Lamina-specific synaptic connections of hippocampal neurons in vitro

By using slice cultures as a model, we demonstrate here that different target selectivities exist among the various afferent fibers to the hippocampus. As in intact animals, septohippocampal cholinergic fibers, provided by a slice culture of septum, innervate a co-cultured slice of hippocampus diffusely, that is, without forming distinct layers of termination. As in vivo, the septal cholinergic fibers establish synapses with a variety of target cells. Conversely, fibers from an entorhinal slice co-cultured to a hippocampal slice display their normal laminar specificity. They preferentially terminate in the outer molecular layer of the fascia dentata, thereby selectively contacting peripheral dendrites of the granule cells. This preferential termination on peripheral dendritic segments is remarkable, since these fibers do not have to compete with commissural fibers, hypothalamic fibers, and septal afferents for dendritic space under these culture conditions. Moreover, in triplet cultures in which first two hippocampal slices were co-cultured and then, with a delay of 5 days, an entorhinal slice was added, the fibers from the entorhinal slice and those from the hippocampal culture terminated in their appropriate layers in the hippocampal target culture. However, in this approach the normal sequence of ingrowth of these two afferents was reversed. In normal ontogenetic development, entorhinal afferents arrive in the hippocampus before the commissural fibers. The results show that there are different degrees of target selectivity of hippocampal afferents and that the characteristic lamination of certain afferent fibers in the hippocampus is not determined by their sequential ingrowth during development. © 1995 John Wiley & Sons, Inc.     

Elevated serum enzyme activity: an explanation-based model.

The well-documented pattern of elevated serum enzyme activity (ESEA) data after a single bout of unaccustomed exercise can very easily be modeled using a biexponential curve. However, the changed pattern of ESEA after a second exercise bout, or after a period of conditioning or during repetitive training, demonstrates that exercise-induced adaptations have been taking place. The mechanism for this is unclear. One plausible explanatory hypothesis is that within the pool of muscle fibers, some fibers are stress susceptible or weak, and the pool becomes diminished as a result of damage induced by earlier exercise. Repair of the muscle damage takes place during the period after exercise but may be incomplete at the time of a subsequent exercise bout, in which case ESEA amplitude is reduced. Frequently repeated bouts may lead to chronic ESEA. These ideas are developed, both mathematically and graphically, by means of a compartment model approach. In so doing, the model explains documented patterns of ESEA response to single and multiple exercise bouts, both closely and widely spaced. Predictions are possible using the model, in particular the previously unreported baseline overshoot (reduction below resting levels) in serum enzyme activity occurring beyond 82 h after very severe exercise. Some directions for experimentation to test the validity of the model are suggested.     

Stem cell transplantation in an in vitro simulated ischemia/reperfusion model

Stem cell transplantation protocols are finding their way into clinical practice. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.     

Pronase modifies synaptic transmission and activity of identifiedLymnaea neurons

Proteolytic enzymes can have significant effects on the physiological properties of neurons. Although several actions of proteolytic enzymes on the physiology of single neurons have been described, the effects of these enzymes on network properties in the central nervous system (CNS) have received less attention. The effects of bath-applied pronase (0.05%) on synaptic connections and spontaneous activity in theLymnaea CNS were examined. Brief application (i.e. 2–3 min) of pronase modified some, but not all, synapses in the CNS. For example, the chemical synapse between two interneurons, RPeD11 and RPeD1, and between the interneuron, RPeD1, and RPA motoneurons were examined. Both these synapses were either biphasic or monophasic (depolarizing) under control conditions. Pronase exposure eliminated the depolarizing phase of the RPeD11→RPeD1 synapse, but had no effect on the connection between RPeD1 and RPA neurons. In addition, the effects of pronase on electrical-coupling between two peptidergic neurons, VD1 and RPD2, in the CNS were investigated. Pronase decreased the total network input resistance and cell input resistances as well as the steady-state coupling ratio. Furthermore, exposure to pronase induced various changes (i.e. depolarization, hyperpolarization, bursting patterns and afterdischarges) in the activity pattern of different identified neurons in the CNS. Collectively, these data show that even brief exposure to a low concentration of pronase can acutely modify both synapses and neuronal activity.     

Creatine protects against excitoxicity in an in vitro model of neurodegeneration

Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H(2)O(2). In good agreement with our in vivo data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H(2)O(2)-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.     

Adenosine preconditions against endothelin-induced constriction of coronary arterioles

Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng. kg(-1). min(-1) administered intracoronary, n = 10) induced progressive constriction over 45 min [25 +/- 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 microgram. kg(-1). min(-1) administered intracoronary) for 20 min and 10 min of washout (n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 +/- 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50-150 micrometer) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM-5 nM) was rightward shifted after preconditioning with adenosine (1 microM) for 20 min and 10 min of washout (n = 11). Blockade of A(2) receptors [8-(3-chlorostyryl)caffeine, 1 microM, n = 9] but not A(1) receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A(2) receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.     

Impulse activity of the nodose ganglion neurons in myocardial ischemia

Standard microelectrode techniques were used to study the impulse activity of different types of nodosal ganglion neurons in the course of the development of myocardial ischemia. The cardiopulmonary late inspiratory and inspiratory-expiratory neuronal responses were estimated upon ligation of the coronary artery during the first respiratory cycle after blood flow stoppage. Spontaneous activity of cardiopulmonary neurons was not dependent on coronary circulation disturbances at the moment of coronary artery ligation. Later on, however, during the development of myocardial ischemia, there occurred substantial changes in all the types of nodosal ganglion neuronal activity, excluding real inspiratory neurons.     

Characterization of synaptic dysfunction in an in vitro corticostriatal model system of Huntington’s disease

Huntington’s disease (HD) is an autosomal-dominant inherited neurodegenerative disease resulting from expanded amino acid (CAG) repeat in the gene that encodes protein huntingtin (Htt). HD remains incurable for now. A lot of evidence implicates aberrant synaptic connection between cortical and striatal neurons, a key component of HD pathophysilogy, which also leads to cognitive decline and motor disorders. In the present work synaptic activity between cortical and striatal neurons was studied on the corticostriatal co-culture model system of HD. Culture was prepared from HD mouse model YAC128. It was shown that first impairment appears on day 14 in vitro. Interestingly, these alterations occur in cortical neurons. Their activity in YAC128 cultures was higher than in cultures of wild-type neurons. At the same time, there were no differences in morphology of spines in striatal neurons. However, using novel optogenetic approach, we demonstrated that synaptic connections are already dysfunctional in YAC128 cultures. On day 19 in vitro the activity of cortical neurons in YAC128 cultures was reduced, which led to alterations on the post-synaptic side. Dendric spines of medium spiny neurons transformed and disappeared, which is possibly the main reason of neurodegenerative mechanisms during the HD development.     

Impulse activity of bulbar respiratory neurons in myocardial ischemia in cats

Acute experiments on cats anesthetized with nembutal, using a microelectrode technique, have demonstrated that pinching of the coronary artery involves changes in the patterns of impulse activity in all types of bulbar respiratory neurons, up to the appearance of ischemic shifts on the ECG. Progression of myocardial ischemia is paralleled by changes in all the bulbar respiratory neurons.     

Neuroprotective effect of GMP in hippocampal slices submitted to an in vitro model of ischemia

1. Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage observed in rat hippocampal slices submitted to an in vitro model of ischemia with or without the presence of the ionotropic glutamate receptor agonist, Kainic acid (KA).2. Cellular injury was evaluated by MTT reduction, lactate dehydrogenase (LDH) release assay, and measurement of intracellular ATP levels.3. In slices submitted to ischemic conditions, 1 mM GMP partially prevented the decrease in cell viability induced by glucose and oxygen deprivation and the addition of KA.4. KA or N-methyl-D-aspartate (NMDA) receptor antagonists, -D-glutamylamino-methylsulfonate (GAMS) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, 20 M) also prevented toxicity in hippocampal slices under ischemic conditions, respectively.5. The association of GMP with GAMS or MK-801 did not induce additional protection than that observed with GMP or that classical glutamate receptor antagonists alone.6. GMP, probably by interacting with ionotropic glutamate receptors, attenuated the damage caused by glucose and oxygen deprivation in hippocampal slices. This neuroprotective action of GMP in this model of excitotoxicity is of outstanding interest in the search for effective therapies against ischemic injury.     

Characterization of an in vitro model of alphavirus infection of immature and mature neurons

Terminally differentiated, mature neurons are essential cells that are not easily regenerated. Neurotropic viruses, such as Sindbis virus (SV), cause encephalomyelitis through their ability to replicate in neurons. SV causes the death of immature neurons, while mature neurons can often survive infection. The lack of a reproducible and convenient neuronal cell culture system has hindered a detailed study of the differences in levels of virus replication between immature and mature neurons and the molecular events involved in virus clearance from mature neurons. We have characterized SV replication in immortalized CSM14.1 rat neuronal cells that can be differentiated into neurons. During differentiation, CSM14.1 cells ceased dividing, developed neuronal morphology, and expressed neuron-specific cell markers. SV infection of undifferentiated CSM14.1 cells was efficient and resulted in high levels of virus replication and cell death. SV infection of differentiated CSM14.1 cells was less efficient and resulted in the production of 10- to 100-fold less virus and cell survival. In undifferentiated cells, SV induced a rapid shutdown of cellular protein synthesis and pE2 was efficiently processed to E2 (ratio of E2 to pE2, 2.14). In differentiated cells, the SV-induced shutdown of cellular protein synthesis was transient and pE2 was the primary form of E2 in cells (ratio of E2 to pE2, 0.0426). We conclude that age-dependent restriction of virus replication is an intrinsic property of maturing neurons and that the CSM14.1 cell line is a convenient model system for investigating the interactions of alphaviruses with neurons at various stages of differentiation.     

In vitro and in vivo activity of an antibacterial peptide analog against uropathogens

The alarming rate of bacterial resistance induction highlights the clinical need for antimicrobial agents that act by novel modes of action. Based on the activity profile, the general tissue distribution and renal clearance of peptide-based drugs, we hypothesized that our newly developed pyrrhocoricin derivative would be able to fight resistant uropathogens in vitro and in vivo. Indeed, the Pip-pyrr-MeArg dimer killed all 11 urinary tract infection-related Escherichia coli and Klebsiella pneumoniae strains we studied in the sub-low micromolar concentration range. Almost all control antibiotics, including the currently leading trimethoprim-sulfametoxazole combination for urinary tract infection, remained without considerable activity against two or more of these bacterial strains. In a mouse ascending urinary tract infection model with E. coli CFT073 as pathogen, two doses of intravenous, subcutaneous or oral treatment with the Pip-pyrr-MeArg derivative reduced the bacterial counts in the kidneys, bladder and urine to varying levels. Statistically significant elimination or reduction of bacteria compared to untreated animals was observed at dual intravenous or subcutaneous doses of 0.4 or 10mg/kg, respectively. Serial passage of the same E. coli strain in the presence of sublethal doses of the designed peptide failed to generate resistant mutants. The Pip-pyrr-MeArg dimer showed no toxicity to COS-7 cells to the highest 500microM concentration studied.     

Temperature dependence of synaptic responses in guinea pig hippocampal CA1 neurons in vitro

1. Temperature-dependent properties of synaptic transmission were studied by recording orthodromic responses of the population spike and excitatory postsynaptic potential in CA1 pyramidal neurons of guinea pig hippocampal slices.2. Increasing the temperature of the perfusing medium from 30 to 43°C resulted in a decrease in the amplitude of the population spike (A-PS) and a reduced slope of the excitatory postsynaptic potential (S-EPSP). Bath application of the -aminobutyric acid receptor antagonist, picrotoxin, or a change in the calcium concentration of the perfusate did not affect the A-PS during heating.3. Increasing the strength of the synaptic input to that eliciting a PS with an amplitude 50, 75, or 100% of maximal at 30°C resulted in a significant increase in the A-PS during the middle phase of hyperthermia (35–39°C).4. The long-term potentiation (LTP) induced at either 30 or 37°C showed the same percentage increase in both the amplitude of the population spike and the S-EPSP after delivery of a tetanus (100 Hz, 100 pulses) to CA1 synapses.5. The results of the present study, therefore, indicate that the decrease in CA1 field potential was linearly related to the temperature of the slice preparation, while LTP was induced in these responses during heating from 30 to 37°C.     

Down-expressed GLT-1 in PSD astrocytes inhibits synaptic formation of NSC-derived neurons in vitro

Little is known about the effect of astroglial GLT-1 of post-stroke depression (PSD) rat model on the function of neural stem cells (NSCs). This study aimed to investigate whether astroglial GLT-1 of PSD rats affect differentiation of NSCs from neonatal rat hippocampus and synaptic formation of NSC-derived neurons. Astrocytes were isolated from the left hippocampus of normal adult SD rats and PSD rats. A lentiviral vector was used to silence the expression of GLT-1 in astrocytes of PSD rats. NSCs were respectively co-cultured with normal (control), PSD, and GLT-1 silenced astrocytes for 7 days. GLT-1, GFAP, MAP2, Synaptophysin (SYN), glutamate (Glu) and glutamine (Gln) were respectively measured by qRT-PCR, western blot, immunofluorescence and efficient mass spectrometry (MS). PSD astrocytes increased the number of NSC-derived astrocytes, but inhibited the expression of GLT-1 of NSC-derived astrocytes and synapses of NSC-derived neurons. On the basis of the low expression of GLT-1 in PSD astrocytes, we further silenced GLT-1 in PSD astrocytes. Interestingly, GLT-1 silenced PSD astrocytes more obviously inhibited synapses of NSC-derived neurons, but increased the number of NSC-derived neurons and reversed the expression of GLT-1 in NSC-derived astrocytes. At the same time, concentration of glutamate in the medium elevated, and glutamine in the medium gradually reduced. In NSC-derived neurons and astrocytes, glutamate metabolism was also affected by changed GLT-1. Down-expressed GLT-1 in PSD astrocytes stimulated NSCs differentiating into astrocytes, but inhibiting the formation of functional synapses by influencing glutamate metabolism in vitro.     

Dexmedetomidine protects against degeneration of dopaminergic neurons and improves motor activity in Parkinson's disease mice model

Parkinson’s disease (PD) is the result of dopaminergic (DA) neuronal death in the substantianigra pars compacta (SNc). Current treatments for PD such as L-dopa are limited in effectiveness and fail to address the cause. Targeted anti-inflammatory therapies, particularly directed at nuclear factor kappa B (NF‐κB) activity in alleviating degeneration of DA-neurons is of evolving interest. In the present study, we hypothesised that dexmedetomidine (DEX), an alpha-2 receptor adrenergic agonist, suppress the inflammatory responses associated with PD and restores dopaminergic levels by alleviating substantia nigral degeneration. Male mice (C57Bl/10, 8–11 months old and of 34–40 g of weight) were divided into: the control, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and MPTP + dexmedetomidine (MPTP + DEX) (n = 26 each group). Dex restored dopamine levels in SNpc of MPTP-induced PD mice model. Results of immunohisto staining revealed that Dex treatment post-MPTP induction restored TH-positive cells, with only 12.37% increase (^##p < 0.01 vs MPTP) on the third day and a steep 55% increase (^###p < 0.001 vs MPTP) following the seventh day of Dex treatment. Moreover, the expressions of proinflammatory markers regulated by NF-κB were diminished in Dex + MPTP group. In addition, cylinder test revealed that Dex treatment improved asymmetric limb usage pattern in MPTP induced mice over the course of 7 days. Hence, in this study, we provided insight on the effect of Dex in the inhibition of NF-κB1 regulated proinflammatory mediators to improve dopamine levels and reduce SNpc dopaminergic neuronal degeneration.     

Biochemical effects of high-frequency synaptic activity studied with in vitro slices

Brain slices have a number of features that may be of value in the analysis of how physiological events affect neuronal chemistry. This paper discusses this topic and describes slice experiments concerned with the chemical events responsible for long-term potentiation (LTP) of synaptic responses found in hippocampus after brief episodes of high-frequency stimulation. Work with two variants of the slice procedure indicated that LTP is accompanied by an increase in the sodium-independent binding of [3H]glutamate to partially purified synaptic membranes; this effect very likely results from an increase in the numbers of a particular postsynaptic receptor. Stimulation that produces long-term potentiation also causes a significant change in the endogenous phosphorylation of pyruvate dehydrogenase (PDH), a key mitochondrial enzyme. Inasmuch as the phosphorylated state of PDH is strongly correlated with calcium sequestration by mitochondria, it is possible that LTP is triggered by a transient perturbation of the calcium buffering function provided by mitochondria. Low micromolecular levels of calcium increase glutamate binding to purified membranes apparently via the activation of a calcium-sensitive thiol proteinase. This mechanism could account for the increase in glutamate binding found in slices exhibiting LTP. These experiments suggest a possible explanation for long-term potentiation and indicate that slices can be used to detect at least some of the biochemical consequences of repetitive synaptic activity.     

Evaluation of synaptic potential dynamics from evoked impulse activity of neurons]

To evaluate intracellular potentials in the region of spike initiation of function lambda (t) is suggested which describes the dependence on time of the density of conditional probability of generation by the neuron of the first impulse in response to the stimulus. Qualitative correspondence of the dynamics of membrane potentials and function lambda (t) is demonstrated on the analog model. The application of the function lambda (t) to the classification of the neurons of auditory system is shown to be promising. The difference between the function lambda (t) and normalized poststimulus histogram allows to evaluate the refracteriness of the neuron.     

Changes in RNA synthesis in neurons depending on the intensity of their synaptic activity

A prolonged synaptic activation of the frog spinal motoneurons increases the intensity of 3H-uridine incorporation into nuclei and nucleoli of the nerve cells. The higher the frequency of neuronal synaptic activation, the higher the incorporation intensity during the post-activation period.     

Preserved synaptic vesicle recycling in hippocampal neurons in a mouse Alzheimer's disease model

A recently described triple-transgenic mouse model (3xTg, PS1(M146V), APP(Swe), and tau(P301L)) develops a neuropathology similar to the brains of Alzheimer's disease patients including progressive deposits of plaques and tangles [Neuron 39 (2003) 409]. These mice also show age-related deficits in hippocampal synaptic plasticity that occurs before the development of plaques and tangles. Here we report unchanged synaptic vesicle recycling, as measured by FM1-43 release, in the hippocampal neurons of the 3xTg mice. Expression levels of presynaptic protein synaptophysin and of proteins involved in synaptic vesicle recycling including AP180, dynamin I, and synaptotagmin I also remain unaffected. These data suggest that the synaptic deficits observed in the 3xTg neurons may not arise from the preserved synaptic vesicle recycling.