that gene therapy approaches employing antisense oligonucleotides represent an exciting therapeutic possibility, there are certain issues that remain to be addressed. These include the choice of cellular targets, the stability of gene silencing, as well as side effects, such as altering off-target gene expression, induction of cellular immune response, and interfering with endogenous mRNA silencing systems. There are also problems associated with viral delivery of therapeutic genes. Transgene expression can be difficult to standardize and regulate, and some of the vectors can be toxic Apart from these particular impediments, there are basic issues typical of all strategies, such as invasiveness of delivery methods associated with the direct administration of therapeutics into the brain due to the presence of blood-brain barrier, restriction of therapeutics distribution only to the area adjacent to the injection or transplantation site, and the need to ensure continuous effect of therapy and appropriate timing with relation to when to intervene in the course of disease.Finally, the main obstacles remaining include our inability to distinguish between primary and secondary disease mechanisms. This raises the question as to which of the many cellular pathways of pathogenesis would be the most effective target in influencing disease onset and progression. Further identification of abnormalities, pathways, and targets that are the most critical for neurodegeneration and discriminating them from the ones that are secondary responses or just related phenomena are required.


Diaphorina citri (Hemiptera: Psyllidae) is the primary vector of the bacterium causing citrus huanglongbing (citrus greening), the most serious disease of citrus worldwide. Psyllids and other hemipterans produce large amounts of honeydew, which has been used previously as an indicator of phloem sap composition and insect feeding or metabolism. Behavioral, ultrastructural and chemical studies on ACP, its honeydew and waxy secretions showed important differences between nymphs, males and females, and suggested some mechanisms by which the psyllids, especially nymphs and adult females, can minimize their contamination with honeydew excretions. The anal opening in ACP, near the posterior end of the abdomen, is on the ventral side in nymphs and on the dorsal side in adult males and females. Video recordings showed that adult males produce clear sticky droplets of honeydew gently deposited behind their body on the leaf surface, whereas adult females produce whitish honeydew pellets powerfully propelled away from the female body, probably to get their excretions away from eggs and newly hatched nymphs. ACP nymphs produce long ribbons or tubes of honeydew that frequently stay attached to the exuviae after molting, or drop when feeding on the lower side of citrus leaves. Furthermore, honeydew excretions of both nymphs and adult females are covered with a thin layer of whitish waxy material ultrastructurally composed of a convoluted network of long fine filaments or ribbons. This material is extruded from intricate arrays of wax pores in the circumanal ring (around the anus) that is found in nymphs and females but not in males of ACP or other psyllid species. Infrared microscopy and mass spectroscopy revealed that, in addition to various sugars, honeydew excretions of ACP nymphs and females are covered with a thin layer of wax similar in profile to ester waxes.The Asian citrus psyllid (ACP),  Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is an invasive species that was found originally in southwestern Asia, but has now spread to many countries in South, Central and North America starting in the 1990 s Candidatus Liberibacter spp.) associated with huanglongbing , currently the world’s most serious disease of citrus The Asian citrus psyllid (ACP), Honeydew excretions by hemipterans are the result of feeding on the phloem sap, which has very high sugar content and osmotic pressure. Sucrose-transglucosidase activity in their gut transforms excess ingested sugar into long-chain oligosaccharides that are voided via honeydew excretion Honeydew quantity or chemical analysis has been used as an indicator of insect feeding or metabolism in various hemipterans Psylla mali nymphs and adult females by Brittain Anomoneura mori, by Waku Psylla mali. However, in some previous investigations on ACP Husain and Nath The present work describes behavioral, ultrastructural and chemical studies of honeydew produced by ACP nymphs, adult males and females, as well as ultrastructure of the circumanal ring and circumanal (wax) gland openings in both nymphs and females of this economically important psyllid species.Citrus macrophylla Wester) in the greenhouse. Anal (honeydew) excretion behavior of ACP was observed and photographed using a stereomicroscope (Leica MZ16) fitted with a Leica DFC 320 camera, or using another stereomicroscope (Leica M60) fitted with a video camera (Leica DFC290 HD) . For these observations, ACP nymphs of various instars were fed in groups (10–20/group) on small pieces of fresh terminal young shoots (8–10 cm long) of sweet orange . ACP adult males and females, separately, were also fed in groups (5–10/group) on excised young Ridge Pineapple sweet orange leaves. The cut end of each terminal shoot or leaf petiole was placed in a small (0.5 ml) microfuge tube filled with water to keep it fresh for 3–7 days. Each shoot or leaf was then placed in a 50-ml polypropylene tube  or in a Petri dish for easier observation under the stereomicroscope ACP nymphs and adults used here were taken from our healthy laboratory colony (not infected with Ca. L. asiaticus) that has been maintained for several generations on young healthy citrus plants .Video recordings (1–2 h each) of anal (honeydew) excretion behavior of ACP males, females and nymphs as well as oviposition by females were undertaken. Three methods were used to prepare ACP nymphs, exuviae and/or adults for SEM to study the ultrastructure of their anal areas. The first method involved fixation and dehydration of live insects in 70% ethanol, 100% ethanol (twice) then air drying before mounting. The second method, used mainly with exuviae, was direct mounting without prior fixation or dehydration. A third method was used to clean the nymphs or adult females from their honeydew and waxy secretions, in order to examine the openings and structure of the circumanal ring (around the anus). This ‘dewaxing/cleaning’ method, modified from Lucchi and Mazzoni In all three SEM preparation methods mentioned above, the insects, exuviae or honeydew were mounted, under a stereomicroscope, on black conductive double-sided adhesive discs (9–12 mm diameter) placed on aluminum stubs , using fine-pointed forceps . Mounted specimens were then sputter coated with Gold-Palladium for 120 sec using Hummer 6.2 Sputter Coater . Coated specimens were then examined at 5 or 10 Kv using a scanning-transmission electron microscope  in the SEM mode at magnifications of 100X to 10,000X. The number of ACP specimens examined by SEM was 8–12 waxed or dewaxed specimens in each of the following categories: males, females and nymphs. All the original electron micrographs digitally obtained in this study were automatically saved on the image management computer program (Quartz PCI version 8) associated with the Hitachi S-4800 electron microscope mentioned above.−1 averaging a total of 128 scans at each position along the map.Spectra of the honeydew produced by ACP nymphs, males and females were obtained using the Thermo Nicolet iN10 FTIR microscope in the reflection mode (for intact honeydew samples), as well as the attenuated total reflectance Fourier Transform Infrared (ATR-FTIR) mode (for crushed samples). Microscope, spectrometer, data acquisition and data processing functions were done using Thermo Nicolet Picta software. To acquire the spectra in the FTIR mode, honeydew samples were placed on an aluminum coated slide, which was mounted on the stage of the microscope. The microscope was focused on the top surface of these honeydew structures, and a linear spectra map was created along the long axis of the top surface of the tube/ribbon. Spectra were acquired at a resolution of 8 cmACP adult males and females, reared separately on citrus leaves or young terminal shoots, were found to feed for long periods at the same feeding site (up to 1–2 hrs), judging by their typical feeding posture, with their body angled ca. 40° with the leaf surface . The colth of a second) but the time that elapsed between producing pellets was much longer (20–30 min or longer). The process of producing anal/honeydew excretions by ACP males and females was recorded by video . Althougrd– 4th instar nymphs this ring measured about 110–130 µm long, and 30–40 µm wide. At the ultrastructural level, SEM showed that the cirucmanal ring is composed of prominent cuticular ridges . The wax pores between each ridge and the next (1.6–1.7 µm wide) are full of small dot-like structures (probable mini-pores) arranged in sets of 3 producing a triangular arrangement  is located on the ventral side near the end of the abdomen . It is sangement . Inside angement . In someangement . The waxst instar, 10–12 setae; 2nd instar, 15–17 setae; 3rd instar, 30–38 setae; 4th and 5th instars, 46–56 setae (with some overlap between the last two instars). One function of these setae appears to be keeping the excreted honeydew tubes/ribbons in a somewhat straight line so that they do not stick to the abdomen’s end  is found on the dorsal side at the anterior third of the dorsal plate of the genital segment . It is oThe anal opening in ACP male is also located on the dorsal side  on top of the anal tube . But it At the ultrastructural level, using SEM with magnifications of 500–10,000x, the outer surface of the honeydew tubes or ribbons of ACP nymphs, was composed of very long, extremely fine, convoluted filaments that apparently came out of the wax pores and cuticular slits described above in the circumanal ring of nymphs . Waxy st−1, attributed to water and hydroxyl groups, and other large bands from 1100 to 1000 cm−1 attributed to the carbon-oxygen single bonds of sugars, including sucrose, fructose and Beta-D-(+)-glucose  spectra of ACP honeydew  showed no sign of wax being present in the honeydew of nymphs, males or females. Typically, ATR-FTIR analysis of these excretions indicated that this material is composed mainly of water and sugars. The spectra are characterized by huge broad bands in the region from 3600–2800 cm-glucose . Peaks i−1 range attributed to the carboxyl C = O of the wax esters as well as two pronounced peaks at 2850 and 2920 cm−1 attributed to C–H bonds of aliphatic hydrocarbons, fatty and ester waxes such as bees wax  also existed in the FTIR reflectance microscopy spectra of the top/surface of the honeydew of males, females and nymphs , spectrabees wax . No pronbees wax .Feeding on the phloem presents certain challenges to hemipteran insects, which normally have certain adaptations to counter these challenges. First, the high sugar content and osmotic pressure of phloem sap is countered by sucrose-transglucosidase activity in their guts, which transforms excess sugar into long-chain oligosaccharides voided as honeydew excretion ACP produces copious amounts of honeydew excretions by nymphs and adults P. mali) were described at the light microscopy level by Brittain A. mori) by transmission electron microscopy, and indicated that these glands consisted of two kinds of cells, derived from epidermal cells: wax cells, which produce and secrete the wax, and flat interstitial cells found among these cells. Each wax cell has a long, wide duct which opens at the cuticle. The openings of the wax glands in the circumanal ring of psyllid nymphs and females were also described at the light microscopy level by Brittain P. mali and D. citri, respectively. It is surprising how accurate these descriptions were, given the resolution of the light microscopy when compared with SEM. However, our SEM work here provided much finer details of the wax pores and cuticular slits found on the circumanal ring of ACP females and/or nymphs. The wax pores we described here, especially in ACP nymphs, are somewhat ultrastructurally similar to openings of the wax glands, described as ‘canaliculated cuticle’, in the nymphal anal ring of the psyllid A. moriMetcalfa pruinosa (Fulgoroidae) The wax  glands in nymphs and female adults of the apple psyllid (Ctenarytaina thysanura) greatly reduced the attractiveness of terminal shoots for oviposition . That ACP nymphs and females have certain mechanisms and adaptations to avoid contamination with honeydew whereas ACP males apparently do not is interesting, and suggests that ACP males and females may occupy different sites on citrus plants, especially with regard to egg laying. ACP females normally lay their eggs on very young, feathery and folded leaves, or in the axillary corners between young terminal shoots and leaf petioles , found in nymphs but not in adults of either sex, appear to keep the long honeydew ribbons or tubes away from their bodies  & 3A, D.The phloem feeding scale insects and mealybugs are known to posses several mechanisms to limit contamination from their own sticky honeydew excretions Video S1Anal (honeydew) excretion behavior of Asian citrus psyllid males and females. This video (1 min 52 sec. long) is composed of 4 short clips showing one male producing two consecutive, clear, honeydew drops, one on top of the other (2 separate clips), followed by one female producing one, yellowish/whitish, honeydew pellet (one clip), and finally another female (at lower right) producing another pellet (one clip). All clips were recorded at real time ; the male clips are played back at normal speed, whereas the female clips are played back at a much slower speed .(WMV)Click here for additional data file.


Developmental and global regulation of mRNA translation plays a major role in regulating gene expression in mammalian spermatogenic cells. Sucrose gradients are widely used to analyze mRNA translation. Unfortunately, the information from sucrose gradient experiments is often compromised by the absence of quantification and absorbance tracings, and confusion about the basic properties of sucrose gradients.The Additional Materials contain detailed protocols for the preparation and analysis of sucrose and Nycodenz gradients, obtaining absorbance tracings of sucrose gradients, aligning tracings and fractions, and extraction of equal proportions of RNA from all fractions.The techniques described here have produced consistent measurements despite changes in personnel and minor variations in RNA extraction, gradient analysis, and mRNA quantification, and describes for the first time potential problems in using gradients to analyze mRNA translation in purified spermatogenic cells.Smcp) mRNA in transgenic mice.Accurate quantification of the proportion of polysomal mRNA is useful in comparing translational activity at different developmental stages, different mRNAs, different techniques and different laboratories. The techniques described here are sufficiently accurate to elucidate the contributions of multiple regulatory elements of variable strength in regulating translation of the sperm mitochondria associated cysteine-rich protein ( Pgk2 mRNA, and all mRNAs in meiotic and haploid spermatogenic cells are partially repressed by a global mechanism(s), some of which appear to undergo little developmental regulation [The regulation of mRNA translation plays an important role in gene expression in spermatogenic cells. The mRNAs encoding many proteins that are first synthesized during the final stages of sperm differentiation are transcribed in early spermatids, stored as translationally repressed free-mRNPs for up to a week in mice, and actively translated in late spermatids . Howevergulation -3.Smcp) 5' and 3'UTR contain multiple elements which regulate translation of the green fluorescent protein (GFP) coding region in transgenic mice to different extents [Sucrose gradients are frequently used to study mRNA translation in spermatogenic cells, because the method can be used to study the translational activity of any mRNA for which probes can be designed. Although sucrose gradients have produced important insights into translational regulation in spermatogenic cells, this body of information is compromised for technical reasons. A common problem is that many sucrose gradient analyses lack absorbance tracings, which are necessary to establish that the translational activity of the cell population under analysis is normal. Another problem is that the polysome loading of mRNAs is rarely quantified. Quantification is necessary to compare polysome loading of different mRNAs, experimental protocols, developmental stages, and laboratories. Quantification has also provided evidence that translation of the sperm-mitochondria cysteine-rich protein mRNA , Smcp (900 nt) and Pabpc1 (3000 nt) free-mRNPs sediment at 20S, 50S and 80S, respectively [Prm1 (153 nt), Smcp (429 nt), Gfp (720 nt) and Ldhc mRNAs (993 nt), sediment respectively with polysomes containing, 3-4, 3-4, 5-6 and 7-8 ribosomes/polysome [Sucrose gradients and Nycodenz gradients have complementary advantages. Sucrose gradients are superior to Nycodenz gradients in providing information about the sizes of free-mRNPs and polysomes. In general, the rate of sedimentation of free-mRNPs is correlated with the length of the mRNA. For example, the peaks of Prm1 50 nt, Smectively ,11. Thispolysome . By compt), Smcp 29 nt, GfSmcp and Ldhc mRNAs and 18S ribosomal RNA was quantified by phosphorimage analysis of Northern blots. Note that all of the gradients in this study were collected manually from the bottom. Figure Smcp and Ldhc free-mRNPs align in Fraction 10, that the peak of polysomal Ldhc mRNA aligns with the peak of 18S rRNA in fraction 4, and that the levels of Smcp mRNA increase continuously in fractions 4-6. Fraction 4 contains ~0.6% of the total Smcp mRNA on the gradient. The absence of a peak of Smcp mRNA in fraction 4, implies that the levels of polysomal Smcp mRNA are negligible at this age, in agreement with the observation that the most advanced spermatids in 26 dpp testis are ~step 9, and therefore lack the step 11-16 spermatids in which the SMCP protein is detected [Nycodenz gradients may also provide more accurate estimates of the levels of polysomal mRNA in cell populations in which mRNA translation is strongly repressed. Figure Several controls are commonly used to establish that mRNAs are associated with polysomes in sucrose gradients.First, absorbance tracings identify regions of sucrose gradients containing mRNAs that sediment faster than monosomes, polysomes, and slower than monosomes, free-mRNPs. The relationship between coding region length and polysome size in sucrose gradients provides another argument supporting the identification of polysomes translating specific mRNAs ,5. AdditPrm1 and Prm2 mRNAs are often used for this purpose because the length of these mRNAs correlates with translational activity: Prm1 and Prm2 mRNAs in free-mRNPs have long, homogenous poly(A) tails, while the polysomal mRNAs have heterogeneous, shortened poly(A) tails [Ldhc mRNA, encoding the testis-specific isoform of lactate dehydrogenase, is another useful control mRNA, because four laboratories report that it exhibits high and essentially constant levels of polysomal mRNA in pachytene spermatocytes, round spermatids, and prepubertal and and adult testis in sucrose gradients [Second, analysis of the distribution of control mRNAs that sediment with free-mRNPs and/or polysomes. The A) tails ,16. The radients ,19-21.++-containing sucrose gradients shift to the mRNP fractions after disruption of the associations of the ribosomal subunits with each other and with mRNA by EDTA. Somewhat surprisingly EDTA-treatment does not shift all polysomal mRNA to the mRNP fractions; a minor fraction of some mRNA species sediment with small polysomes [Third, mRNAs that sediment with polysomes in Mgolysomes .These methods usually seem to work well. In most cases, inferences of translational activity based on expression of various testicular proteins, the proportions of mRNAs sedimenting with polysomes and free-mRNPs in sucrose and Nycodenz gradients, and dissociation by EDTA are in complete agreement ,4.Drosophila cell-free translation systems [Gfp 5'UTR, the Gfp coding region and the Smcp 3'UTR in 21 dpp mouse testis [Smcp-Gfp mRNA sediments with polysomes in sucrose gradients, despite the fact that GFP fluorescence is undetectable and 3% of the mRNA equilibrates with polysomes in Nycodenz gradients [Ldhc mRNA appears to be in large, inactive mRNPs based on findings that 56% and 35% is polysomal in sucrose and Nycodenz gradients, respectively, in both 21 dpp and adult testis [However, the identification of polysomal mRNAs is complicated by several recent reports of translationally repressed mRNAs that sediment with polysomes in sucrose gradients. These large, inactive mRNPs include "pseudopolysomes" induced by microRNAs in  systems ,23, and e testis . 55% of radients . In addit testis . The frebona fide polysomes in sucrose gradients. The large, inactive complexes mentioned above are dissociated by EDTA [Smcp-Gfp mRNA mentioned above sediment with polysomes in sucrose and Nycodenz gradients, respectively, suggests that Nycodenz gradients may be a more reliable control than dissociation with EDTA to establish that a mRNA is associated with polysomes [Unfortunately, there are no simple tests that reliably distinguish between large inactive complexes and  by EDTA ,5,22, th by EDTA , Cataldoolysomes . Howeverolysomes  report tolysomes , but wouPrm1 mRNA is the most commonly used control mRNA to establish the positions of free-mRNPs and polysomes in sucrose gradients. This mRNA has potential problems because its coding region is one of the smallest in mammalian cells, 153 nt. For example, an experiment in which polysomal Prm1 mRNA sediments near the bottom of a sucrose gradient is one in which large polysomes have pelleted. Thus, failure to analyze the mRNA in the pellet could lead to the erroneous conclusion that a large mRNA is translationally inactive. We know of no evidence that the common practice of placing a 60% sucrose pad on the bottom of sucrose gradients effectively prevents large polysomes from pelleting.The Differences in the sizes of mRNPs and polysomes containing mRNAs of varying sizes also create difficulties in dividing sucrose gradients into two fractions  for microarray studies of global mRNA translation.Prm1 and Prm2 mRNAs may create another potential problem. For many years it was generally believed that the number of ribosomes associated with specific mRNAs is usually proportional to the length of the coding region and that the ribosomes are spaced ~100 nt apart on coding regions [Prm1 mRNA in sucrose gradients accurately reflects the translation of all mRNAs. Now, it is unclear whether this is true: the size and levels of Prm1 polysomes may be less sensitive to stress than those of mRNAs with longer coding regions.The small size of the  regions ,25 demon regions  extend t regions  it seemeAnother misconception is that the all of the proteins detected by Western blots that sediment slower than single ribosomes are in free-mRNPs. In reality, most free-proteins sediment between 2 and 7S in sucrose gradients , while mAccurate quantification of polysomal mRNA requires attention to details in the preparation of the cytoplasmic extract for the gradients, extraction of RNA from the gradient fractions, and quantification of mRNA levels.Quantitative analysis of mRNA translation in sucrose gradients should begin with absorbance tracings which demonstrate normal levels of polysomes for testis. Four laboratories have reported similar absorbance tracings of extracts of adult testis with a major peak of 80S single ribosomes and a broad hump of polysomes with maximum absorbance in polysomes containing 5-7 ribosomes/polysome ,9,27-29.The distribution of a control mRNA in sucrose gradients is often used to argue that translation is normal. However, this argument is invalid in the absence of quantification: some sucrose and Nycodenz gradients have been "validated" with control mRNAs that exhibit virtually no polysomal mRNA.6 pachytene spermatocytes contain sufficient ribosomes to obtain absorbance tracings. In addition, the tracing in Figure Another problem is illustrated in Figure Ldhc mRNA that sediment with polysomes, ~60%, are similar to those in freshly dissected adult and prepubertal testis, ~56%  is reputed to be less variable and nearly as sensitive as RT-qPCR .Smcp mRNA that sediment in the polysomal region of sucrose and Nycocdenz gradients in 21 dpp testes are polysomal mRNA and free-mRNP contaminants [Smcp mRNA in the polysomal regions should not obscure the importance of this question. Premature mRNA translation in spermatids reduces male fertility [Second, it is unclear what proportions of the 3.9% of the aminants . The lowertility ,36, a trertility . Thus, sertility .Finally, translational regulation of individual mRNAs can analyzed by a recently developed technique, ribosome profiling, in which ribosome-protected RNase digestion fragments are deeply sequenced . This teThe authors declare that they have no competing interests.KCK conceived of the project, assisted in several experiments, and wrote the manuscript; SKH and JB did most of the experiments, and LC performed the analysis of mRNA translation in purified pachytene spermatocytes. All authors have read and approved the manuscript.Methods for analysis of mRNA translation using sucrose and Nycodenz gradients.Click here for fileFigure S1: Equipment for pouring sucrose gradients.Click here for fileFigure S2: Equipment for collecting fractions from sucrose and Nycodenz gradients.Click here for fileFigure S3: Changes in absorbance during the analysis of a sucrose gradient.Click here for file


Sharp wave-ripples (SWRs) are population oscillatory patterns in hippocampal LFPs during deep sleep and immobility, involved in the replay of memories acquired during wakefulness. SWRs have been extensively studied, but their exact generation mechanism is still unknown. A computational model has suggested that fast perisomatic inhibition may generate the high frequency ripples (~200 Hz). Another model showed how replay of memories can be controlled by various classes of inhibitory interneurons targeting specific parts of pyramidal cells (PC) and firing at particular SWR phases. Optogenetic studies revealed new roles for interneuronal classes and rich dynamic interplays between them, shedding new light in their potential role in SWRs. Here, we integrate these findings in a conceptual model of how dendritic and somatic inhibition may collectively contribute to the SWR generation. We suggest that sharp wave excitation and basket cell (BC) recurrent inhibition synchronises BC spiking in ripple frequencies. This rhythm is imposed on bistratified cells which prevent pyramidal bursting. Axo-axonic and stratum lacunosum/moleculare interneurons are silenced by inhibitory inputs originating in the medial septum. PCs receiving rippling inhibition in both dendritic and perisomatic areas and excitation in their apical dendrites, exhibit sparse ripple phase-locked spiking. In recent years a wealth of knowledge about the anatomical, physiological, molecular, and synaptic properties of the various cell types in the hippocampus has accumulated. Apart from the numerous different identified classes of hippocampal interneurons targeting specific parts of pyramidal cells (PC)  from rodents, primates and humans, during deep sleep, anaesthesia, and awake immobility. They are observed synchronously throughout the hippocampus , through the perforant path, and in their proximal dendrites from the CA3 Schaffer collaterals, as a portion of the trisynaptic loop. PC axons target mainly subicular and neocortical areas, and recurrent excitation is very low (less than 1%) in CA1 (Amaral and Witter, The different CA1 excitatory and inhibitory neurons display diverse firing patterns during SWRs (Klausberger et al., Early theoretical studies (Traub and Bibbig, Based on the observations that inhibition is necessary for SWR generation (Ellender et al., Nevertheless, the model does not address the variable roles of the different identified classes of hippocampal interneurons targeting specific parts of PCs. Cutsuridis and Hasselmo  were thein vivo by Royer et al. (Moreover, recent experimental reports, all based on combined optogenetic, juxtacellular, and pharmacological approaches, shed new light on the role of various interneuronal classes in shaping the CA1 spiking output (Lapray et al., r et al. , on micer et al.  producedr et al.  also shoWe attempt to incorporate these new findings in a conceptual model on how various forms of somatic and dendritic inhibition may collectively contribute to the generation and maintenance of SWRs in region CA1, while at the same time providing functional roles for the various CA1 and MS cells during SWRs Figure  model: CDuring the onset of the CA3 population burst, AACs are the first to respond by increasing their spiking (Klausberger et al., 2+ dendritic spikes are rarely observed during SWRs (Kamondi et al., BSCs are the second interneuronal class to respond to the strong CA3 excitation (Klausberger et al., BCs are the third interneuronal class to respond to the CA3 population bursts, increasing their firing slightly after BSCs (Klausberger et al., As a result, from the first stages of the SWR on, CA1 PCs receive a barrage of excitatory inputs throughout their apical and basal dendrites via Schaffer collaterals, combined with a ripple-frequency oscillating inhibition that is synchronous throughout their dendritic arborization and their soma, reflected in intracellular voltage oscillations Figure . In accoAlthough BC axons have been shown to make synaptic contacts to cells located in stratum oriens (e.g., OLM cells, Klausberger et al., What functions do CA1 PCs serve when they produce bursts as opposed to when they fire regular spikes? If PCs would turn to busters by silencing of BSCs during SWRs, what would the effect be on the fidelity of pattern replays and consequently on memory-task performance?What is the functional role of the AAC turn-off on the transfer of the PC output to its synaptic targets?How is the activity of MS GABAergic cells controlled by SWRs and what is its functional role in CA1? How would the silencing of type 1A MS cells, during SWRs, affect their CA1 interneuronal targets and consequently SWRs?What effect does the BSC inhibition have in synaptic plasticity properties of CA1 PC thin oblique dendrites during SWRs?Assuming that spike sequence replays are generated within CA3, what are the exact synaptic/network mechanisms within CA1 controlling their transfer to extrahippocampal targets?What is the functional role of the plethora of other interneuronal classes during SWRs, not addressed here?Which intrinsic cell properties and network features need to be incorporated in a computational model to simulate the characteristics of our conceptual framework? What would such a computational model predict about CA1 functionalities in non-SWR hippocampal states?How could our model be combined with recent modeling studies on the role of extracellular spikes in high-frequency LFPs (Schomburg et al., This theoretical model combines the computational approaches of Cutsuridis and Hasselmo  and TaxiNew optogenetic, juxtacellular, pharmacological and imaging experiments (Lapray et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.