Properties of the Nucleo-Olivary Pathway: An In Vivo Whole-Cell Patch Clamp Study

The inferior olivary nucleus (IO) forms the gateway to the cerebellar cortex and receives feedback information from the cerebellar nuclei (CN), thereby occupying a central position in the olivo-cerebellar loop. Here, we investigated the feedback input from the CN to the IO in vivo in mice using the whole-cell patch-clamp technique. This approach allows us to study how the CN-feedback input is integrated with the activity of olivary neurons, while the olivo-cerebellar system and its connections are intact. Our results show how IO neurons respond to CN stimulation sequentially with: i) a short depolarization (EPSP), ii) a hyperpolarization (IPSP) and iii) a rebound depolarization. The latter two phenomena can also be evoked without the EPSPs. The IPSP is sensitive to a GABA_A receptor blocker. The IPSP suppresses suprathreshold and subthreshold activity and is generated mainly by activation of the GABA_A receptors. The rebound depolarization re-initiates and temporarily phase locks the subthreshold oscillations. Lack of electrotonical coupling does not affect the IPSP of individual olivary neurons, nor the sensitivity of its GABA_A receptors to blockers. The GABAergic feedback input from the CN does not only temporarily block the transmission of signals through the IO, it also isolates neurons from the network by shunting the junction current and re-initiates the temporal pattern after a fixed time point. These data suggest that the IO not only functions as a cerebellar controlled gating device, but also operates as a pattern generator for controlling motor timing and/or learning.     

Spontaneous Activity and Responses to Sensory Stimulation in Ventrobasal Thalamic Neurons in the Rat: An In Vivo Intracellular Recording and Staining Study

Spontaneous activity and responses to sensory stimulation in ventrobasal (VB) thalamic neurons were studied in barbiturate-anesthetized rats through intracellular recordings. The recordings were carried out with micropipettes filled with K acetate, KCl plus horseradish peroxidase (HRP), our KCl plus biocytin. Two types of spontaneous depolarizing events were observed: fast potentials (FPs), characterized by a low amplitude (5.3 ± 1.8 mV [mean and standard deviation]), a fast rising slope (1.15 ± 0.19 msec), and a short duration (8.47 ± 0.89 msec); and slow potentials (SPs), characterized by a larger and more variable amplitude (9.1 ± 5.6 mV) and a longer duration (62.5 ± 27.2 msec), with a slower rising slope (26.2 ± 6.4 msec). The potential changes elicited by sensory stimuli delivered manually were similar to those elicited by electronically gated short air jets to the receptive fields. FPs were evoked by sensory stimulation in 62.7% of the recorded neurons, and SPs in the remaining 37.3%. Both types of events could occur spontaneously in the same neuron, but only one of them was triggered by stimulation of the receptive field. Five neurons that were successfully stained with either HRP or biocytin were studied in detail. AH were medium-sized stellate cells, with spine-like appendages sparsely distributed along slender radiating dendrites. The axons took a rostrolateral course across the VB, and all but one left one or two thin collaterals in the reticular thalamic nucleus. No overt morphological differences were observed between VB neurons that responded with FPS or SPs to sensory stimulation.     

Correlative In Vivo 2 Photon and Focused Ion Beam Scanning Electron Microscopy of Cortical Neurons

Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information. Here we describe a semi-automated technique whereby volumes of brain tissue containing axons and dendrites, previously studied in vivo, are subsequently imaged in three dimensions with focused ion beam scanning electron microcopy. These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms. The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.     

Reorganization of Receptive Fields of Cortical Field 21a Neurons and Formation of Responses to Moving Visual Stimuli

Using extracellular recording of spike activity from single neurons of field 21a of the cat neocortex, we examined in detail the spatial organization of receptive fields (RFs) of such cells after conditions of presentation of an immobile blinking light spot (a static RF) and moving visual stimuli (dynamic RFs). As was shown, the excitability of different RF subfields of a group of neurons possessing homogeneous on–off organization of the static RF changes significantly depended on the contrast, shape, dimension, orientation, and direction of movement of the applied mobile visual stimulus. This is manifested in changes in the number of discharge centers and shifts of their spatial localization. A hypothesis on the possible role of synchronous activation of the neurons neighboring the cell under study in the formation of an additional neuronal mechanism providing specialization of neuronal responses is proposed.     

PINP: A New Method of Tagging Neuronal Populations for Identification during In Vivo Electrophysiological Recording

Neural circuits are exquisitely organized, consisting of many different neuronal subpopulations. However, it is difficult to assess the functional roles of these subpopulations using conventional extracellular recording techniques because these techniques do not easily distinguish spikes from different neuronal populations. To overcome this limitation, we have developed PINP (Photostimulation-assisted Identification of Neuronal Populations), a method of tagging neuronal populations for identification during in vivo electrophysiological recording. The method is based on expressing the light-activated channel channelrhodopsin-2 (ChR2) to restricted neuronal subpopulations. ChR2-tagged neurons can be detected electrophysiologically in vivo since illumination of these neurons with a brief flash of blue light triggers a short latency reliable action potential. We demonstrate the feasibility of this technique by expressing ChR2 in distinct populations of cortical neurons using two different strategies. First, we labeled a subpopulation of cortical neurons—mainly fast-spiking interneurons—by using adeno-associated virus (AAV) to deliver ChR2 in a transgenic mouse line in which the expression of Cre recombinase was driven by the parvalbumin promoter. Second, we labeled subpopulations of excitatory neurons in the rat auditory cortex with ChR2 based on projection target by using herpes simplex virus 1 (HSV1), which is efficiently taken up by axons and transported retrogradely; we find that this latter population responds to acoustic stimulation differently from unlabeled neurons. Tagging neurons is a novel application of ChR2, used in this case to monitor activity instead of manipulating it. PINP can be readily extended to other populations of genetically identifiable neurons, and will provide a useful method for probing the functional role of different neuronal populations in vivo.     

The Involvement of Autophagy in the Response of Neurons and Glial Cells to Photodynamic Treatment

Photodynamic therapy is used for selective destruction of pathological tissue in oncology; this method is also employed for ablation of brain tumors. Cell death upon photodynamic treatment results from oxidative stress that develops in photosensitized cells upon exposure to light in the presence of oxygen. We studied the development of autophagy during photo-induced oxidative stress in a crayfish stretch receptor that consists of two sensory neurons in a glial envelope by measuring the number and distribution of lysosomes using a LysoTracker Red fluorescent probe. Photodynamic treatment with radachlorin (500 nM) for 15 min decreased the number of lysosomes, probably due to their photodestruction. However, 1 h after the photodynamic treatment with radachlorin (250 nM) the LysoTracker Red fluorescence increased with larger granules occurring, which may be indicative of autophagic processes in the cell. Photodynamic treatment is capable of inducing necrosis of neurons and glial cells and glial apoptosis in a crayfish stretch receptor. Modulation of autophagy by application of specific inhibitors and activators of AMPK (AICAR, dorsomorphin) and mTOR (rapamycin, KU0063794) revealed that autophagy could protect glia against photo-induced cell death.     

Susceptibility of Hippocampal and Cortical Neurons to Argon-Mediated In Vitro Ischemia

Abstract: Neurons from cerebral cortex and hippocampal CA1 sector exhibit a striking difference in vulnerability to transient ischemia. To establish whether this difference is due to the inherent (pathoclitic) properties of these neurons, the ischemic susceptibility was studied in primary cortical and hippocampal cultures by using a new model of argon-induced in vitro ischemia. Neuronal cultures were exposed at 37°C for 10–30 min to argon-equilibrated glucose-free medium. During argon equilibration, P o ₂ declined to <2.5 torr within 1 min and stabilized shortly later at ∼1.3 torr. After 30 min of in vitro ischemia, total adenylate was <45% and ATP content <15% of control in both types of culture. Cytosolic calcium activity increased from 15 to 50 n M . Reoxygenation of cultures after in vitro ischemia led to delayed neuronal death, the severity of which depended on the duration of in vitro ischemia but not on the type of neuronal cultures. Energy charge of adenylate transiently returned to ∼90% of control after 3 h, but ATP content recovered only to 40% and protein synthesis to <35%. Cytosolic calcium activity continued to rise after ischemia and reached values of ∼500 n M after 3 h. The new argon-induced in vitro ischemia model offers major advantages over previous methods, but despite this improvement it was not possible to replicate the differences in cortical and hippocampal vulnerability observed in vivo. Our study does not support the hypothesis that selective vulnerability is due to an inherent pathoclitic hypersensitivity.     

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional,Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.     

Learning-Induced Expression of Meningeal Ependymin mRNA and Demonstration of Ependymin in Neurons and Glial Cells

Abstract: The turnover of a CNS-specific cell adhesion glycoprotein, ependymin, has earlier been found to increase during periods of neuronal plasticity. Here, ependymin mRNA expression was analyzed by semiquantitative in situ hybridization in goldfish. Learning of an active avoidance response resulted in a significant increase in ependymin mRNA expression 20 min to 4 h after acquisition of the task. In contrast, yoked control animals that were exposed to the same numbers of conditioned and unconditioned stimuli in a random, unpaired manner exhibited a strong down-regulation of ependymin mRNA. Hybridization signals were also increased by injection of anti-ependymin antiserum into brain ventricles. Ependymin mRNA was exclusively localized to reticular-shaped fibroblasts of the inner endomeningeal cell layer. Immunoelectron microscopic investigation, however, revealed ependymin also in distinct neuronal and glial cell populations in which no ependymin mRNA had been detected. Uptake of meningeal protein factors into glial and neuronal cells may therefore be of functional importance for plastic adaptations of the CNS.     

Mesenchymal Stromal Cells Rescue Cortical Neurons from Apoptotic Cell Death in an In Vitro Model of Cerebral Ischemia

Cell therapy with mesenchymal stromal cells (MSCs) was found to protect neurons from damage after experimental stroke and is currently under investigation in clinical stroke trials. In order to elucidate the mechanisms of MSC-induced neuroprotection, we used the in vitro oxygen–glucose deprivation (OGD) model of cerebral ischemia. Co-culture of primary cortical neurons with MSCs in a transwell co-culture system for 48 h prior to OGD-reduced neuronal cell death by 30–35%. Similar protection from apoptosis was observed with MSC-conditioned media when added 48 h or 30 min prior to OGD, or even after OGD. Western blot analysis revealed increased phosphorylation of STAT3 and Akt in neuronal cultures after treatment with MSC-conditioned media. Inhibition of the PI3K/Akt pathway completely abolished the neuroprotective potential of MSC-conditioned media, suggesting that MSCs can improve neuronal survival by an Akt-dependent anti-apoptotic signaling cascade. Using mass spectrometry, we identified plasminogen activator inhibitor-1 as an active compound in MSC-conditioned media. Thus, paracrine factors secreted by MSCs protect neurons from apoptotic cell death in the OGD model of cerebral ischemia.     

Postnatal Development of Neurons,Interneurons and Glial Cells in the Substantia Nigra of Mice

We investigated postnatal alterations of neurons, interneurons and glial cells in the mouse substantia nigra using immunohistochemistry. Tyrosine hydroxylase (TH), neuronal nuclei (NeuN), parvalbumin (PV), neuronal nitric oxide synthase (nNOS), glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba 1), CNPase (2′,3′-cyclic nucleotide 3′-phosphodiesterase), brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF) immunoreactivity were measured in 1-, 2-, 4- and 8-week-old mice. In the present study, the maturation of NeuN-immunopositive neurons preceded the production of TH in the substantia nigra during postnatal development in mice. Furthermore, the maturation of nNOS-immunopositive interneurons preceded the maturation of PV-immunopositive interneurons in the substantia nigra during postnatal development. Among astrocytes, microglia and oligodendrocytes, in contrast, the development process of oligodendrocytes is delayed in the substantia nigra. Our double-labeled immunohistochemical study suggests that the neurotrophic factors such as BDNF and GDNF secreted by GFAP-positive astrocytes may play some role in maturation of neurons, interneurons and glial cells of the substantia nigra during postnatal development in mice. Thus, our findings provide valuable information on the development processes of the substantia nigra.     

In Vivo Optogenetic Control of Striatal and Thalamic Neurons in Non-Human Primates

Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 10⁹ particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.     

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 µm) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.     

Assessment of In Vivo and In Vitro Genotoxicity of Glibenclamide in Eukaryotic Cells

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.     

Intracellular Localization of the HCS2 Gene Products in Identified Snail Neurons In Vivo and In Vitro

SUMMARY 1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca²⁺ concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy.2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system.3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons.4. The immunogold particles were practically absent in the bodies of cultured neurons.5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.     

An In Vivo Model for Studying Function of Brain Tissue Temporarily Devoid of Glial Cell Metabolism: The Use of Fluorocitrate

The effect of intrastriatal injection of fluorocitrate on amino acid pattern, cell enzyme markers, and ultrastructural appearance was investigated. A dose of 1 nmol of fluorocitrate resulted in temporarily decreased levels of glutamine, glutamate, and aspartate, whereas the level of alanine was increased. The glutamine level was severely reduced after 4 h but was reversed after 24 h. The activity of different cellular enzyme markers did not change markedly after this dose. Ultrastructural changes in glial cells were observed, concomitant with the biochemical changes. A dose of greater than or equal to 2 nmol of fluorocitrate resulted in more marked and irreversible changes in amino acid levels. By 24-72 h after the injection of this dose, several marker enzyme activities decreased markedly. The ultrastructural changes affected the neurons as well as the glial cells and were not reversible. The use of microinjection of 1 nmol of fluorocitrate into the neostriatum of the rat to provide a model for studying transmitter amino acid metabolism in brain devoid of glial cell activity is discussed.     

The HIV-1 Tat Protein Induces the Activation of CD8+ T Cells and Affects In Vivo the Magnitude and Kinetics of Antiviral Responses

T cells are functionally compromised during HIV infection despite their increased activation and proliferation. Although T cell hyperactivation is one of the best predictive markers for disease progression, its causes are poorly understood. Anti-tat natural immunity as well as anti-tat antibodies induced by Tat immunization protect from progression to AIDS and reverse signs of immune activation in HIV-infected patients suggesting a role of Tat in T cell dysfunctionality. The Tat protein of HIV-1 is known to induce, in vitro, the activation of CD4⁺ T lymphocytes, but its role on CD8⁺ T cells and how these effects modulate, in vivo, the immune response to pathogens are not known. To characterize the role of Tat in T cell hyperactivation and dysfunction, we examined the effect of Tat on CD8⁺ T cell responses and antiviral immunity in different ex vivo and in vivo models of antigenic stimulation, including HSV infection. We demonstrate for the first time that the presence of Tat during priming of CD8⁺ T cells favors the activation of antigen-specific CTLs. Effector CD8⁺ T cells generated in the presence of Tat undergo an enhanced and prolonged expansion that turns to a partial dysfunctionality at the peak of the response, and worsens HSV acute infection. Moreover, Tat favors the development of effector memory CD8⁺ T cells and a transient loss of B cells, two hallmarks of the chronic immune activation observed in HIV-infected patients. Our data provide evidence that Tat affects CD8⁺ T cell responses to co-pathogens and suggest that Tat may contribute to the CD8⁺ T cell hyperactivation observed in HIV-infected individuals.