Disruption of CaV2.3 channels in the brain of the 5xFAD mice

The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

There is controversy concerning whether orthodromic action potentials originate from the apical or basal dendrites of CA1 pyramidal cells in vivo. The participation of the dendrites in the initialization and propagation of population spikes in CA1 of urethan-anesthetized rats in vivo was studied using simultaneously recorded field potentials and current source density (CSD) analysis. CSD analysis revealed that the antidromic population spike, evoked by stimulation of the alveus, invaded in succession, the axon initial segment (stratum oriens), cell body and approximately 200 microm of the proximal apical dendrites. Excitation of the basal dendrites of CA1, following stimulation of CA3 stratum oriens, evoked an orthodromic spike that started near the cell body or initial segment and then propagated approximately 200 microm into the proximal apical dendrites. In contrast, the population spike that followed excitation of the apical dendrites of CA1 initiated at the proximal apical dendrites, 50-100 microm distal to the cell body layer, and then propagated centripetally to the cell body and the proximal basal dendrites. A late apical dendritic spike may arise in the mid-apical dendrites (250-300 microm from the cell layer) and propagated distally. The origin or the pattern of propagation of each population spike type was similar for near-threshold to supramaximal stimulus intensities. In summary, population spikes following apical dendritic and basal dendritic excitation in vivo appeared to originate from different locations. Apical dendritic excitation evoked a population spike that initiated in the proximal apical dendrites while basal dendritic excitation evoked a spike that started near the initial segment or cell body. An original finding of this study is the propagation of the population spike from basal to apical dendrites in vivo or vice versa. This backpropagation from one dendritic tree to the other may play an important role in the synaptic plasticity among a network of CA3 to CA1 neurons.

Event Abstract Back to Event Differential distribution of KCC2 along the axo-somato-dendritic axis of CA1 pyramidal cells Rita Baldi1*, Csaba Varga1 and Gabor Tamas1 1 Hungarian Academy of Sciences, University of Szeged, Hungary The neuron-specific K-Cl cotransporter (KCC2) plays a crucial role in adjusting intracellular chloride concentrations. Thus, the relative abundance of KCC2 along neuronal plasma membrane compartments can influence the reversal potential of chloride and GABAA receptor mediated synapses as shown for perisomatic and axo-axonic GABAergic synapses. We reported the lack of KCC2 in the axon compared to the soma in the cerebral cortex and extended the quantification of KCC2 to the dendrites of CA1 pyramidal cells with high resolution preembedding immunolocalization. Our results show that KCC2 has an uneven distribution profile along the axo-somato-dendritic axis of CA1 pyramidal neurons. Confirming our results on neocortical pyramidal cells, the axon initial segment contained ~ 5 % of somatic KCC2 cocentrations on CA1 pyramids. Compared to the proximal apical dendrites, the concentration of KCC2 decreased by ~ 37 % in the distal part of stratum radiatum (p<0.0001). Interestingly, the density of gold particles indicating KCC2 concentration was higher in lacunosum moleculare than in the distal part of stratum radiatum, but still ~ 22 % lower than in the proximal dendritic membranes (p<0.03). Basal dendrites showed KCC2 concentrations similar to proximal dendrites of str. radiatum and str. lacunosum moleculare and thus basal dendrites contained more KCC2 than dendrites in the distal str. radiatum. In conclusion, KCC2 is present in the soma as well as in the basal and apical dendritic shafts and spines of CA1 pyramidal cells. Moreover, the density of KCC2 seems to correlate with the relative abundance of GABAergic synapses innervating different segments of the apical dendrites in CA1 pyramidal cells. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Research on the cerebral cortex and related structures Citation: Baldi R, Varga C and Tamas G (2009). Differential distribution of KCC2 along the axo-somato-dendritic axis of CA1 pyramidal cells. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.187 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Mar 2009; Published Online: 06 Mar 2009. * Correspondence: Rita Baldi, Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary, baldi.rita@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rita Baldi Csaba Varga Gabor Tamas Google Rita Baldi Csaba Varga Gabor Tamas Google Scholar Rita Baldi Csaba Varga Gabor Tamas PubMed Rita Baldi Csaba Varga Gabor Tamas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Fast sodium action potentials are generated in the distal apical dendrites of rat hippocampal CA1 pyramidal cells

Alzheimer's Disease (AD) is the leading cause of dementia and has a higher incidence in post-menopausal women than men by poorly understood mechanisms. AD is associated with amyloid-β (Aβ) plaques in the brain, in particular pyroglutamatylated Aβ (Aβ-pE3), which forms the core of the plaques, promoting further Aβ deposition. Accumulation of Aβ on the vasculature can lead to vascular rarefication, ultimately contributing to AD dementia. However, it is unknown if these parameters are altered in 5x-FAD females, a mouse model of AD, which can account for increased incidence of dementia. Thus, we hypothesized that female 5x-FAD mice would have a greater accumulation of Aβ and Aβ-pE3 plaques, as well as vascular rarefication, than males. Data are means ± SEM. Using histochemical methods, we labeled brain slices from 3-months-old male (n=8) and female (n=7) 5x-FAD mice with thioflavin-T, a fluorescent dye that binds to Aβ, and an antibody specific to Aβ-pE3. Plaque numbers were counted in the cortex and hippocampus. Thioflavin-T plaque density, expressed as plaques per mm², was higher in 5x-FAD females compared to males in the cortex (69.8 ± 21.8 vs 26.8 ± 4.8; p<0.05, one-tailed Mann-Whitney test), but not in the hippocampus (59.8 ± 22.0 vs 17.7 ± 2.1; p=0.095, one-tailed Mann-Whitney test). Aβ-pE3 plaque density was significantly higher in 5x-FAD females compared to males in both cortex (133.4 ± 31.2 vs 63.4 ± 7.9; p<0.05, one-tailed Mann-Whitney test) and hippocampus (89.9 ± 23.3 vs 35.6 ± 7.3; p<0.05, one-tailed Mann-Whitney test). To examine the vasculature, brain slices of 5x-FAD males (n=8) and females (n=7) and wild-type (WT) males (n=6) and females (n=4) were immunolabeled for collagen IV. Capillaries were then counted and expressed as capillary number per field of view. No significant differences in capillary numbers were observed between male 5x-FAD and WT mice (29.3 ± 1.3 vs 31.3 ± 0.9; p>0.05, one-tailed Student's t-test) or female 5x-FAD and WT mice (29.8 ± 2.6 vs 31.5 ± 1.3; p>0.05, one-tailed Student's t-test). Co-labeling these same sections with an Aβ-pE3 antibody showed little parenchymal microvascular Aβ in 5x-FAD males or females, expressed as percent of collagen IV volume covered by Aβ-pE3 (2.0 ± 0.3 vs 2.3 ± 0.7; p>0.05, one-tailed Student's t-test), and no sex differences. Aβ plaques were more prevalent in pial vessels of 5x-FAD males (n=4) and females (n=5) without sex differences, expressed as percent of pial vessels that are Aβ-positive (47.0 ± 10.7 vs 41.4 ± 8.2, one-tailed Student's t-test). These data suggest that Aβ and Aβ-pE3 accumulation is larger in females than males 5x-FAD, without differences in vascular Aβ. Contrary to our hypothesis, at 3-months of age, 5x-FAD females seem to be protected from parenchymal Aβ accumulation. Ongoing studies in the laboratory are investigating the cognitive status of these mice.

Apical dendritic depolarizations and field interactions evoked by stimulation of afferent inputs to rat hippocampal ca1 pyramidal cells

BackgroundThe deposition of protein tau (Tau) and amyloid beta (Aβ) aggregates during Alzheimer disease (AD) is accompanied by other processes, mostly controlled by glial activation. Despite numerous attempts, there is little information on the role of these processes in the disease mechanism. Exact mapping and correlation of pathological changes would give information that could help reveal underlying causes for the onset of the disease. We set off to longitudinally investigate and correlate disease related processes in the brains of 5xFAD and P301S mice models.MethodNeuroinflammation and oxidative stress in P301S and 5xFAD transgenic (tg) mice were monitored longitudinally by PET‐CT imaging and compared to age‐matched healthy controls (WT). PET imaging using [18F]DPA‐714, [18F]FSPG, [18F]florbetaben, and [18F]EFAPEM1 was performed to assess levels of neuroinflammation, oxidative stress, Aβ load (5xFAD mice) and Tau aggregates (P301S) load, respectively. Uptake in the cortex (CTX) was presented as SUV or SUVr when a reference region was available. IHC staining of brain slices for Aβ, Tau deposits, neuroinflammation and oxidative stress is currently in progress.ResultPET‐CT studies showed increase of levels of Tau deposits at 8 month‐old P301S mice and Aβ levels at 6, 8 and 10 month‐old 5xFAD mice. For 5xFAD mice, [18F]DPA‐714 and [18F]FSPG uptake followed the trend of Aβ deposition, peaking at 8 months. Compared to WT mice, four‐fold increase in [18F]DPA‐714 (p&lt;0.0001) and three‐fold increase in [18F]FSPG (p=0.0019) SUV was observed, suggesting protein deposits‐induced increase in inflammation and oxidative stress. Surprisingly, neuroinflammation and oxidative stress did not follow the trend of Tau deposition in P301S mice.ConclusionLongitudinal deposition of Aβ and Tau was in accordance with reported ex vivo data for the two animal models. Neuroinflammation and oxidative stress correspond well with the levels of Aβ, longitudinally. The two processes also occur in P301S mice brain but correspond poorly with Tau deposition. These results suggest that either Aβ and Tau aggregates affect neuroinflammation and oxidative stress differently, or that neuroinflammation and oxidative stress are independent processes. Further studies are in progress to confirm the observed data.

Perinatal undernutrition attenuates field excitatory postsynaptic potentials and influences dendritic spine density and morphology in hippocampus of male rat offspring

Dual specificity protein phosphatase 6 (DUSP6) was recently identified as a key hub gene in a causal VGF gene network that regulates late-onset Alzheimer's disease (AD). Importantly, decreased DUSP6 levels are correlated with an increased clinical dementia rating (CDR) in human subjects, and DUSP6 levels are additionally decreased in the 5xFAD amyloidopathy mouse model. To investigate the role of DUSP6 in AD, we stereotactically injected AAV5-DUSP6 or AAV5-GFP (control) into the dorsal hippocampus (dHc) of both female and male 5xFAD or wild type mice, to induce overexpression of DUSP6 or GFP. Barnes maze testing indicated that DUSP6 overexpression in the dHc of 5xFAD mice improved memory deficits and was associated with reduced amyloid plaque load, Aß1-40 and Aß1-42 levels, and amyloid precursor protein processing enzyme BACE1, in male but not in female mice. Microglial activation, which was increased in 5xFAD mice, was significantly reduced by dHc DUSP6 overexpression in both males and females, as was the number of "microglial clusters," which correlated with reduced amyloid plaque size. Transcriptomic profiling of female 5xFAD hippocampus revealed upregulation of inflammatory and extracellular signal-regulated kinase pathways, while dHc DUSP6 overexpression in female 5xFAD mice downregulated a subset of genes in these pathways. Gene ontology analysis of DEGs (p < 0.05) identified a greater number of synaptic pathways that were regulated by DUSP6 overexpression in male compared to female 5xFAD. In summary, DUSP6 overexpression in dHc reduced amyloid deposition and memory deficits in male but not female 5xFAD mice, whereas reduced neuroinflammation and microglial activation were observed in both males and females, suggesting that DUSP6-induced reduction of microglial activation did not contribute to sex-dependent improvement in memory deficits. The sex-dependent regulation of synaptic pathways by DUSP6 overexpression, however, correlated with the improvement of spatial memory deficits in male but not female 5xFAD.

Women are disproportionately affected by Alzheimer's disease (AD), even after adjusting for age and socioeconomic status. Sex-based biological differences in immune response, including hormonal and X-linked immune genes, may contribute to differences in disease progression. However, we have limited understanding of the role of sex in modulating immune response and how these differences are affected by AD pathology. Here, we hypothesized that female and male mice would exhibit distinctive neural immune signaling in response to challenge with the pro-inflammatory stimulus lipopolysaccharides (LPS), and that these differences would be exacerbated in model of AD pathology compared to wild-type controls.Female and male six-month-old 5xFAD amyloid beta (Aβ) and wild type (WT) littermate mice were interperitoneally injected with LPS or saline vehicle once daily for 0,1, or 4 days (N=4). Luminex multiplexed ELISAs was used to quantify inflammatory cytokines, MAPK intracellular signaling phospho-proteins in cortical tissues.In the saline group, we observed no differences between females and males in either 5xFAD or WT mice. Additionally, in wild type mice, we observed no significant differences between females and males after either one or four daily injections of LPS. In contrast, in 5xFAD mice injected with LPS, we found significant differences between females and males after either one or four once daily injections. In particular, female 5xFAD mice after a single injection exhibited significantly increased profile (p=0.014, two tailed t-test) of phospho-proteins and cytokines, including MEK, cJUN, IL-13, IL-17, IL-10, and IL-1α compared to males. After four once-daily LPS injections, female 5xFAD mice exhibited a significantly different profile (p=0.016, two tailed t-test) consisting of increased phosphorylation of ATF2, Mek, cJUN, and HSP-27, together with decreased expression of multiple proinflammatory cytokines including IL-9, VEGF, MIP-1β compared to males.Our data reveal that although sex is not a significant determinant of neural immune response in WT mice, it significantly affects neural immune response to LPS challenge in 5xFAD mice. These data suggest distinctive immune processes in female vs male 5xFAD mice that are not present in healthy controls. Future work will consider the effects of Aβ pathology on these results.

Dendritic mechanisms have been implied to play a key role in the formation of epileptic discharges. However, presently only a handful of direct dendritic recordings have been reported during epileptic discharges. In this study, I performed simultaneous voltage recordings from the soma and apical dendrite of the same neuron combined with calcium-imaging measurements to investigate inter-ictal- and ictal-like epileptic discharges in dendrites of layer 5 pyramidal neurons. Neocortical brain slices treated with bicuculline (BCC) produced both isolated "inter-ictal" paroxysymal depolarization shift (PDS) responses and electrographic seizures. Concomitant voltage recordings from the soma and apical dendrite revealed that PDS responses developed in both the apical dendrites and soma. However, the two responses differed from one another. In apical dendrites, the PDS was significantly higher in amplitude and shorter in duration compared with the somatic PDS. The PDS response in dendrites had a peak amplitude of 68.9 +/- 2.2 (SD) mV, peak voltage value of 9.3 +/- 2.7 mV, and half-width of 203.8 +/- 38.4 ms. In contrast, the somatic PDS had a peak amplitude of 48.7 +/- 2.7 mV, peak voltage value of -11.9 +/- 3.1 mV, and half-width of 247.8 +/- 57.3 ms (P < 0.01, n = 18). In addition the apical dendritic PDS always preceded the somatic counterpart in all 18 neurons examined. Concomitant calcium-imaging measurements showed the PDS evoked large calcium influx into the entire dendritic tree including the apical tuft, basal, and oblique dendrites. The PDS evoked [Ca(2+)](i) were not uniform along the dendritic tree, being highest in the oblique dendrites (71.3 +/- 14.5 microM) and lowest at the distal tuft branches (9.3 +/- 0.7 microM). The PDS responses persisted after blockade of voltage-gated sodium channels by intracellular QX-314 but became narrower (by 69.6 +/- 9.7%) following intracellular administration of the voltage-gated calcium channel blocker D600. Electrographic seizures recorded in the soma and apical dendrites were composed of recurrent bursts. The initial bursts represented PDS responses. During the seizure the amplitude of bursts gradually attenuated and reached an average value of 26 +/- 13% of the initial ictal PDS burst. Double recordings during electrographic seizures revealed the initial one to four ictal bursts appeared first at the apical dendrite while later ictal bursts were always observed first at the soma. In conclusion, the results of this study show "inter-ictal" PDS responses originated in the apical dendritic tree, were partially mediated by voltage-gated calcium channels and spread throughout the dendritic tree including the fine tuft, basal, and oblique dendrites. During electrographic seizures the origin of epileptic bursts shifted from the apical dendritic tree to the soma-basal region.

Most hippocampal physiologic studies in Alzheimer disease (AD) have focused on memory circuit dysfunction in dorsal CA1. Ventral CA1 (vCA1) has received little attention, despite evidence supporting its increased susceptibility and its role in mood and affective symptoms, which play a prominent role in AD. We aimed to study the effect of amyloid pathology on circuit dysfunction in vCA1 using 5xFAD mice. We evaluated intrinsic excitability of vCA1 pyramidal neurons (PNs) as well as the impact of two distinct inputs onto their apical dendrite: entorhinal cortex (EC) input onto the distal compartment and CA3 inputs onto the proximal compartment. Acute ventral hippocampal slices were prepared from 12-14 month old 5xFAD and age-matched wild type (WT) mice. Whole cell patch clamp recordings were performed on vCA1 PNs. EC and CA3 input responses were elicited with extracellular stimulation of stratum lacunosum moleculare and stratum radiatum. We also addressed presynaptic function (paired pulse ratio), dendritic excitability (decay time constant of synaptic response), and intrinsic excitability (resting membrane potential, input resistance, sag ratio, firing rate). The EC stimulus-response curve reduced in 5xFAD vs. WT mice (n=6-10 PNs; p=0.0096) with a peak reduction of ∼33% (1.5mV vs. 1.0mV). In these same cells, the CA3 stimulus-response curve was also reduced (p<0.0001) but to a much larger extent of ∼67% at the peak (4.3mV vs. 1.4mV). The paired pulse ratio and response decay time constant were not affected with either input. Lastly, there were no statistically significant changes in intrinsic excitability measures. In 5xFAD mice, vCA1 PN circuit dysfunction is dominated by synaptic not intrinsic excitability changes. These changes are not in presynaptic function or postsynaptic ion currents, suggesting glutamate receptor changes or spine loss as alternative mechanisms. Moreover, these alterations are much more evident in CA3 versus EC input responses, perhaps driven by the inherent differences of the proximal and distal dendritic compartment. As such, with amyloidosis, vCA1 PN output shifts from being heavily dependent on CA3 input to being nearly equivalently dependent on EC input. These changes are important for understanding the circuit basis of mood and affect dysfunction in AD.

Gamma-aminobutyric acid type A receptor (GABA(A)-R) activation leads to depolarization of pyramidal cells during the first postnatal week and produces hyperpolarization from the second week. However, immunohistochemical evidence has suggested that during the second and third postnatal weeks the NKCC1 cotransporter relocates from the soma to the dendrites of CA3 pyramidal cells. We hypothesized that this leads to depolarizing responses in apical dendrites. Here we show that the activation of GABA(A)-R in the distal dendrites of CA3 pyramidal cells at P15 by restricted application of muscimol or synaptic activation by stimulation of interneurons in stratum radiatum (SR) causes depolarizing postsynaptic potentials (PSPs), which are blocked by NKCC1 cotransporter antagonists. By contrast, activation of proximal GABA(A)-R by muscimol application or by stimulation of interneurons in s. oriens (SO) leads to hyperpolarizing PSPs. Activation of the dentate gyrus (DG) in the presence of glutamatergic blockers evokes hyperpolarizing responses during the second postnatal week; however, the reversal potential of the DG-evoked inhibitory (I)PSPs is more depolarized than that of IPSPs evoked by activation of SO interneurons. Despite the shift of GABA action from depolarizing to hyperpolarizing, DG-evoked field potentials (f-PSPs) recorded in s. lucidum/radiatum (SL/R) do not change in polarity until the third week. Current source density analysis yielded results consistent with depolarizing actions of GABA in the dendritic compartment. Our data suggest that GABAergic input to apical dendrites of pyramidal cells of CA3 evokes depolarizing PSPs long after synaptic inhibition has become hyperpolarizing in the somata, in the axon initial segments and in basal dendrites.

Motor function deficits in the 12 month-old female 5xFAD mouse model of Alzheimer’s disease

Physiological variability in brain-derived neurotrophic factor expression predicts dendritic spine density in the mouse dentate gyrus