Functional expression of TrkB receptors on interneurones and pyramidal cells of area CA3 of the rat hippocampus.

The dentate gyrus and hippocampal area CA3 region of the mammalian brain contains the highest levels of brain-derived neurotrophic factor (BDNF) and its canonical membrane receptor, tropomyosin-related kinase B (TrkB). Therefore, the present study examines the expression and physiological responses triggered by activation of TrkB on hippocampal area CA3 interneurones and pyramidal cells of the rat hippocampus. Triple immunolabelling for TrkB, glutamate decarboxylase 67, and the calcium-binding proteins parvalbumin, calbindin or calretinin confirms the somatic expression of TrkB in all CA3 sublayers. TrkB-positive interneurones with fast-spiking discharge are restricted to strata oriens and lucidum, whereas regular-spiking interneurones are found in the strata lucidum, radiatum and lacunosum-moleculare. Activation of TrkB receptors with 7,8-dihydroxyflavone (DHF) modulates amplitude and frequency of spontaneous synaptic currents recorded from CA3 interneurones. Furthermore, the isolated excitatory postsynaptic currents (EPSC) of CA3 interneurones evoked by the mossy fibres (MF) or commissural/associational (C/A) axons, show input-specific synaptic potentiation in response to TrkB stimulation. On CA3 pyramidal cells, stimulation with DHF potentiates the MF synaptic transmission and increases the MF-EPSP - spike coupling. The latter exhibits a dramatic increase when picrotoxin is bath perfused after DHF, indicating that local interneurones restrain the excitability mediated by activation of TrkB. Therefore, we propose that release of BDNF on area CA3 reshapes the output of this hippocampal region by simultaneous activation of TrkB on GABAergic interneurones and pyramidal cells.

Structural Correlates of CA2 and CA3 Pyramidal Cell Activity in Freely-Moving Mice.

Plasticity within hippocampal circuits is essential for memory functions. The hippocampal CA2/CA3 region is thought to be able to rapidly store incoming information by plastic modifications of synaptic weights within its recurrent network. High-frequency spike-bursts are believed to be essential for this process, by serving as triggers for synaptic plasticity. Given the diversity of CA2/CA3 pyramidal neurons, it is currently unknown whether and how burst activity, assessed in vivo during natural behavior, relates to principal cell heterogeneity. To explore this issue, we juxtacellularly recorded the activity of single CA2/CA3 neurons from freely-moving male mice, exploring a familiar environment. In line with previous work, we found that spatial and temporal activity patterns of pyramidal neurons correlated with their topographical position. Morphometric analysis revealed that neurons with a higher proportion of distal dendritic length displayed a higher tendency to fire spike-bursts. We propose that the dendritic architecture of pyramidal neurons might determine burst-firing by setting the relative amount of distal excitatory inputs from the entorhinal cortex.SIGNIFICANCE STATEMENT High-frequency spike-bursts are thought to serve fundamental computational roles within neural circuits. Within hippocampal circuits, spike-bursts are believed to serve as potent instructive signals, which increase the efficiency of information transfer and induce rapid modifications of synaptic efficacies. In the present study, by juxtacellularly recording and labeling single CA2/CA3 neurons in freely-moving mice, we explored whether and how burst propensity relates to pyramidal cell heterogeneity. We provide evidence that, within the CA2/CA3 region, neurons with higher proportion of distal dendritic length display a higher tendency to fire spike-bursts. Thus, the relative amount of entorhinal inputs, arriving onto the distal dendrites, might determine the burst propensity of individual CA2/CA3 neurons in vivo during natural behavior.

Nonspecific Expression in Limited Excitatory Cell Populations in Interneuron-Targeting Cre-driver Lines Can Have Large Functional Effects.

Transgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. The investigation of interneurons in particular has benefitted from the ability to genetically target specific cell types. However, the specificity of some Cre driver lines has been called into question. Here, we show that nonspecific expression in a subset of hippocampal neurons can have substantial nonspecific functional effects in a somatostatin-Cre (SST-Cre) mouse line. Nonspecific targeting of CA3 pyramidal cells caused large optogenetically evoked excitatory currents in remote brain regions. Similar, but less severe patterns of nonspecific expression were observed in a widely used SST-IRES-Cre line, when crossed with a reporter mouse line. Viral transduction on the other hand yielded more specific expression but still resulted in nonspecific expression in a minority of pyramidal layer cells. These results suggest that a careful analysis of specificity is mandatory before the use of Cre driver lines for opto- or chemogenetic manipulation approaches.

Extracellular pH modulation of excitatory synaptic transmission in hippocampal CA3 neurons.

In this study, the effect of extracellular pH on glutamatergic synaptic transmission was examined in mechanically dissociated rat hippocampal CA3 pyramidal neurons using a whole-cell patch-clamp technique under voltage-clamp conditions. Native synaptic boutons were isolated without using any enzymes, using a so-called "synapse bouton preparation," and preserved for the electrical stimulation of single boutons. Both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were found to decrease and increase in response to modest acidic (~pH 6.5) and basic (~pH 8.5) solutions, respectively. These changes in sEPSC frequency were not affected by the addition of TTX but completely disappeared by successive addition of Cd2+. However, changes in sEPSC amplitude induced by acidic and basic extracellular solutions were not affected by the addition of neither TTX nor Cd2+. The glutamate-induced whole-cell currents were decreased and increased by acidic and basic solutions, respectively. Acidic pH also decreased the amplitude and increased the failure rate (Rf) and paired-pulse rate (PPR) of glutamatergic electrically evoked excitatory postsynaptic currents (eEPSCs), while a basic pH increased the amplitude and decreased both the Rf and PPR of eEPSCs. The kinetics of the currents were not affected by changes in pH. Acidic and basic solutions decreased and increased voltage-gated Ca2+ but not Na+ channel currents in the dentate gyrus granule cell bodies. Our results indicate that extracellular pH modulates excitatory transmission via both pre- and postsynaptic sites, with the presynaptic modulation correlated to changes in voltage-gated Ca2+ channel currents.NEW & NOTEWORTHY The effects of external pH changes on spontaneous, miniature, and evoked excitatory synaptic transmission in CA3 hippocampal synapses were examined using the isolated nerve bouton preparation, which allowed for the accurate regulation of extracellular pH at the synapses. Acidification generally reduced transmission, partly via effects on presynaptic Ca2+ channel currents, while alkalization generally enhanced transmission. Both pre- and postsynaptic sites contributed to these effects.

Multimodal Chemical Analysis of the Brain by High Mass Resolution Mass Spectrometry and Infrared Spectroscopic Imaging.

The brain functions through chemical interactions between many different cell types, including neurons and glia. Acquiring comprehensive information on complex, heterogeneous systems requires multiple analytical tools, each of which have unique chemical specificity and spatial resolution. Multimodal imaging generates complementary chemical information via spatially localized molecular maps, ideally from the same sample, but requires method enhancements that span from data acquisition to interpretation. We devised a protocol for performing matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance-mass spectrometry imaging (MSI), followed by infrared (IR) spectroscopic imaging on the same specimen. Multimodal measurements from the same tissue provide precise spatial alignment between modalities, enabling more advanced image processing such as image fusion and sharpening. Performing MSI first produces higher quality data from each technique compared to performing IR imaging before MSI. The difference is likely due to fixing the tissue section during MALDI matrix removal, thereby preventing analyte degradation occurring during IR imaging from an unfixed specimen. Leveraging the unique capabilities of each modality, we utilized pan sharpening of MS (mass spectrometry) ion images with selected bands from IR spectroscopy and midlevel data fusion. In comparison to sharpening with histological images, pan sharpening can employ a plethora of IR bands, producing sharpened MS images while retaining the fidelity of the initial ion images. Using Laplacian pyramid sharpening, we determine the localization of several lipids present within the hippocampus with high mass accuracy at 5 µm pixel widths. Further, through midlevel data fusion of the imaging data sets combined with k-means clustering, the combined data set discriminates between additional anatomical structures unrecognized by the individual imaging approaches. Significant differences between molecular ion abundances are detected between relevant structures within the hippocampus, such as the CA1 and CA3 regions. Our methodology provides high quality multiplex and multimodal chemical imaging of the same tissue sample, enabling more advanced data processing and analysis routines.

Biospectroscopic Imaging Provides Evidence of Hippocampal Zn Deficiency and Decreased Lipid Unsaturation in an Accelerated Aging Mouse Model.

Western society is facing a health epidemic due to the increasing incidence of dementia in aging populations, and there are still few effective diagnostic methods, minimal treatment options, and no cure. Aging is the greatest risk factor for memory loss that occurs during the natural aging process, as well as being the greatest risk factor for neurodegenerative disease such as Alzheimer's disease. Greater understanding of the biochemical pathways that drive a healthy aging brain toward dementia (pathological aging or Alzheimer's disease), is required to accelerate the development of improved diagnostics and therapies. Unfortunately, many animal models of dementia model chronic amyloid precursor protein overexpression, which although highly relevant to mechanisms of amyloidosis and familial Alzheimer's disease, does not model well dementia during the natural aging process. A promising animal model reported to model mechanisms of accelerated natural aging and memory impairments, is the senescence accelerated murine prone strain 8 (SAMP8), which has been adopted by many research group to study the biochemical transitions that occur during brain aging. A limitation to traditional methods of biochemical characterization is that many important biochemical and elemental markers (lipid saturation, lactate, transition metals) cannot be imaged at meso- or microspatial resolution. Therefore, in this investigation, we report the first multimodal biospectroscopic characterization of the SAMP8 model, and have identified important biochemical and elemental alterations, and colocalizations, between 4 month old SAMP8 mice and the relevant control (SAMR1) mice. Specifically, we demonstrate direct evidence of Zn deficiency within specific subregions of the hippocampal CA3 sector, which colocalize with decreased lipid unsaturation. Our findings also revealed colocalization of decreased lipid unsaturation and increased lactate in the corpus callosum white matter, adjacent to the hippocampus. Such findings may have important implication for future research aimed at elucidating specific biochemical pathways for therapeutic intervention.

Npas4 Is a Critical Regulator of Learning-Induced Plasticity at Mossy Fiber-CA3 Synapses during Contextual Memory Formation.

Synaptic connections between hippocampal mossy fibers (MFs) and CA3 pyramidal neurons are essential for contextual memory encoding, but the molecular mechanisms regulating MF-CA3 synapses during memory formation and the exact nature of this regulation are poorly understood. Here we report that the activity-dependent transcription factor Npas4 selectively regulates the structure and strength of MF-CA3 synapses by restricting the number of their functional synaptic contacts without affecting the other synaptic inputs onto CA3 pyramidal neurons. Using an activity-dependent reporter, we identified CA3 pyramidal cells that were activated by contextual learning and found that MF inputs on these cells were selectively strengthened. Deletion of Npas4 prevented both contextual memory formation and this learning-induced synaptic modification. We further show that Npas4 regulates MF-CA3 synapses by controlling the expression of the polo-like kinase Plk2. Thus, Npas4 is a critical regulator of experience-dependent, structural, and functional plasticity at MF-CA3 synapses during contextual memory formation.

Alterations of L-type voltage dependent calcium channel alpha 1 subunit in the hippocampal CA3 region during and after pilocarpine-induced epilepsy.

Voltage-dependent calcium channels (VDCC) have been shown to regulate neuronal excitability and their antagonists have been used clinically for the control of seizures. While functional studies of VDCC in epileptogenesis in the CA1 area of hippocampus or the dentate gyrus have been done, few studies were carried out in the CA3 area. Given the bursting characteristics of the CA3 neurons, we speculated that VDCC in the CA3 area might play an important role in the epileptogenesis. In the present study in the mouse pilocarpine model of temporal lobe epilepsy, we investigated the alterations of alpha 1 subunits of L-type VDCC in the CA3 area of the hippocampus at different stages of epileptogenesis, i.e., acute stage from 10 min to 1 day during and after pilocapine-induced status epilepticus (SE), latent period at 1 week, and chronic stage with spontaneous recurrent seizures at 2 months after SE. We found that an immediate redistribution of alpha 1 subunits in the CA3 area occurred during SE which might be involved in the seizure occurrence indicated by the Racine score record. Alterations of alpha 1 subunits were also demonstrated in the latent period and chronic stage, which might be related to the epileptogenesis and occurrence of epilepsy. Cav1.3, but not Cav1.2, was expressed in reactive astrocytes of the CA3 area, indicating the involvement of Cav1.3 in the modulation of astrocytic Ca2+ homeostasis during epileptogenesis.

A study of bio-hybrid silsesquioxane/yeast: Biosorption and neuronal toxicity of lead.

Lead is a heavy metal of high impact for the environment as well as for human health, being cause of several diseases. Considering the importance of obtaining an effective treatment for lead removal, a new hybrid material was developed for sorption of Pb2+ from aqueous solution. The effect of pH, temperature, liquid/solid ratio (g/cm3) and lead concentration on the sorption capacity of yeasts chemically modified with cubic silsesquioxane (YS) was analyzed. Additionally, the toxicity of lead on the neuronal activity was also investigated in order to assess whether the damage caused by the Pb2+ ion is reversible or not. The YS is highly promissory as sorbent of lead in high concentrations (100 and 500ppm), reaching high efficiency in short contact times (15min), and at the natural pH (4) of the Pb2+ solution and room temperature. The best sorption obtained was 82% removal and 248mg/g with 500cm3/g sorbent, pH 4, room temperature and contact time of 15min. Besides, such high efficiencies are obtained with low quantities of biosorbent, when compared with other similar materials. The impact of lead on neuronal function was studied by measuring autofluorescence signals, associated with changes in cellular metabolism, at the hippocampal CA3 area in brain slices. In this toxicity tests, the effect of low concentrations of lead (1 and 3µM) on neuronal activity was evaluated. After removal of the lead, the irreversibility of the observed changes can be verified, which suggests the existence of neuronal damages.

Metabolomics approach to explore the effects of Kai-Xin-San on Alzheimer's disease using UPLC/ESI-Q-TOF mass spectrometry.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease that influences elderly populations, with no effective method for its treatment so far. To improve its diagnosis and treatment, changes of small molecule metabolite during AD should be elucidated. Kai-Xin-San (KXS) is an herbal formulae that has been widely used to treat mental disorders, especially amnesia and depression in China. Experimental AD was induced in rats by an intraperitoneal injection of d-galactose (d-gal) and administered intragastrically with aluminum chloride (AlCl3) simultaneously for 105 days. Morris water maze task as a behavior test was used for testing the effects of KXS on AD model and pathological changes to the brain were assessed by hematoxylin-eosin staining and immunohistochemistry. The levels of Bcl-2 and ChAT in hippocampus were evaluated by western-blot. Furthermore, metabolite profiling of AD was performed through ultra-performance liquid chromatography/electrospray ionization quadruple time-of- flight-high-definition mass spectrometry (UPLC/ESI-Q-TOF/HDMS) combined with pattern recognition approaches and pathway analysis. d-gal and AlCl3-treated caused a decline in spatial learning and memory, hippocampal histopathological abnormalities and increased Aß1-40 levels in the brain cortex and hippocampus along with decreased Bcl-2 and ChAT expression in the hippocampus. KXS significantly improved the cognitive impairment induced by d-gal and AlCl3, attenuated hippocampal histopathological abnormalities, reduced Aß1-40 levels and increased Bcl-2 and ChAT expression in the hippocampus. A total of 48 metabolites were considered as potential biomarkers of AD, and 36 metabolites may correlate with the regulation of KXS treatment on AD. Changes in AD metabolic profiling were close to normal states through regulating multiple perturbed pathways after KXS treatment. This study has revealed the potential biomarkers and metabolic networks of AD, illuminated the biochemistry mechanism of AD and the metabolic pathways influenced by KXS.

Expression of c-Fos protein in medial septum/diagonal band of Broca and CA3 region, associated with the temporary inactivation of the supramammillary area.

The supramammillary (SuM) area is part of the diencephalic nuclei comprising the mammillary bodies, and is a key structure in the memory and spatial learning processes. It is a critical region in the modulation/generation of hippocampal theta rhythm. In addition, many papers have recently shown a clear involvement of this structure in the processes of spatial learning and memory in animal models, although it is still not known how it modulates spatial navigation and response emotional. The aim of the present research was to study the effect of the temporary inactivation of the SuM area on synaptic plasticity of crucial structures in the formation of spatial memory and emotional response. Sprague-Dawley rats were asigned in three groups: a control group where the animals were not subjected to any treatment, and two groups where the rats received microinjections of tetrodotoxin (TTX) in the SuM area (5ng diluted in 0.5µl of saline) or saline (0.5µl). The microinjections were administered 90min before the perfusion. Later, cellular activity in medial septum/diagonal band of Broca (MS/DBB) and CA3 region of the dorsal hippocampus was assessed, by measuring the immediate early gene c-fos. The results show a clear hiperactivity cellular in medial septum/diagonal band of Broca and a clear hypoactivity cellular in the CA3 region of the hippocampus when there was a functional inactivation of the SuM area. It suggests that the SuM area seems to be part of the connection and information input pathways to CA3 region of the hippocampal formation, key for proper functioning in spatial memory and emotional response.

ß-Asarone prevents autophagy and synaptic loss by reducing ROCK expression in asenescence-accelerated prone 8 mice.

ß-Asarone is an active component of the Acori graminei rhizome that is a traditional Chinese medicine clinically used in treating dementia in China. However, the cognitive effect of ß-asarone and its mechanism has remained elusive. Here, we used asenescence-accelerated prone 8 (SAMP8) mice, which mimic many of the salient features of Alzheimer׳s disease (AD), to further investigate whether modulation of the ROCK signaling pathway and/or autophagy, synaptic loss is involved in the effects of ß-asarone on learning and memory. SAMP8 mice at the age of 6 months were intragastrically administered by ß-asarone or a vehicle daily for 2 months. Senescence-accelerated-resistant (SAMR1) mice were used as the control. Our results demonstrate that autophagy and ROCK expression were increased significantly in 8 months SAMP8 mice, which were concomitant with that SAMP8 mice at the same age displayed a significant synaptic loss and cognitive deficits. The up-regulation of ROCK expression and autophage in the hippocampus of SAMP8 were significantly reduced by ß-asarone, and prevents synaptic loss and improved cognitive function of the SAMP8 mice. ß-asarone decreased neuronophagia and lipofuscin in the hippocampus of SAMP8 mice, but did not reduce Aß42 levels and malondialdehyde levels and superoxide dismutase activities. Moreover, suppression of ROCK2 by siRNA significantly reduced the effects of ß-asarone on the autophage and synaptic proteins expression in PC12 cells damage induced by Aß1-40. Taken together, ß-asarone prevents autophagy and synaptic loss by reducing ROCK expression in SAMP8 mice.

AMIGO2 mRNA expression in hippocampal CA2 and CA3a.

AMIGO2, or amphoterin-induced gene and ORF (open reading frame) 2, belongs to the leucine-rich repeats and immunoglobulin superfamilies. The protein is a downstream target of calcium-dependent survival signals and, therefore, promotes neuronal survival. Here, we describe the mRNA distribution pattern of AMIGO2 throughout the mouse brain with special emphasis on the hippocampus. In the Ammon's horn, a detailed comparison between the subregional mRNA expression patterns of AMIGO2 and Pcp4 (Purkinje cell protein 4)--a known molecular marker of hippocampal CA2 (Cornu Ammonis 2)--revealed a prominent AMIGO2 mRNA expression level in both the CA2 and the CA3a (Cornu Ammonis 3a) subregion of the dorsal and ventral hippocampus. Since this CA2/CA3a region is particularly resistant to neuronal injury and neurotoxicity [Stanfield and Cowan (Brain Res 309(2):299­307 1984); Sloviter (J Comp Neurol 280(2):183­196 1989); Leranth and Ribak (Exp Brain Res 85(1):129­136 1991); Young and Dragunow (Exp Neurol 133(2):125­137 1995); Ochiishi et al. (Neurosci 93(3):955­967 1999)], we suggest that the expression pattern of AMIGO2 indeed fits with its involvement in neuroprotection.

Free chelatable zinc modulates the cholinergic function during hypobaric hypoxia-induced neuronal damage: an in vivo study.

The deregulation of cholinergic system and associated neuronal damage is thought to be a major contributor to the pathophysiologic sequelae of hypobaric hypoxia-induced memory impairment. Uniquely, the muscarinic receptors also play a role in zinc uptake. Despite the potential role of muscarinic receptors in the development of post hypoxia cognitive deficits, no studies to date have evaluated the mechanistic relationship between memory dysfunction and zinc homeostasis in brain. In the present study, we evaluated the effect of Ca(2)EDTA, a specific zinc chelator in the spatial working and associative memory deficits following hypobaric hypoxia. Our results demonstrate that accumulation of intracellular free chelatable zinc in the hippocampal CA3 pyramidal neurons is accompanied with neuronal loss and memory impairment in hypobaric hypoxic condition. Chelation of this free zinc with Ca(2)EDTA (1.25 mM/kg) ameliorated the hippocampus-dependent spatial as well as associative memory dysfunction and neuronal damage observed on exposure to hypobaric hypoxia. The zinc chelator significantly alleviated the downregulation in expression of choline acetyltransferase, muscarinic receptor 1 and 4, and acetylcholinesterase activity due to hypobaric hypoxia. Our data suggest that the free chelatable zinc released during hypobaric hypoxia might play a critical role in the neuronal damage and the alteration in cholinergic function associated with hypobaric hypoxia-induced memory impairment. We speculate that zinc chelation might be a potential therapy for hypobaric hypoxia-induced cognitive impairment.

Nipkow confocal imaging from deep brain tissues.

One of the problems in imaging from brain tissues is light-scattering. Thus, multiphoton laser scanning microscopy is widely used to optically access fluorescent signals located deeply in tissues. Here we report that Nipkow-type spinning-disk one-photon confocal microscopy, which embodies high temporal resolution and slow photobleaching, is also capable of imaging tissues to a depth of up to 150 µm. Using a Nipkow-disk microscope, we conducted functional multi-cell calcium imaging of CA3 neurons from in toto intact hippocampal preparations and astrocytes from in vivo neocortical layer 1. This novel application of Nipkow-disk microscopy expands the potential usefulness of this type of microscopy and will contribute to our understanding of natural neuronal microcircuitry.

In vitro measurements of extracellular L-glutamate level in region CA3 of mouse hippocampal slices under chemical stimulation.

The concentration level of extracellular L-glutamate released from region CA3 of mouse hippocampal slices under tetraethylammonium (TEA) chloride and KCl stimulation was measured with independent methods, i.e., a capillary-based enzyme sensor, a patch sensor, and an enzyme-based imaging method. The L-glutamate level was compared with those at regions CA1 and DG. It was found that the enhanced concentration level at CA3 by TEA stimulation is very similar to that at CA1, but it is much lower than that at DG. The order of the regional distribution of L-glutamate, i.e., DG > CA1 ≈ CA3, was the same as that obtained by K(+) stimulation. However, in the presence of an uptake inhibitor, DL-TBOA, KCl stimulation showed the strongest L-glutamate flux at CA1, while TEA stimulation exhibited the strongest flux at CA3. The usefulness of the present approach for knowing the extracellular L-glutamate level in acute hippocampal slices is discussed.

Hippocampal expression and distribution of CB1 receptors in the Amazonian rodent Proechimys: an animal model of resistance to epilepsy.

Proechimys, a rodent living in the Amazon region, has shown resistance to developing chronic epilepsy when submitted to different experimental models. Recently, many studies have attributed a potent anticonvulsant action to cannabinoid receptor CB1. This study investigated the distribution and expression of the CB1 receptor in the hippocampal formation of Proechimys using immunohistochemistry and Western blotting techniques. Results were compared with values obtained from adult Wistar rats. The immunoreactivity for CB1 was evident throughout the Ammon's horn and in the hilar region of both animal species. However, the distribution of these receptors was higher in the stratum lucidum of CA3 and in the hilar region of Proechimys. In addition, higher expression of CB1 receptors was observed in the Proechimys hippocampus. These data could explain, at least partially, the natural resistance of this animal species to developing spontaneous seizures following epileptogenic precipitating events.