Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

Temporal Characteristics of Gustatory Responses in Rat Parabrachial Neurons Vary by Stimulus and Chemosensitive Neuron Type

It has been demonstrated that temporal features of spike trains can increase the amount of information available for gustatory processing. However, the nature of these temporal characteristics and their relationship to different taste qualities and neuron types are not well-defined. The present study analyzed the time course of taste responses from parabrachial (PBN) neurons elicited by multiple applications of “sweet” (sucrose), “salty” (NaCl), “sour” (citric acid), and “bitter” (quinine and cycloheximide) stimuli in an acute preparation. Time course varied significantly by taste stimulus and best-stimulus classification. Across neurons, the ensemble code for the three electrolytes was similar initially but quinine diverged from NaCl and acid during the second 500ms of stimulation and all four qualities became distinct just after 1s. This temporal evolution was reflected in significantly broader tuning during the initial response. Metric space analyses of quality discrimination by individual neurons showed that increases in information (H) afforded by temporal factors was usually explained by differences in rate envelope, which had a greater impact during the initial 2s (22.5% increase in H) compared to the later response (9.5%). Moreover, timing had a differential impact according to cell type, with between-quality discrimination in neurons activated maximally by NaCl or citric acid most affected. Timing was also found to dramatically improve within-quality discrimination (80% increase in H) in neurons that responded optimally to bitter stimuli (B-best). Spikes from B-best neurons were also more likely to occur in bursts. These findings suggest that among PBN taste neurons, time-dependent increases in mutual information can arise from stimulus- and neuron-specific differences in response envelope during the initial dynamic period. A stable rate code predominates in later epochs.     

Morphology,Classification, and Distribution of the Projection Neurons in the Dorsal Lateral Geniculate Nucleus of the Rat

The morphology of confirmed projection neurons in the dorsal lateral geniculate nucleus (dLGN) of the rat was examined by filling these cells retrogradely with biotinylated dextran amine (BDA) injected into the visual cortex. BDA-labeled projection neurons varied widely in the shape and size of their cell somas, with mean cross-sectional areas ranging from 60–340 µm². Labeled projection neurons supported 7–55 dendrites that spanned up to 300 µm in length and formed dendritic arbors with cross-sectional areas of up to 7.0×10⁴ µm². Primary dendrites emerged from cell somas in three broad patterns. In some dLGN projection neurons, primary dendrites arise from the cell soma at two poles spaced approximately 180° apart. In other projection neurons, dendrites emerge principally from one side of the cell soma, while in a third group of projection neurons primary dendrites emerge from the entire perimeter of the cell soma. Based on these three distinct patterns in the distribution of primary dendrites from cell somas, we have grouped dLGN projection neurons into three classes: bipolar cells, basket cells and radial cells, respectively. The appendages seen on dendrites also can be grouped into three classes according to differences in their structure. Short “tufted” appendages arise mainly from the distal branches of dendrites; “spine-like” appendages, fine stalks with ovoid heads, typically are seen along the middle segments of dendrites; and “grape-like” appendages, short stalks that terminate in a cluster of ovoid bulbs, appear most often along the proximal segments of secondary dendrites of neurons with medium or large cell somas. While morphologically diverse dLGN projection neurons are intermingled uniformly throughout the nucleus, the caudal pole of the dLGN contains more small projection neurons of all classes than the rostral pole.     

Mitigation of Lead-Induced Neurotoxicity by the Naringin: Erythrocytes as Neurons Substitute Markers

This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with l-arginine (l-Arg), N ^ω-nitro-l-Arginine methyl ester ( l-NAME), NAR, PbAc, PbAc + l-Arg, PbAc + NAR, or PbAc + l-Arg +NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of l-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of l-Arg + NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.     

Biochemical and Temporal Analysis of Events Associated with Apoptosis Induced by Lowering the Extracellular Potassium Concentration in Mouse Cerebellar Granule Neurons

Abstract: We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K⁺ concentration ([K⁺]_o), a significant part of the genomic DNA was already cleaved to high-molecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K⁺]_o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning ～4 h after the removal of high [K⁺]_o and developed to nuclear condensation 4 h later. Six hours after high [K⁺]_o deprivation, the DNA was fragmented into oligonucleosome-sized fragments. Within 6 h after removal of the extracellular K⁺, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K⁺]_o. Similar to high [K⁺]_o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K⁺]_o removal. An ～40% decrease in RNA and protein synthesis was detected by 6 h of [K⁺]_o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome-sized fragments, and the reduction in mitochondrial activity—events that occurred within the critical time—may indicate that these processes have an important part in the mechanism that committed the neurons to die.     

Neurons produce FGF2 and VEGF and secrete them at least in part by shedding extracellular vesicles

We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood-brain barrier. We then looked, by immunofluorescence and western analysis, for factors, produced by neurons, with the potential to influence growth and differentiation of endothelial cells. In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well-known angiogenic factors. More interestingly, we gained evidence that both factors are released by neurons, at least in part, by shedding of extracellular vesicles, that contain beta1 integrin, a membrane protein already known to be part of extracellular vesicles released by tumour cells. Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.     

Membrane Properties of Striatal Direct and Indirect Pathway Neurons in Mouse and Rat Slices and Their Modulation by Dopamine

D1 and D2 receptor expressing striatal medium spiny neurons (MSNs) are ascribed to striatonigral (“direct”) and striatopallidal (“indirect”) pathways, respectively, that are believed to function antagonistically in motor control. Glutamatergic synaptic transmission onto the two types is differentially affected by Dopamine (DA), however, less is known about the effects on MSN intrinsic electrical properties. Using patch clamp recordings, we comprehensively characterized the two pathways in rats and mice, and investigated their DA modulation. We identified the direct pathway by retrograde labeling in rats, and in mice we used transgenic animals in which EGFP is expressed in D1 MSNs. MSNs were subjected to a series of current injections to pinpoint differences between the populations, and in mice also following bath application of DA. In both animal models, most electrical properties were similar, however, membrane excitability as measured by step and ramp current injections consistently differed, with direct pathway MSNs being less excitable than their counterparts. DA had opposite effects on excitability of D1 and D2 MSNs, counteracting the initial differences. Pronounced changes in AP shape were seen in D2 MSNs. In direct pathway MSNs, excitability increased across experimental conditions and parameters, and also when applying DA or the D1 agonist SKF-81297 in presence of blockers of cholinergic, GABAergic, and glutamatergic receptors. Thus, DA induced changes in excitability were D1 R mediated and intrinsic to direct pathway MSNs, and not a secondary network effect of altered synaptic transmission. DAergic modulation of intrinsic properties therefore acts in a synergistic manner with previously reported effects of DA on afferent synaptic transmission and dendritic processing, supporting the antagonistic model for direct vs. indirect striatal pathway function.     

The Distribution of Afferent Neurons in the Trigeminal Mesencephalic Nucleus and the Central Projection of Afferent Fibers of the Mylohyoid Nerve in the Rat

Horseradish peroxidase conjugated to wheatgerm agglutinin (HRP:WGA) was injected into the proximal cut ends of three branches of the mylohyoid nerve in rats: the branch to the mylohyoid muscle (BrMh), the branch to the anterior belly of the digastricus muscle (BrDg), and the cutaneous branch (BrCu). HRP-labeled cells were detected in the ipsilateral caudal portion of the trigeminal mesencephalic nucleus (Vmes) and the ipsilateral ventromedial division of the trigeminal motor nucleus, except when HRP:WGA was applied to the BrCu. Morphologically, all labeled Vmes cells were of the pseudounipolar type.

Projections of the primary afferents of the BrMh were observed in the ipsilateral trigeminal nucleus caudalis, the upper cervical dorsal horns of laminae I -III, and the dorsolateral recticular formation (Rf), whereas the primary afferents of the BrDg terminated in the ipsilateral trigeminal nucleus principalis and Rf. These observations suggest that the role of the afferent inputs of the mylohyoid muscle differs from that of those of the anterior belly of the digastricus muscle in terms of several functions associated with jaw-closing and infrahyoid muscles.     

Morphological Characteristics of Motor Neurons Do Not Determine Their Relative Susceptibility to Degeneration in a Mouse Model of Severe Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, resulting primarily from the degeneration and loss of lower motor neurons. Studies using mouse models of SMA have revealed widespread heterogeneity in the susceptibility of individual motor neurons to neurodegeneration, but the underlying reasons remain unclear. Data from related motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), suggest that morphological properties of motor neurons may regulate susceptibility: in ALS larger motor units innervating fast-twitch muscles degenerate first. We therefore set out to determine whether intrinsic morphological characteristics of motor neurons influenced their relative vulnerability to SMA. Motor neuron vulnerability was mapped across 10 muscle groups in SMA mice. Neither the position of the muscle in the body, nor the fibre type of the muscle innervated, influenced susceptibility. Morphological properties of vulnerable and disease-resistant motor neurons were then determined from single motor units reconstructed in Thy.1-YFP-H mice. None of the parameters we investigated in healthy young adult mice – including motor unit size, motor unit arbor length, branching patterns, motor endplate size, developmental pruning and numbers of terminal Schwann cells at neuromuscular junctions - correlated with vulnerability. We conclude that morphological characteristics of motor neurons are not a major determinant of disease-susceptibility in SMA, in stark contrast to related forms of motor neuron disease such as ALS. This suggests that subtle molecular differences between motor neurons, or extrinsic factors arising from other cell types, are more likely to determine relative susceptibility in SMA.     

Glucose Utilization Rates in Single Neurons and Neuropil Determined by Injecting Nontracer Amounts of 2-Deoxyglucose

Abstract: A nontracer amount of 2-deoxyglucose (DG) was intravenously injected into rats, which were frozen 2 and 4 min later in liquid nitrogen. The freeze-dried samples of cell bodies of anterior horn cells, dorsal root ganglion cells, and cerebellar Purkinje cells, as well as the neuropil adjacent to anterior horn cell bodies, were prepared. Their contents of glucose, glucose 6-phosphate, DG, and 2-deoxyglucose 6-phosphate were microassayed using an enzymatic amplification reaction, NADP cycling. Based on the resulting data and theoretical equations previously described, glucose utilization rate (GUR) and apparent distribution volumes (DVs) of glucose and DG were determined. Anterior horn cell bodies had the highest GUR and their neuropil the lowest, although apparent DVs of glucose and DG were similar in both. This indicates that the glucose supply was equally balanced in all, but that the cell bodies had higher functional activity supported by hexokinase (and other enzymes) related to their energy demands. Dorsal root ganglion cells showed the lowest 2-deoxyglucose 6-phosphate formation rate, but their GUR was slightly higher than that of neuropil because of their markedly large DV of glucose, thus demonstrating that the abundant glucose supply supports the neuronal function. Purkinje cells indicated GUR and apparent DVs similar to molecular and granular layers.     

Effects of Single and Repeated Electroconvulsive Shock Administration on Inositol Phosphate Accumulation in Rat Brain Slices

Accumulation of inositol-1-phosphate after labeling with [3H]inositol and stimulation with noradrenaline, carbachol, and serotonin was measured in rat cortical, caudate nucleus, and hippocampal slices. The response to noradrenaline was significantly increased in cortical slices from animals that had received either a single electroconvulsive shock (ECS) or a series of 10 daily ECS but was unchanged in caudate nucleus or hippocampal slices. The response to carbachol, a muscarinic cholinergic agonist, was unchanged in cortical or caudate nucleus slices but was significantly reduced in hippocampal slices from animals that had received chronic ECS. The response to serotonin in cortical slices was not affected by the treatment. The results are correlated with changes in receptor number, which have been demonstrated to occur after administration of ECS.     

Imaging of the Degeneration of Neurons and Their Processes in Rat or Cat Brain by 45CaCl2 Autoradiography or 55CoCl2 Positron Emission Tomography

The possibility of using radiolabeled divalent cations to visualize nerve cell degeneration in the brain was investigated after intoxication with neurotoxins. At different survival times after the intracerebral injection of kainic acid or 6-hydroxydopamine, autoradiographs were made from brain sections of rats that had received 45CaCl2 intravenously 24 h before death. Brain sections, adjacent to those used for autoradiography, of the 6-hydroxydopamine-treated rats were used for histofluorescence of catecholamines to check the neurochemical effect of the treatment. These experiments show that radioactive Ca accumulates in brain tissue during a particular phase of degeneration. Not only could degenerating cell bodies be traced by 45Ca autoradiography, but also degenerating nerve terminals in the striato-nigral and nigro-striatal projection systems. In positron emission tomography (PET) studies, 55CoCl2 was used as a marker for Ca2+. Unilateral lesions of the cat forebrain, produced by kainic acid, could be imaged in vivo by PET with 55CoCl2. PET with this radiolabel may provide diagnostic potentials for human neurodegenerative disorders.     

Variation in Activity State,Axonal Projection,and Position Define the Transcriptional Identity of Individual Neocortical Projection Neurons

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Malonate-Induced Degeneration of Basal Forebrain Cholinergic Neurons: Attenuation by Lamotrigine, MK-801, and 7-Nitroindazole

Abstract: Previously, we have reported that intranigral infusions of malonate, an inhibitor of mitochondrial function, lead to the degeneration of the dopaminergic neurons of the nigrostriatal pathway that is mediated, at least in part, through NMDA receptor activation and nitric oxide formation. In the present study, unilateral focal infusions of malonate into the nucleus basalis magnocellularis (nbM) of male Sprague-Dawley rats (weighing 250–300 g) resulted in a dose-related depletion in ipsilateral cortical and amygdaloid choline acetyltransferase (ChAT) activity. Infusion of a 3 µmol dose of malonate into the nbM of vehicle-treated animals resulted in a 41 and 54% decrease in cortical and amygdaloid ChAT activity, respectively. Systemic pretreatment with lamotrigine (16 mg/kg, i.p.) and MK-801 (5 mg/kg, i.p.) attenuated the depletions in cortical and amygdaloid ChAT activity that resulted from an infusion of this dose of malonate into the nbM. Acetylcholinesterase (AChE) histochemistry of the nbM following focal infusion of malonate (3 µmol) showed a marked decrease in the number of AChE-positive neurons that was partially prevented by MK-801 pretreatment. Before examining the role of nitric oxide formation in malonate-induced toxicity, the ability of systemic administration of N^ω-nitro-l -arginine (l -NA) to inhibit nitric oxide synthase (NOS) activity in the nbM and cerebellum was investigated. l -NA (2, 10, and 20 mg/kg, i.p.) produced a dose-related inhibition of nbM and cerebellar NOS activity that was maximal following a dose of 10 mg/kg l -NA. This level of NOS inhibition persisted for at least 13 h following l -NA (10 mg/kg) administration. Subsequently, the effect of l -NA pretreatment on malonate toxicity was evaluated. Following pretreatment with l -NA (2 and 10 mg/kg, i.p.), the toxic action of malonate on cortical and amygdaloid ChAT activity was not altered. In addition, infusion of a lower dose of malonate (2 µmol) into the nbM resulted in decreases in cortical and amygdaloid ChAT activity that were not altered by pretreatment with l -NA (2 and 10 mg/kg, i.p.). In 7-nitroindazole (7-NI; 25 and 50 mg/kg, i.p.)-pretreated animals, malonate (3 µmol) produced decreases in cortical and amygdaloid ChAT activity that were attenuated by both doses of 7-NI. Thus, malonate-induced destruction of the basal forebrain cholinergic neurons was attenuated by systemic pretreatment with lamotrigine, MK-801, and 7-NI but not by l -NA.     

Bovicins: The Bacteriocins of Streptococci and Their Potential in Methane Mitigation

Probiotics and Antimicrobial Proteins - Bovicin is a type AII lantibiotic, possessing two β-methyllanthionine and a disulfide bridge encoded by bovA gene hitherto unknown a couple of decades...