Early excitability changes in a novel acute model of transient focal ischemia and reperfusion in the in vitro isolated guinea pig brain

The study of the early events that characterize cerebral ischemia is limited in available experimental models. The study of neurophysiological network changes that occur in brain tissue during the early minutes that follow focal ischemia induction is restricted in the in vivo condition. Very simplified systems, such as in vitro brain slices and in isolated neurons, have been utilized for this type of studies. We describe here a new model of transient focal ischemia and reperfusion developed in the isolated guinea pig brain, maintained in vitro by arterial perfusion with a complex saline solution without blood cells. In this preparation, that combines the advantage of an in vitro preparation with the functional preservation of both vascular and neuronal compartments, the arteries of the Willis circle are directly accessible by visual control. To induce transitory focal ischemia, one medial cerebral artery (MCA) was transiently tied for 30 min, while brain activity was recorded with multiple electrodes positioned in brain areas within and outside MCA territory. Anoxic depression in ischemic areas propagated to the surrounding tissue and was associated with the abolition of evoked responses due to both functional impairment of afferent olfactory input and tissue depression. Recovery of evoked responses was obtained after MCA reperfusion. The spatial distribution of hypoxic depressions was characterized and was correlated with the extension of brain damage, defined by immunohistochemical analysis with antibodies against microtubule-associated protein (MAP-2). We propose that the present model can be utilized to analyze brain activity changes that occur in early stages of focal brain ischemia and reperfusion.     

The Isolated and Perfused Brain of the Guinea-pig In Vitro

We describe here an isolated and perfused in vitro adult guinea-pig whole brain preparation which is an extension of the previously described in vitro brainstem–cerebellum preparation, Viability was tested by the analysis of trans-synaptic responses along the visual pathways following the electrical stimulation of the optic nerve or the optic radiations. The evoked field potentials were recorded in the dorsal lateral geniculate, the superior colliculus and the visual cortex. The distribution of extracellular currents was studied using current source density analysis, in order to determine the amplitude, time course and spatial organization of the synaptic activity at these sites. The study indicates that field potentials were very similar to those described in vivo. These data demonstrate the survival of a complex adult sensory system in vitro and suggest that this preparation can be used for the analysis of multisynaptic circuits in the mammalian brain.     

Expression of adhesion factors induced by epileptiform activity in the endothelium of the isolated guinea pig brain in vitro

PURPOSE: Brain inflammation has been recently considered in the pathogenesis of focal epilepsies. Synthesis of pro-inflammatory mediators in the brain was described both in experimental models of seizures and in human postsurgical tissue. Inflammatory mediators may up-regulate endothelial adhesion molecules, therefore promoting adhesion and homing of leucocytes into the brain. In the present study, expression of inducible adhesion factors in brain endothelium was verified after pharmacological induction of seizure-like activity in specific brain areas of the in vitro isolated guinea pig brain. METHODS: Experiments were performed in isolated guinea-pig brains maintained in vitro by arterial perfusion. In this preparation, brief application of the GABAa receptor-antagonist, bicuculline, consistently induced focal ictal discharges in the limbic region that secondarily diffuse to the neocortex, as verified by simultaneous electrophysiological recording of extracellular activity. At the end of the electrophysiological experiment (after 5 h in vitro), brains were fixed and immunostaining for adhesion molecules P-selectin and ICAM-1 and for Fos protein was evaluated. RESULTS: Immunohistochemical analysis of isolated brains in which seizure-like activity was induced revealed expression of inducible adhesion factors P-selectin and ICAM-1 in the endothelium of small-medium size brain vessels. In particular, the expression of these molecules was consistently observed in all areas involved in epileptic seizure-like ictal activity (limbic cortices and neocortex), and was infrequently found in regions that generated interictal spiking (piriform cortex), suggesting a trigger role played by seizures for endothelial activation. An increase in Fos protein expression was evident in all analyzed limbic areas and in the neocortex, indicating a correlation between the areas of neuronal and endothelial activation. In control brains maintained in vitro for comparable times without induction of epileptiform activity, no immunoreactivity for Fos and adhesion molecules was observed. CONCLUSIONS: Seizure-like activity in an in vitro isolated brain preparation induces the expression of adhesion molecules in the cerebral endothelium. These observations indicate that local endothelial activation may represent a crucial step for the development of an inflammatory response induced by seizures, and suggest a possible novel pathogenic mechanism during the process of epileptogenesis.     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

The electrophysiology of the olfactory-hippocampal circuit in the isolated and perfused adult mammalian brain in vitro.

The viability and general electrophysiological properties of the limbic system in the adult mammalian brain isolated and maintained in vitro by arterial perfusion are described. The isolated brain preparation combines the advantages of intact synaptic connectivity and accessibility of different areas of the encephalic mass with those of the in vitro approach, i.e., stability and control of the ionic environment. Extracellular field potential as well as intracellular recordings were performed at different levels in the limbic system of isolated adult guinea pig brains. The results demonstrate that in the piriform, entorhinal, and hippocampal cortices, the intrinsic electrical properties of individual cells as well as the spontaneous and evoked electrical activity in the neuronal ensembles they comprise, were virtually identical to those observed in vivo. The properties of the limbic system loop were determined.     

正常成人脑海马区多潜能神经前体细胞的体外分离鉴定

目的 从正常成人脑海马分离鉴定多潜能神经前体细胞并探讨体外培养条件.方法 材料取自8例非神经系统疾病的死亡者.分离正常成人脑海马细胞,在培养基中添加生长因子体外培养.通过细胞培养成球和BrdU染色鉴定培养细胞的增殖能力.利用免疫细胞化学三标染色来鉴定分化神经细胞的表型.结果 从正常人海马分离的细胞培养2周时,开始增殖成簇,致一个月时,形成细胞球,这些细胞能与BrdU结合,并能在体外分化成神经系统不同谱系的细胞.结论 与在啮齿类动物和猴的研究中结果一致,从正常成人脑海马区分离的细胞是多潜能神经前体细胞.     

Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry

Following intrathecal injection of fluorescent tracers, ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain. Tracer penetration into the brain is usually quantified by image-processing, even though this approach requires much time and manual operation. Here, we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry. At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers, certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue. The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves. Using this approach, the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed, and the clearance of tracers from the brain was also determined. Furthermore, the impairment of glymphatic influx in the brains of old mice was confirmed using our approach. Our method is more accurate and efficient than the imaging approach in terms of the quantitative determination of glymphatic activity, and this will be useful in preclinical studies.     

Intracellular recording from respiratory neurones in the perfused 'in situ' rat brain

The study describes an arterially perfused in situ rat brain preparation, which uses an 'open circuit' flow of blood substitute with or without an oxygen carrier (2% perfluorotributylamine). The respiratory motor output was recorded from the phrenic and hypoglossal nerves, and could be maintained for up to 11 h from the start of perfusion (temperature of perfusate: 27-30 degrees C). The preparation allowed stable intracellular recordings from respiratory neurons in the brain stem and cervical spinal cord, and should be suitable for other studies which cannot be performed in standard whole animal models. The advantages of this approach compared with other in vitro or perfused in situ preparations are discussed.     

HSV-1 H129-Derived Anterograde Neural Circuit Tracers: Improvements,Production, and Applications

Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.     

Blood-brain barrier preservation in the in vitro isolated guinea pig brain preparation

The morphofunctional preservation of the blood-brain barrier (BBB) was evaluated in the isolated guinea pig brain maintained in vitro by arterial perfusion. Electron microscopy evaluation after 5 hr in vitro demonstrated that cerebral capillaries and BBB specializations in this preparation retain features compatible with structural integrity. BBB-impermeable and -permeable atropine derivatives arterially perfused to antagonize carbachol-induced fast oscillatory activity confirmed the functional preservation of the BBB in vitro. To study BBB function further, changes in extracellular K+ concentration during arterial perfusion of a high-K+ solution were measured with K+-sensitive electrodes positioned in the cortex and, as control, at the brain venous outlet, where the solution perfused through the brain arterial system was collected. After 5 hr in vitro, the [K+](o) values measured during high-K+ perfusion in the piriform and entorhinal cortices were 5.02 +/- 0.17 mM (mean +/- SE) and 5.2 +/- 0.21 mM, respectively (n = 6). Coperfusion of the high-K+ solution with the Na+/K+ pump blocker ouabain (10 microM; n = 4) induced consistently spreading depression preceded by a rise in [K+](o). Finally, sporadic, isolated spots of extravasation of the fluorescent marker fluorescein isothiocyanate (FITC)-dextran preferentially circumscribed to deep cortical layers was observed in brains perfused with FITC-dextran after 5 hr in vitro. The study demonstrates that the in vitro isolated guinea pig brain is viable for studying cerebrovascular interactions and BBB permeability of compounds active in the central nervous system.     

Learning: Neural analysis in the isolated brain of a previously trained mollusc,Pleurobranchaea californica

The neural manifestations of food avoidance learning in the mollusc, Pleurobranchaea, survive the surgical reduction of the preparation to the nearly isolated brain. These manifestations include increased synaptic inhibition and reduced synaptic excitation of the phasic paracerebral feeding command interneurons (PCps) in the brain in response to food stimulation of chemosensory structures left attached to the brain. The same changes are not evident, however, in brains removed from naive, control or satiated specimens. Therefore the nearly isolated brain preparation permits analysis of the cellular substrates of learning in relative isolation from non-associative motivational variables. The isolated brain preparation is here used to show that the increased synaptic inhibition consequent to associative training is distributed not only to the PCps but also to their identified central presynaptic inputs, including other identified feeding command interneurons (PSEs and ETIIs; ref. 21). The decrease in PCp excitation is explained in part by a training-induced inhibition of excitatory inputs to the PCps, and in part by a training-induced reduction in the efficacy of an identified polysynaptic excitatory pathway presynaptic to the PCps.     

Kinetics and distribution volumes for tracers of different sizes in the brain plasma space

Regional brain and plasma concentrations were determined for a series of radiotracers that differ in molecular weight and size in pentobarbital-anesthetized rats at 1, 5 and 30 min after i.v. injection. The tracers, [3H]inulin (mol. wt. 5000 Da, radius 1.5 nm), 5 [3H]dextrans (10,000-200,000 Da, 2.3-9.5 nm) and [51Cr]transferrin (79,000 Da, 3.8 nm), are not taken up into erythrocytes and do not measurably cross the blood-brain barrier in 30 min. Results were expressed as a brain distribution volume, defined as (dpm/g brain)/(dpm/ml plasma). Within 1 min after injection, all tracers attained an initial distribution volume which varied regionally from 0.4 to 1.6 X 10(-2) ml/g. The volumes remained constant between 1 and 30 min for tracers with radii greater than or equal to 3.8 nm, whereas the volumes increased up to 90% for tracers with radii less than or equal to 3.1 nm. Rates of equilibration for tracers with radii less than or equal to 3.1 nm were size dependent with smaller tracers equilibrating before larger tracers. These results indicate that the brain distribution volume for plasma tracers consists of two compartments: one which is quickly filled (less than or equal to 1 min) by all tracers and comprises approximately 60% of the total volume, and one which allows only tracers with radii less than or equal to 3.1 nm and comprises 40% of the total volume. The inverse relation between the rate of equilibration in the second compartment and molecular size may indicate a diffusion limitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic changes in cAMP concentration and phosphodiesterase activity in a mammalian circadian clock studied in vitro.

The mammalian suprachiasmatic nuclei (SCN) contain a circadian pacemaker that continues to keep 24-h time when isolated in vitro. We are investigating the role of cAMP in the cellular mechanisms underlying SCN function. We have previously shown that increasing intracellular cAMP during the subjective day resets the SCN pacemaker in the in vitro rat brain slice preparation. We now report that the level of cAMP fluctuates within the rat SCN under constant conditions in vitro. The level of endogenous cAMP is high during late day and late night, and low during early night. These changes in cAMP concentration are accompanied by opposite changes in phosphodiesterase activity; we detected no significant change in adenylate cyclase activity. These results provide further support for the hypothesis that cAMP is involved in circadian function in the SCN.     

Characterization of radioiodinated ligand binding to amyloid β plaques

Several novel series of iodinated compounds based on the thioflavin backbone structure have been developed and characterized. These iodinated compounds showed high specific binding to amyloid β (Aβ) aggregates with subnanomolar to nanomolar affinities. Probes like IMPY and MIPA display high brain uptakes and fast washout in normal mice, resulting in low background signals (presumably no amyloid plaques present in normal mouse brain), whereas TZDM shows long brain retention in normal mice suggesting high nonspecific in vivo binding. It is likely that tracers, that is, IMPY or MIPA, with desirable in vivo properties, will provide the highest target to non-target ratio; therefore, they are most likely to be successful as imaging agents targeting Aβ plaques in the brain.     

Characterization of radioiodinated ligand binding to amyloid beta plaques

Several novel series of iodinated compounds based on the thioflavin backbone structure have been developed and characterized. These iodinated compounds showed high specific binding to amyloid beta (Abeta) aggregates with subnanomolar to nanomolar affinities. Probes like IMPY and MIPA display high brain uptakes and fast washout in normal mice, resulting in low background signals (presumably no amyloid plaques present in normal mouse brain), whereas TZDM shows long brain retention in normal mice suggesting high nonspecific in vivo binding. It is likely that tracers, that is, IMPY or MIPA, with desirable in vivo properties, will provide the highest target to non-target ratio; therefore, they are most likely to be successful as imaging agents targeting Abeta plaques in the brain.     

The amiloride-sensitive Na/H exchange antiporter and control of intracellular pH in hippocampal brain slices

The intracellular pH, 7.54 ± 0.03 (mean ± S.D., n = 15), determined with the Neutral red method, of the hippocampal brain slice preparation under baseline incubation conditions is considerably more alkaline than the bath buffer pH. Neutralization by amiloride suggests that the alkalinity was due to Na⁺/H⁺ exchange antiporter activation. To characterize the brain Na⁺/H⁺ exchange antiporter we compared the inhibitory effects of MIA, amiloride and other 5-N substituted analogues on proton extrusion after acid loading by transient exposure to ammonium chloride in the isolated hippocampal brain slice preparation. The potencies of amiloride compounds on the initial recovery rate of intracellular pH after acid-loading were DMA > MIA > HMA = MHA ≥ IPA-HCl > IPA > MNPA = Amil > Benzamil. The greater potency of the 5-N substituted analogs of amiloride over amiloride and benzamil strongly suggest that Na⁺/H⁺ exchange antiporter is the mechanism responsible for alkalinization in the isolated hippocampal brain slice in vitro.     

Synthesis, characterization, and first successful monkey imaging studies of metabotropic glutamate receptor subtype 5 (mGluR5) PET radiotracers

Three metabotropic glutamate receptor subtype 5 (mGluR5) PET tracers have been labeled with either carbon-11 or fluorine-18 and their in vitro and in vivo behavior in rhesus monkey has been characterized. Each of these tracers share the common features of high affinity for mGluR5 (0.08-0.23 nM vs. rat mGluR5) and moderate lipophilicity (log P 2.8-3.4). Compound 1b was synthesized using a Suzuki or Stille coupling reaction with [11C]MeI. Compounds 2b and 3b were synthesized by a SNAr reaction using a 3-chlorobenzonitrile precursor. Autoradiographic studies in rhesus monkey brain slices using 2b and 3b showed specific binding in cortex, caudate, putamen, amygdala, hippocampus, most thalamic nuclei, and lower binding in the cerebellum. PET imaging studies in monkey showed that all three tracers readily enter the brain and provide an mGluR5-specific signal in all gray matter regions, including the cerebellum. The specific signal observed in the cerebellum was confirmed by the autoradiographic studies and saturation binding experiments that showed tracer binding in the cerebellum of rhesus monkeys. In vitro metabolism studies using the unlabeled compounds showed that 1a, 2a, and 3a are metabolized slower by human liver microsomes than by monkey liver microsomes. In vivo metabolism studies showed 3b to be long-lived in rhesus plasma with only one other more polar metabolite observed.     

Viruses as transneuronal tracers.

Tracing chains of neurones requires the use of transneuronal tracers, which are transferred between connected neurones. The conventional transneuronal tracers used so far produce weak labelling of recipient neurones, probably because only a small amount of tracer is transferred. Live neurotropic viruses are beginning to be used as transneuronal tracers. The viruses are replicated in recipient neurones after transneuronal transfer. This replication, which is a unique characteristic of viruses, produces strong transneuronal labelling. The findings indicate that herpes-viruses in particular represent powerful tools for demonstrating neuronal connections across synapses, for example between peripheral nerves and neurones in the brain.     

Neuroimaging methods applied in Parkinson’s disease

Abstract. Radiotracer methods provide regional in vivo quantified information about specific biochemical activities in brain tissue. The understanding of the principles governing radiotracer uptake into brain tissue determines the potential value of these tracers in assessing pathophysiology of brain diseases. Too often a reductionist view of images is taken to directly point to clinical features or even diagnoses of brain diseases. Parkinsons disease like many other neurodegenerative brain diseases is a multisystem disorder of considerable biological and clinical complexity while the information given by regional cerebral tracer uptake points to a momentary biochemical local tissue feature. Examples applying to the well-known dopaminergic tracers are given.     

Multiunit activity recordings in the suprachiasmatic nuclei: in vivo versus in vitro models

The suprachiasmatic nuclei (SCN) of the hypothalamus continue to oscillate when they are isolated in a brain slice preparation. We recorded multiunit activity in the SCN of the rat both in vivo and in vitro to determine the circadian discharge pattern. The variability of the discharge pattern is larger and the amplitude of the rhythm is smaller in vivo than in vitro. Moreover we found evidence for a direct effect of the animal's behavioural activity on electrical activity of the SCN in vivo. These findings may provide an electrophysiological basis for the known effects of behavioural stimuli on the circadian pacemaker. This study underscores the importance of recordings in intact preparations in addition to in vitro work when generalisations to physiological conditions are to be made.