Coupled map lattice approximations for spatially explicit individual-based models of ecology

Spatially explicit individual-based models are widely used in ecology but they are often difficult to treat analytically. Despite their intractability they often exhibit clear temporal and spatial patterning. We demonstrate how a spatially explicit individual-based model of scramble competition with local dispersal can be approximated by a stochastic coupled map lattice. The approximation disentangles the deterministic and stochastic element of local interaction and dispersal. We are thus able to understand the individual-based model through a simplified set of equations. In particular, we demonstrate that demographic noise leads to increased stability in the dynamics of locally dispersing single-species populations. The coupled map lattice approximation has general application to a range of spatially explicit individual-based models. It provides a new alternative to current approximation techniques, such as the method of moments and reaction-diffusion approximation, that captures both stochastic effects and large-scale patterning arising in individual-based models.     

Trafficking of astrocytic vesicles in hippocampal slices

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.     

Characteristics of GABA-activated chlorine channels in hippocampal slices

A new preparation for patch clamp study of ion channels is reported. Transverse hippocampal slices were incubated in normal physiological solution for 8 h. When necessary they were transferred into the experimental chamber and torn immediately on the level of pyramidal layer. The neurons, visible on the torn surface, were capable of forming tight glass-membrane contacts. Preliminary measurements of GABA induced chlorine currents were performed by patch-lamp technique. There were at least two types of patches with different properties, activated by low and high GABA concentrations. The influence of diazepam on GABA sensitivity was also examined. The conclusion was made that diazepam enhanced the channel activity without affecting channel properties.     

Dynamics of epileptiform activity in mouse hippocampal slices

Increase of the extracellular K⁺ concentration mediates seizure-like synchronized activities in vitro and was proposed to be one of the main factors underlying epileptogenesis in some types of seizures in vivo. While underlying biophysical mechanisms clearly involve cell depolarization and overall increase in excitability, it remains unknown what qualitative changes of the spatio-temporal network dynamics occur after extracellular K⁺ increase. In this study, we used multi-electrode recordings from mouse hippocampal slices to explore changes of the network activity during progressive increase of the extracellular K⁺ concentration. Our analysis revealed complex spatio-temporal evolution of epileptiform activity and demonstrated a sequence of state transitions from relatively simple network bursts into complex bursting, with multiple synchronized events within each burst. We describe these transitions as qualitative changes of the state attractors, constructed from experimental data, mediated by elevation of extracellular K⁺ concentration.     

Cryopreservation of rat hippocampal slices by vitrification

Although much interest has attended the cryopreservation of immature neurons for subsequent therapeutic intracerebral transplantation, there are no reports on the cryopreservation of organized adult cerebral tissue slices of potential interest for pharmaceutical drug development. We report here the first experiments on cryopreservation of mature rat transverse hippocampal slices. Freezing at 1.2 degrees C/min to -20 degrees C or below using 10 or 30% v/v glycerol or 20% v/v dimethyl sulfoxide yielded extremely poor results. Hippocampal slices were also rapidly inactivated by simple exposure to a temperature of 0 degree C in artificial cerebrospinal fluid (aCSF). This effect was mitigated somewhat by 0.8 mM vitamin C, the use of a more "intracellular" version of aCSF having reduced sodium and calcium levels and higher potassium levels, and the presence of a 25% w/v mixture of dimethyl sulfoxide, formamide, and ethylene glycol ("V(EG) solutes"; Cryobiology 48, pp. 22-35, 2004). It was not mitigated by glycerol, aspirin, indomethacin, or mannitol addition to aCSF. When RPS-2 (Cryobiology 21, pp. 260-273, 1984) was used as a carrier solution for up to 50% w/v V(EG) solutes, 0 degree C was more protective than 10 degrees C. Raising V(EG) concentration to 53% w/v allowed slice vitrification without injury from vitrification and rewarming per se, but was much more damaging than exposure to 50% w/v V(EG). This problem was overcome by using the analogous 61% w/v VM3 vitrification solution (Cryobiology 48, pp. 157-178, 2004) containing polyvinylpyrrolidone and two extracellular "ice blockers." With VM3, it was possible to attain a tissue K(+)/Na(+) ratio after vitrification ranging from 91 to 108% of that obtained with untreated control slices. Microscopic examination showed severe damage in frozen-thawed slices, but generally good to excellent ultrastructural and histological preservation after vitrification. Our results provide the first demonstration that both the viability and the structure of mature organized, complex neural networks can be well preserved by vitrification. These results may assist neuropsychiatric drug evaluation and development and the transplantation of integrated brain regions to correct brain disease or injury.     

Membrane and network theta-rhythm generation in hippocampal slices

The hippocampal rhythms observed in vivo are the result of a complex interplay between cellular and synaptic properties within the hippocampus, and extra-hippocampal tonic as well as periodic inputs. For the stable rhythm to occur, the hippocampal circuitry should have the potential to oscillate at the specific frequencies. The in vitro studies revealed multiple mechanisms supporting the generation of the theta rhythm, which is the main operational mode of the hippocampus. In the hippocampus and related structures cellular membranes can oscillate at theta rhythm when they are depolarized to near-threshold membrane potentials; membranes are also adjusted to resonate with the external signal applied at theta frequency. Synaptically connected hippocampal network alone can generate theta rhythm when a necessary tonic excitation is provided. Finally, rhythmic inputs in theta range from the septum and entorhinal cortex have a propensity to synchronize oscillations in the whole hippocampal formation and associated structures to operate in a unified mode of activity. Based on the results obtained in slices and slice cultures, the present review shows this multilevel hierarchy, which serves to guarantee easy occurrence and reliable maintenance of the theta rhythm in the hippocampus.     

Halothane suppresses slow inward currents in hippocampal slices

Single-electrode voltage-clamp experiments were made on CA1 neurons in the presence of tetrodotoxin and K channel blockers. Applications of halothane (1-3% v/v) for 3-10 min caused a similar marked and reversible depression of slow inward currents (probably Ca currents) evoked by depolarizing pulses from a holding potential near -80 or near -40 mV. The peak amplitudes of the inward currents were much reduced, in a concentration-dependent manner, and they decayed more rapidly (half-decay time was shortened by a quarter). In most cases, leak conductances were diminished by halothane, making it unlikely that the suppression of inward currents was primarily caused by enhancement of outward currents. A similar inactivation of Ca currents in presynaptic terminals would explain why halothane depresses synaptic transmission.     

Quantitative analysis of acetylcholine release in depolarized hippocampal slices

Time course of the hippocampal slice acetylcholine content and the rate of acetylcholine release were studied during high K⁺-induced depolarization for 4 to 60 min. At the end of the potassium exposure, both the acetylcholine remaining in the tissue and appearing in the incubation medium were quantitatively determined by gas chromatography using a nitrogen-sensitive detector. During prolonged K⁺ incubation, the acetylcholine content of the slices decreased by 60%, reaching a steady state after 16 min. The increase in the acetycholine concentration of the depolarizing medium showed a biphasic pattern, with rate constants of 1.40 and 0.69 nmol/min/g in the early (0–16 min) and late (16–60 min) phase, respectively. K⁺-evoked acetylcholine release was Cal⁺-dependent, but addition of choline did not alter tissue levels of acetylcholine or the pattern of K⁺-evoked acetylcholine release. The rate of acetylcholine release was markedly decreased by inhibition of choline uptake with hemicholinium-3 or by addition of 4-(1-naphthylvinyl)pyridine which inhibits both ACh producing enzyme, choline acetyltransferase and choline uptake mechanism. These data confirm the essential role during depolarization of extracellular choline transport into the cholinergic terminals utilizing choline released by the slices during the incubation. It is concluded that drugs which can influence the processes of choline uptake and acetylcholine sythesis can alter the rate of acetylcholine release measured under similar conditions.     

Ghrelin inhibited serotonin release from hippocampal slices

Ghrelin (Ghr) is a peptide produced peripherally and centrally. It participates in the modulation of different biological processes. In our laboratory we have shown that (a) Ghr administration, either intracerebroventricular or directly into the hippocampus enhanced memory consolidation in a step down test in rats (b) the effect of Ghr upon memory decreases in animals pretreated with a serotonin (5-HT) reuptake inhibitor, Fluoxetine, suggesting that Ghr effects in the hippocampus could be related to the availability of 5-HT. It has been demonstrated that Ghr inhibits 5-HT release from rat hypothalamic synaptosomes. Taking in mint these evidences, we studied the release of radioactive 5-HT to the superfusion medium from hippocampal slices treated with two doses of Ghr (0.3 and 3 nm/μl). Ghr inhibited significantly the 5-HT release in relation to those superfused with artificial cerebrospinal fluid (ACSF) (H = 9.48, df = 2, p ≤ 0.05). In another set of experiments, Ghr was infused into the CA1 area of hippocampus of the rats immediately after training in the step down test and the 5-HT release from slices was studied 24 h after Ghr injection showing that in this condition also the 5-HT release was inhibited (H = 11.72, df = 1, p ≤ 0.05). In conclusion, results provide additional evidence about the neurobiological bases of Ghr action in hippocampus.     

Patch-clamp recording from mossy fiber terminals in hippocampal slices

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size. Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2-5 microm. The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting. Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice. Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.     

Caspase-3 inhibition blocks long-term potentiation in hippocampal slices

Incubation of hippocampal slices with Z-DEVD-FMK, a specific inhibitor of caspase-3, elicits a time dependent decrease in long-term potentiation (LTP). After 4 hours or later after the incubation with Z-DEVD-FMK the tetanization fails to induce LTP. However, Z-DEVD-FMK does not affect basal indices of synaptic plasticity and short-term plasticity (population spike amplitudes and paired pulse facilitation). The results are the first evidence for the involvement of caspase-3-mediated mechanisms in long-term potentiation phenomenon.     

Cytochalasin B inhibits phosphoinositide hydrolysis in rat hippocampal slices

The effect of cytochalasin B on phosphoinositide (PI) hydrolysis was examined in rat hippocampal slices. Pretreatment of the slices with cytochalasin B caused a significant decrease in PI hydrolysis elicited by carbachol, norepinephrine, or by high K⁺. This effect was cytochalasin B dose- and time-dependent and was not mimicked by cytochalasin D, vinblastine, colchicine, or phloretin. In contrast, in [³H]inositol-prelabeled hippocampal membranes, cytochalasin B did not affect PI hydrolysis elicited by GTPS and GTPS plus carbachol. Similar result was obtained using the membranes prepared from the slices pretreated with cytochalasin B. The inhibitory effect of cytochalasin B on the carbachol-response was observed in SK-N-SH human neuroblastoma cells, but not in cultured rat astrocytes. These results indicate that cytochalasin B inhibits PI hydrolysis in neuron-specific manner and that its action may be an indirect cellular mechanism other than interaction with cytoskeleton elements.     

A profile of the insulin receptor in cultured hippocampal slices

OBJECTIVES: Insulin receptors (IRs) are distributed in a region-specific fashion throughout the brain, and may play a role in processes related to learning and memory. The hippocampus, which participates in spatial memory formation, is one region in which the IR is abundantly expressed. Organotypic hippocampal slice cultures (OHSCs) are an in vitro model that permits the easy manipulation of growth conditions, yet retains much of the source structure's cytoarchitecture. To assess OHSCs as a model for the study of hippocampal IRs, ligand-binding and the expression and cellular distribution of the β-subunit (which transduces the insulin signal) were examined over time in culture. Design & Results: Fluorescently conjugated insulin was used to assess neural insulin receptor binding, and revealed that labelling remained similar over three weeks in culture (a typical length of OHSC maintenance). Cross-linking of surface proteins helped to show that approximately half of β-subunits were found at the cell surface, and that this relative proportion remained stable over several weeks. In contrast, expression of the β-subunit protein progressively declined to a plateau approximately 60% less than that seen when the cultures were prepared. Conclusions: Our results provide a foundation for subsequent studies to employ OHSCs to explore neural IRs; for instance, the dissonance between the progressive decline in expression of the IR β-subunit and the relative stability of receptor-mediated binding suggests the presence of an active process to hold steady the ability of cells to respond to insulin stimulation.     

Structural plasticity of synapses in hippocampal slices after oxygen-glucose deprivation

We analyzed structural rearrangements of synaptic contacts in the stratum radiatum of the CA1 area of cultured rat hippocampal slices under conditions of the development of potentiation of synaptic transmission induced by short-term (10 min) oxygen-glucose deprivation (OGD). Studies were carried out using electron microscopy and 3D reconstruction of cellular compartments. Within the 1st h after OGD, we observed increases in the volume of pre-synaptic terminals and post-synaptic spines and also in the area of postsynaptic densities (PSDs) in both asymmetric excitatory and symmetric inhibitory synapses, especially in the case were the PSD was perforated. We also observed significant activation of glial cells (increases in their volume and area of contacts of their processes with the components of synapses). Therefore, OGD results in activationassociated structural rearrangements of both excitatory and inhibitory synapses of the hippocampal CA1 area. Such rearrangements are accompanied by a clearly pronounced reaction of the glia, which correlates with an important role of the latter in modulation of the functioning of neurons.     

Anoxia reversibly suppresses neuronal calcium currents in rat hippocampal slices

Intracellular recording from CA1 neurons confirmed that short periods of anoxia (95% N2 + 5% CO2 for 2-4 min) have a hyperpolarizing action, caused by a rise in K conductance. After blockage of K channels with extracellular Cs+ and tetraethylammonium (or intracellular Cs+), large inward currents of Ca were evoked by depolarizing pulses: transient currents at a holding potential near -70 mV, and more sustained ones near -50 mV. Both types of Ca current were much reduced or fully suppressed after 1-3 min of anoxia, but they largely (or fully) recovered within 1-10 min of starting reoxygenation.     

Spontaneous synchronous discharges in hippocampal slices. Simulation and experiment

Chaotic oscillations of extracellular potential of field-type nerve tissues are simulated by a 2D coupled map lattice. These tissues, say, the fields of the hippocampus, are represented by neural mass sheets consisting of current sources. The relationship between the source-sink ensembles and the extracellular field potential at each discrete instant of time t = 1, 2, ... is described by a single-site map creating chaos. The 2D coupled map lattice is viewed as a network of diffusively coupled the maps creating spatiotemporal chaos. The conversion of chaotic oscillations into synchronous ones, which are typical for epileptiform discharges, is studied. The results obtained are in good agreement with those derived from hippocampal slices treated with picrotoxin.     

Mossy fiber pathfinding in multilayer organotypic cultures of rat hippocampal slices

1. Using a novel technique of organotypic cultures, in which two hippocampal slices were cocultured in a bilayer style, we found that the mossy fibers arising from the dentate gyrus grafted onto another dentate tissue grew along the CA3 stratum lucidum of the host hippocampal slice. The same transplantation of a CA1 microslice failed to form a network with the host hippocampus.2. Thus, the type of grafted neurons is important to determine whether they can form an appropriate network after transplantation.     

GABA对大鼠海马脑片缺氧损伤的保护作用

目的：研究GABA对大鼠海马脑片急性缺氧损伤的保护机制。方法：采用成年大鼠离体海马脑片,用胞外记录的电生理技术,观察GABA对急性缺氧后海马脑片诱发电位的影响。结果：（1）GABA可明显延迟PV的消失,但对PS却无影响;（2）给予GABAA受体拮抗剂荷包牡丹碱（bicuculine)以及Cl^-通道阻抗剂NPPB可阻断GABA的保护作用。结论：GAB可提高海马脑片耐缺氧能力,其机制可能与GABA通过GABAA受体提高Cl^-内流有关。     

Pharmacological induction of ischemic tolerance in hippocampal slices by sarcosine preconditioning

Brain ischemic tolerance is a protective mechanism induced by a preconditioning stimulus, which prepare the tissue against harmful insults. Preconditioning with N-methyl-d-aspartate (NMDA) agonists induces brain tolerance and protects it against glutamate excitotoxicity. Recently, the glycine transporters type 1 (GlyT-1) have been shown to potentiate glutamate neurotransmission through NMDA receptors suggesting an alternative strategy to protect against glutamate excitotoxicity. Here, we evaluated the preconditioning effect of sarcosine pre-treatment, a GlyT-1 inhibitor, in rat hippocampal slices exposed to ischemic insult. Sarcosine (300mg/kg per day, i.p.) was administered during seven consecutive days before induction of ischemia in hippocampus by oxygen/glucose deprivation (OGD). To access the damage caused by an ischemic insult, we evaluated cells viability, glutamate release, nitric oxide (NO) production, lactate dehydrogenase (LDH) levels, production of reactive oxygen species (ROS), and antioxidant enzymes as well as the impact of oxidative stress in the tissue. We observed that sarcosine reduced cell death in hippocampus submitted to OGD, which was confirmed by reduction on LDH levels in the supernatant. Cell death, glutamate release, LDH levels and NO production were reduced in sarcosine hippocampal slices submitted to OGD when compared to OGD controls (without sarcosine). ROS production was reduced in sarcosine hippocampal slices exposed to OGD, although no changes were found in antioxidant enzymes activities. This study demonstrates that preconditioning with sarcosine induces ischemic tolerance in rat hippocampal slices submitted to OGD.