Optogenetic stimulation: Understanding memory and treating deficits.

Technology allowing genetically targeted cells to be modulated by light has revolutionized neuroscience in the past decade, and given rise to the field of optogenetic stimulation. For this, non-native, light activated proteins (e.g., channelrhodopsin) are expressed in a specific cell phenotype (e.g., glutamatergic neurons) in a subset of central nervous system nuclei, and short pulses of light of a narrow wavelength (e.g., blue, 473 nm) are used to modulate cell activity. Cell activity can be increased or decreased depending on which light activated protein is used. We review how the greater precision provided by optogenetics has transformed the study of neural circuits, in terms of cognition and behavior, with a focus on learning and memory. We also explain how optogenetic modulation is facilitating a better understanding of the mechanistic underpinnings of some neurological and psychiatric conditions. Based on this research, we suggest that optogenetics may provide tools to improve memory in neurological conditions, particularly diencephalic amnesia and Alzheimer's disease.

Anterior thalamic nuclei neurons sustain memory.

A hippocampal-diencephalic-cortical network supports memory function. The anterior thalamic nuclei (ATN) form a key anatomical hub within this system. Consistent with this, injury to the mammillary body-ATN axis is associated with examples of clinical amnesia. However, there is only limited and indirect support that the output of ATN neurons actively enhances memory. Here, in rats, we first showed that mammillothalamic tract (MTT) lesions caused a persistent impairment in spatial working memory. MTT lesions also reduced rhythmic electrical activity across the memory system. Next, we introduced 8.5 Hz optogenetic theta-burst stimulation of the ATN glutamatergic neurons. The exogenously-triggered, regular pattern of stimulation produced an acute and substantial improvement of spatial working memory in rats with MTT lesions and enhanced rhythmic electrical activity. Neither behaviour nor rhythmic activity was affected by endogenous stimulation derived from the dorsal hippocampus. Analysis of immediate early gene activity, after the rats foraged for food in an open field, showed that exogenously-triggered ATN stimulation also increased Zif268 expression across memory-related structures. These findings provide clear evidence that increased ATN neuronal activity supports memory. They suggest that ATN-focused gene therapy may be feasible to counter clinical amnesia associated with dysfunction in the mammillary body-ATN axis.

Myelinated, synapsing cultures of murine spinal cord--validation as an in vitro model of the central nervous system.

Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.

Activation of ion channels in tumor cells by leukoregulin, a cytostatic lymphokine.

We used the whole-cell patch-clamp recording technique to study the effects of leukoregulin (LR), a cytostatic lymphokine produced by activated human peripheral blood lymphocytes, on the electrical properties of K562 tumor cells. LR induced changes in the membrane excitability in 33 of 55 cells studied. A minute or more after application, LR elicited a complex and reversible electrical response. The response lasted for several more minutes in the continued presence of LR. It consisted of (a) moderate-conductance (50 pS), cation-selective, ion-channel activity, (b) a shift of the zero-current membrane potential close to 0 mV, and (c) a suppression of a depolarization-activated K+ conductance. All of these changes in tumor cell excitability act coordinately to depolarize the tumor cells and may be important in the cytostatic actions of LR.

Leukoregulin-increased plasma membrane permeability and associated ionic fluxes.

The role of ion fluxes in the increased plasma membrane permeability of tumor cells exposed to the anticarcinogenic and tumor cell proliferation inhibitory lymphokine, leukoregulin, was examined by flow cytometic analysis of cell surface perturbations indicative of membrane destabilization. The pI 5.3 form of leukoregulin was isolated by ion exchange, isoelectric focusing, and molecular sizing chromatography of lymphokines from phytohemagglutinin-stimulated normal human lymphocytes. Membrane permeability increased within 5 min of leukoregulin exposure of tumor cells and was quantified by following the efflux of fluorescein or influx of propidium iodide. Membrane permeability increased in proportion to leukoregulin concentration. By 2 h, 0.25-30 units/ml induces a 10-90% change in K562 erythroleukemia cell permeability. Similar changes are effected by Ca2+ ionophores A23187 and X-537A but not by the Na+ ionophore monensin or the K+ ionophore valinomycin. The Ca2+ ionophores and the intracellular Ca2+ mobilizers ouabain and amphotericin B enhance, whereas calmodulin inhibits leukoregulin action. Ca2+ channel blockers nifedipine and verapamil and the Na+ and K+ ion transport inhibitors amiloride and atractyloside, respectively, neither alter membrane permeability nor influence leukoregulin activity. Further evidence for the role of increased Ca2+ flux in leukoregulin's action is provided by detection of increased intracellular free Ca2+ in leukoregulin-treated cells with the Ca2+-sensitive fluorescent probe 2-[2-bis(carboxymethyl)amino-5-methylphenoxy]methyl-6-methoxy-8-bis (carboxymethyl)aminoquinoline. Kinetic analysis of cell volume, forward and right angle light scatter, fluorescein efflux, and propidium iodide influx, moreover, reveals that the action of leukoregulin is unique. Membrane perturbations may be critical initial steps in the ability of leukoregulin to directly prevent the development of carcinogenesis as well as inhibit the continued proliferation of neoplastic cells.

HPV-16 E7 functions at the G1 to S phase transition in the cell cycle.

Previous reports have demonstrated that E7 is the major transforming gene of HPV-16 and that continued expression of the gene is required to maintain the transformed phenotype of primary baby rat kidney cells transformed by HPV-16 E7 and EJ-ras. To investigate the point of action of E7 in the cell cycle we have utilised a system of inducible expression of the E7 gene. The studies reported here show that stimulation of cellular DNA synthesis by E7 is distinct from that observed with calf serum. In combination with cytofluorimetric analyses these results indicate that E7 functions at the transition from G1 to S phase of the cell cycle. This adds further support to the hypothesis of a common pathway of transformation shared by the DNA tumour viruses HPV, SV40 and Adenovirus.

Studies of mammary carcinoma metastasis in a mouse model system. I: Derivation and characterization of cells with different metastatic properties during tumour progression in vivo.

The biological and metastatic properties of cells from a murine mammary adenocarcinoma, MT1, were studied during serial transplantation in syngeneic hosts. Over 35 generations the tumour progressed from a well-differentiated, poorly metastatic neoplasm to an anaplastic highly metastatic state. At early passages the tumour yielded uniform cultures of cuboidal epithelial cells, at passage 17 both epitheloid and spindle type cells were present, and by passage 30 only spindle type cells were obtained. Epithelioid cell lines and clones when injected intravenously into syngeneic hosts produced lung colonies only, whereas spindle cell lines were capable of extensive extrapulmonary colonisation. Similar patterns of dissemination and growth were seen in spontaneous metastasis assays. In spite of the marked phenotypic differences in these 'subpopulations', their comparable ultrastructural features, oestrogen receptor levels, expression of MMTV antigens, DNA content and lectin binding profiles suggested a common cell lineage. It is proposed that these cell lines will be of use in the determination of tumour and host factors influencing tumour progression and the evolution of metastatic potential.

Studies of mammary carcinoma metastasis in a mouse model system. II: Lectin binding properties of cells in relation to the incidence and organ distribution of metastases.

A panel of fluorescein-conjugated lectins was used to investigate the cell surface carbohydrates of cell lines isolated from a mouse mammary adenocarcinoma which differ markedly in their morphological and metastatic properties. The lectin-binding profiles of the cells showed them to express generally similar cell surface characteristics; however, two minor differences were evident. Galactose moieties recognized by peanut lectin were expressed on all highly metastatic fusiform cell types examined, but only on 50-60 per cent of the polygonal cells of limited metastatic capacity. Similarly, N-acetylgalactosamine moieties were demonstrated on fusiform cell types by soya bean lectin binding but were not expressed on intact polygonal cells. In both cases pretreatment of polygonal cells with neuraminidase allowed lectin binding comparable with that of fusiform cells suggesting that Gal and GalNAc sugars were abundantly present but masked by sialic acid residues. Using a novel technique in which tumour cells were incubated on cryostat sections of normal tissues, it was found that the cell lines exhibited different adhesion patterns which to some extent reflected their preferential sites for spontaneous metastasis and organ colonization in vivo. Thus the adherence of fusiform cells to liver was five times as great as that of polygonal cells, whereas the latter bound preferentially to lung tissue. Prior treatment of polygonal cells with neuraminidase doubled their frequency of attachment to liver sections, but had no effect on their binding to other tissues. Also, the presence of 100 mM N-acetylgalactosamine during incubation specifically inhibited the adherence of fusiform cells to liver tissues, but did not significantly influence other cell-tissue interactions. The data suggest that the expression of galactosyl or N-acetylated galactosyl groups on the fusiform cells facilitates their attachment to lectin-like receptors on liver cells and contributes to their superior capacity, compared with polygonal cells, for growth and metastasis in this organ.

The differentiation of glial cell progenitor populations following transplantation into non-repairing central nervous system glial lesions in adult animals.

The non-repairing nature of the locally x-irradiated ethidium bromide (EB)-induced demyelinating white matter lesion has been further validated by showing that injections of two cultures which promote host remyelination of EB lesions in normal tissue do not do so in x-irradiated lesions. The behaviour of an oncogene-immortalized glial cell line and a growth-factor-expanded glial progenitor population have been examined following transplantation into the non-repairing EB lesion. Our studies indicate that the selected glial cell populations were each capable of establishing glial environments around demyelinated axons. Extensive oligodendrocyte remyelination with little astrocytic presence was observed in lesions transplanted with growth-factor-expanded optic nerve progenitors, while less extensive oligodendrocyte remyelination with the establishment of astrocyte-like cells was found in lesions transplanted with ts A58-SV40T immortalized glial cells. Prolonged expansion of both populations resulted in a loss of differentiation to normal glial phenotypes.

Adhesion molecule expression and phenotype of glial cells in the olfactory tract.

These data illustrate that OBECs have a highly plastic nature in keeping with their need to respond rapidly to changing environmental cues. This relates to their required function in supporting axonal extension throughout life. Future studies using antibodies to PSA-NCAM and L-NGFr together with FACS sorting to purify the two types of OBEC should give us a clearer understanding of the lineage relationship of the two phenotypes. With purified populations of the astrocyte-like and Schwann cell-like OBEC we should be able to determine if these cells have different functions in vivo, using several approaches namely: i) identifying the growth factors that regulate their growth and differentiation, ii) measuring the ability of the purified cells to remyelinate the experimentally-created CNS lesions and iii) carry out more detailed cellular and molecular comparisons of the two phenotypes.

Epac and the high affinity rolipram binding conformer of PDE4 modulate neurite outgrowth and myelination using an in vitro spinal cord injury model.

BACKGROUND AND PURPOSE: cAMP and pharmacological inhibition of PDE4, which degrades it, are promising therapeutic targets for the treatment of spinal cord injury (SCI). Using our previously described in vitro SCI model, we studied the mechanisms by which cAMP modulators promote neurite outgrowth and myelination using enantiomers of the PDE4-specific inhibitor rolipram and other modulators of downstream signalling effectors. EXPERIMENTAL APPROACH: Rat mixed neural cell myelinating cultures were cut with a scalpel and treated with enantiomers of the PDE4-specific inhibitor rolipram, Epac agonists and PKA antagonists. Neurite outgrowth, density and myelination were assessed by immunocytochemistry and cytokine levels analysed by qPCR. KEY RESULTS: Inhibition of the high-affinity rolipram-binding state (HARBS), rather than the low-affinity rolipram binding state (LARBS) PDE4 conformer promoted neurite outgrowth and myelination. These effects were mediated through the activation of Epac and not through PKA. Expression of the chemokine CXCL10, known to inhibit myelination, was markedly elevated in astrocytes after Rho inhibition and this was blocked by inhibition of Rho kinase or PDE4. CONCLUSIONS AND IMPLICATIONS: PDE4 inhibitors targeted at the HARBS conformer or Epac agonists may provide promising novel targets for the treatment of SCI. Our study demonstrates the differential mechanisms of action of these compounds, as well as the benefit of a combined pharmacological approach and highlighting potential promising targets for the treatment of SCI. These findings need to be confirmed in vivo.

KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals.

The Salvador/Warts/Hippo (Hippo) signaling pathway defines a novel signaling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but functional consequences of these biochemical changes have not been previously addressed. We show that in MCF10A cells, loss of KIBRA expression displays epithelial-to-mesenchymal transition (EMT) features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the serine 127 phosphorylation site and we show that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Finally, reduced KIBRA expression in primary breast cancer specimens correlates with the recently described claudin-low subtype, an aggressive sub-group with EMT features and a poor prognosis.

Repair of central nervous system lesions by transplantation of olfactory ensheathing cells.

Clinical conditions affecting the central nervous system (CNS) fall into two main categories - degenerative conditions in which nerve cells are lost (Alzheimer's, Parkinson's, Huntington's disease, etc.), and traumatic insults which sever nerve fibers but leave their cell bodies and initial parts of the severed axons intact (spinal cord injury, cerebrovascular accidents, or tumors affecting fiber tracts). After injuries of this second type, the survival of the nerve cell bodies and the local sprouting at the severed ends of the proximal stumps of the axons raise the tantalizing possibility of one day learning how to induce these severed fibers to regenerate to their original targets and restore lost functions. This chapter gives an overview of current research into the strategy of transplantation of olfactory ensheathing cells into axotomizing injuries.

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture.

In tissue engineering, chemical and topographical cues are normally developed using static cell cultures but then applied directly to tissue cultures in three dimensions (3D) and under perfusion. As human cells are very sensitive to changes in the culture environment, it is essential to evaluate the performance of any such cues in a perfused environment before they are applied to tissue engineering. Thus, the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scaled-down bioreactor system, which allows evaluation of the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (a) quick and firm cell adhesion in the 3D scaffold was critical for cell survival in perfusion culture compared with static culture; thus, cell-seeding procedures for static cultures might not be applicable, therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures; (b) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion; (c) micro-grooves still maintained their influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture.

Implantation of pure cultured olfactory ensheathing cells in an animal model of parkinsonism.

BACKGROUND: Implantation of neural cells has been proposed as a therapeutic strategy for repairing the injured or diseased brain. In the present study we have examined the potential of olfactory ensheathing cells (OEC) to promote brain repair after surgical implantation in a rodent model of parkinsonism. METHODS: Neonatal OECs were implanted in the striatum after a 6-hydroxydopamine lesion of the ipsilateral substantia nigra. Amphetamine-induced rotational asymmetry scores were determined 48 hours before and 4, 6 and 8 weeks after OEC implantation. The density of immunostaining for tyrosine hydroxylase and synaptophysin in the striatum and the number of tyrosine hydroxylase-positive cells remaining in the substantia nigra were also determined. RESULTS: Rotational asymmetry scores were similar in OEC-implanted and vehicle-treated groups at all time points examined, and at each time were similar to those observed prior to implantation. Levels of striatal tyrosine-hydroxylase and synaptophysin immunoreactivity were similar in OEC- and vehicle-treated groups. The number of tyrosine-hydroxylase-positive cells in the substantia nigra was similar in both groups indicating that severity of the lesion was similar. Visualisation of GFP-labelled OECs one week after implantation in a separate group of animals revealed the cells to be located in the area immediately surrounding the needle tract. CONCLUSION: This study demonstrates that implantation of OECs alone is not sufficient to promote tissue repair and functional recovery in a rodent model of parkinsonism. The results add to a growing number of studies that propose a caveat for the use of pure OECs as a neurosurgical strategy for the treatment of brain disease or injury.

Influence of extracellular calcium on cell permeabilization and growth regulation by the lymphokine leukoregulin.

Permeablization of human K562 leukemia cells was measured in the presence and absence of extracellular ionic calcium to examine the relationship of ionic calcium to increased membrane permeability and the inhibition of cell proliferation by this lymphokine. In the absence of extracellular calcium, the ability of leukoregulin to permeabilize the cell membrane is diminished but is fully restored by addition of 1 mM extracellular Ca++ as shown flow cytometrically by loss of intracellular fluorescein. Membrane permeability is also increased by calcium ionophore A23187 but permeablization is completely blocked in calcium-free medium despite the intramembrane presence of the calcium ionophore. Membrane permeablization by the lectin phytohemagglutinin, in contrast, is independent of extracellular calcium. A similar divergence in cell proliferation activity of the three modulators of calcium flux and membrane permeability occurs in the absence of extracellular calcium. Leukoregulin inhibition of cell proliferation is abolished, inhibition by calcium ionophore A23817 is greatly reduced, and inhibition by phytohemagglutinin is unchanged. Leukoregulin permeabilized K562 cells isolated by fluorescence activated cell sorting resume proliferation after 72 h. In contrast cells permeablized by calcium ionophore A23187 or phytohemagglutinin fail to resume proliferation by 7 days. The membrane permeablizing action of leukoregulin is, therefore, partially dependent upon extracellular calcium. It is also effected through a mechanism other than calcium ionophore transport or lectin type transmembrane signaling, and is accompanied by a reversible inhibition of cell proliferation.