Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

Functions of the SLC36 transporter Pathetic in growth control of Drosophila sensory neurons

Thesis (Ph.D.)--University of Washington, 2016-08

Simulating the formation of a sigmoidal flux rope in AR10977 from SOHO/MDI magnetograms

The modeling technique of Mackay et al. is applied to simulate the coronal magnetic field of NOAA active region AR10977 over a seven day period (2007 December 2-10). The simulation is driven with a sequence of line-of-sight component magnetograms from SOHO/MDI and evolves the coronal magnetic field though a continuous series of non-linear force-free states. Upon comparison with Hinode/XRT observations, results show that the simulation reproduces many features of the active region's evolution. In particular, it describes the formation of a flux rope across the polarity inversion line during flux cancellation. The flux rope forms at the same location as an observed X-ray sigmoid. After five days of evolution, the free magnetic energy contained within the flux rope was found to be 3.9 × 1030 erg. This value is more than sufficient to account for the B1.4 GOES flare observed from the active region on 2007 December 7. At the time of the observed eruption, the flux rope was found to contain 20% of the active region flux. We conclude that the modeling technique proposed in Mackay et al.—which directly uses observed magnetograms to energize the coronal field—is a viable method to simulate the evolution of the coronal magnetic field.

Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination

Clustering through post inhibitory rebound in synaptically coupled neurons

Post inhibitory rebound is a nonlinear phenomenon present in a variety of nerve cells. Following a period of hyper-polarization this effect allows a neuron to fire a spike or packet of spikes before returning to rest. It is an important mechanism underlying central pattern generation for heartbeat, swimming and other motor patterns in many neuronal systems. In this paper we consider how networks of neurons, which do not intrinsically oscillate, may make use of inhibitory synaptic connections to generate large scale coherent rhythms in the form of cluster states. We distinguish between two cases i) where the rebound mechanism is due to anode break excitation and ii) where rebound is due to a slow T-type calcium current. In the former case we use a geometric analysis of a McKean type model to obtain expressions for the number of clusters in terms of the speed and strength of synaptic coupling. Results are found to be in good qualitative agreement with numerical simulations of the more detailed Hodgkin-Huxley model. In the second case we consider a particular firing rate model of a neuron with a slow calcium current that admits to an exact analysis. Once again existence regions for cluster states are explicitly calculated. Both mechanisms are shown to prefer globally synchronous states for slow synapses as long as the strength of coupling is sufficiently large. With a decrease in the duration of synaptic inhibition both systems are found to break into clusters. A major difference between the two mechanisms for cluster generation is that anode break excitation can support clusters with several groups, whilst slow T-type calcium currents predominantly give rise to clusters of just two (anti-synchronous) populations.

Low doses of a neonicotinoid insecticide modify pheromone response thresholds of central but not peripheral olfactory neurons in a pest insect

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Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

Neural fields with sigmoidal firing rates: approximate solutions

Many tissue level models of neural networks are written in the language of nonlinear integro-differential equations. Analytical solutions have only been obtained for the special case that the nonlinearity is a Heaviside function. Thus the pursuit of even approximate solutions to such models is of interest to the broad mathematical neuroscience community. Here we develop one such scheme, for stationary and travelling wave solutions, that can deal with a certain class of smoothed Heaviside functions. The distribution that smoothes the Heaviside is viewed as a fundamental object, and all expressions describing the scheme are constructed in terms of integrals over this distribution. The comparison of our scheme and results from direct numerical simulations is used to highlight the very good levels of approximation that can be achieved by iterating the process only a small number of times.

Regulation of electrical transmission by protein kinase C in Aplysia bag cell neurons

Electrical synapses are composed of gap junctions, made from paired hemi-channels that allow for the transfer of current from one neuron to another. Gap junctions mediate electrical transmission in neurons, where they synchronize spiking and promote rapid transmission, thereby influencing the coordination, pattern, and frequency of firing. In the marine snail, Aplysia calfornica, two clusters of neuroendocrine bag cell neurons use electrical synapses to synchronize a 30-min burst of action potentials, known as the afterdischarge, which releases egg-laying hormone and induces reproduction. In culture, paired bag cell neurons present a junctional conductance that is non-rectifying and largely voltage-independent. During the afterdischarge, PKC is activated, which is known to increase voltage-gated Ca2+ current; yet, little is understood as to how this pathway impacts electrical transmission. The transfer of presynaptic spike-like waveforms (generated in voltage-clamp) to the postsynaptic cell (measured in current-clamp) was monitored with or without PKC activation. It was found that pretreatment with the PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced junctional conductance between bag cell neurons. Furthermore, in control, presynaptic action potential waveforms mainly evoked postsynaptic electrotonic potentials at both -60 and -40 mV. However, with PKC activation the presynaptic stimulus consistently elicited postsynaptic action potentials from resting potentials of -40 mV, and would occasionally result in firing from repetitive input at -60 mV. Moreover, to assess whether this enhanced electrical transmission genuinely reflects a greater junctional conductance or a change in postsynaptic responsiveness, a fast-phase junctional-like current was applied to single bag cell neurons. Neurons in PMA always fired action potentials in response to current injection as opposed to control, which were less likely to spike. This outcome did not change when the junctional-like current was artificially enhanced in control conditions. Also, in response to fast- and slow-phase electrotonic potential (ETP) waveforms, Ca2+ current was markedly larger in single PMA-treated neurons. These findings suggest that PKC activation may contribute to afterdischarge fidelity by recruiting postsynaptic Ca2+ current to promote synchronous network firing. Finally, Aplysia gap junction genes (innexins) were transfected into mouse N2A cells and characterized. This revealed a biophysical and pharmacological profile similar to native gap junctions.

Neurodegeneration and the m-AAA protease: pathogenic cascades in neurons and myelinating cells

The m-AAA protease is a hexameric complex involved in processing of specific substrates and turnover of misfolded polypeptides in the mitochondrial inner membrane. In humans, the m-AAA protease is composed of AFG3L2 and paraplegin. Mutations in AFG3L2 have been implicated in dominant spinocerebellar ataxia (SCA28) and recessive spastic ataxia-neuropathy syndrome (SPAX5). Mutations of SPG7, encoding paraplegin, are linked to hereditary spastic paraplegia. In the mouse, a third subunit AFG3L1 is expressed. Various mouse models recapitulate the phenotype of these neurodegenerative disorders, however, the pathogenic mechanism of neurodegeneration is not completely understood. Here, we studied several mouse models and focused on cell-autonomous role of the m-AAA protease in neurons and myelinating cells. We show that lack of Afg3l2 triggers mitochondrial fragmentation and swelling, tau hyperphosphorylation and pathology in Afg3l2 full-body and forebrain neuron-specific knockout mice. Moreover, deletion of Afg3l2 in adult myelinating cells causes early-onset mitochondrial abnormalities as in the neurons, but the survival of these cells is not affected, which is a contrast to early neuronal death. Despite the fact that myelinating cells have been previously shown to survive respiratory deficiency by glycolysis, total ablation of the m-AAA protease by deleting Afg3l2 in an Afg3l1 null background (DKO), leads to myelinating cell demise and subsequently progressive axonal demyelination. Interestingly, DKO mice show premature hair greying due to loss of melanoblasts. Together, our data demonstrate cell-autonomous survival thresholds to m-AAA protease deficiency, and an essential role of the m-AAA protease to prevent cell death independent from mitochondrial dynamics and the oxidative capacity of the cell. Thus, our findings provide novel insights to the pathogenesis of diseases linked to m-AAA protease deficiency, and also establish valuable mitochondrial dysfunctional mouse models to study other neurodegenerative diseases, such as tauopathies and demyelinating diseases.

Ikkε Is Key To Induction Of Insulin Resistance In The Hypothalamus, And Its Inhibition Reverses Obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

The Intrahippocampal Infusion Of Crotamine From Crotalus Durissus Terrificus Venom Enhances Memory Persistence In Rats.

Previous research has shown that crotamine, a toxin isolated from the venom of Crotalus durissus terrificus, induces the release of acetylcholine and dopamine in the central nervous system of rats. Particularly, these neurotransmitters are important modulators of memory processes. Therefore, in this study we investigated the effects of crotamine infusion on persistence of memory in rats. We verified that the intrahippocampal infusion of crotamine (1 μg/μl; 1 μl/side) improved the persistence of object recognition and aversive memory. By other side, the intrahippocampal infusion of the toxin did not alter locomotor and exploratory activities, anxiety or pain threshold. These results demonstrate a future prospect of using crotamine as potential pharmacological tool to treat diseases involving memory impairment, although it is still necessary more researches to better elucidate the crotamine effects on hippocampus and memory.

Epilepsies Associated With Hippocampal Sclerosis.

Hippocampal sclerosis (HS) is considered the most frequent neuropathological finding in patients with mesial temporal lobe epilepsy (MTLE). Hippocampal specimens of pharmacoresistant MTLE patients that underwent epilepsy surgery for seizure control reveal the characteristic pattern of segmental neuronal cell loss and concomitant astrogliosis. However, classification issues of hippocampal lesion patterns have been a matter of intense debate. International consensus classification has only recently provided significant progress for comparisons of neurosurgical and clinic-pathological series between different centers. The respective four-tiered classification system of the International League Against Epilepsy subdivides HS into three types and includes a term of gliosis only, no-HS. Future studies will be necessary to investigate whether each of these subtypes of HS may be related to different etiological factors or with postoperative memory and seizure outcome. Molecular studies have provided potential deeper insights into the pathogenesis of HS and MTLE on the basis of epilepsy-surgical hippocampal specimens and corresponding animal models. These include channelopathies, activation of NMDA receptors, and other conditions related to Ca(2+) influx into neurons, the imbalance of Ca(2+)-binding proteins, acquired channelopathies that increase neuronal excitability, paraneoplastic and non-paraneoplastic inflammatory events, and epigenetic regulation promoting or facilitating hippocampal epileptogenesis. Genetic predisposition for HS is clearly suggested by the high incidence of family history in patients with HS, and by familial MTLE with HS. So far, it is clear that HS is multifactorial and there is no individual pathogenic factor either necessary or sufficient to generate this intriguing histopathological condition. The obvious variety of pathogenetic combinations underlying HS may explain the multitude of clinical presentations, different responses to clinical and surgical treatment. We believe that the stratification of neuropathological patterns can help to characterize specific clinic-pathological entities and predict the postsurgical seizure control in an improved fashion.

Metabolic Memory Of ß-cells Controls Insulin Secretion And Is Mediated By Camkii.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of metabolic memory, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca(2+)-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.

Fractalkine (cx3cl1) Is Involved In The Early Activation Of Hypothalamic Inflammation In Experimental Obesity.

Hypothalamic inflammation is a common feature of experimental obesity. Dietary fats are important triggers of this process, inducing the activation of toll-like receptor-4 (TLR4) signaling and endoplasmic reticulum stress. Microglia cells, which are the cellular components of the innate immune system in the brain, are expected to play a role in the early activation of diet-induced hypothalamic inflammation. Here, we use bone marrow transplants to generate mice chimeras that express a functional TLR4 in the entire body except in bone marrow-derived cells or only in bone marrow-derived cells. We show that a functional TLR4 in bone marrow-derived cells is required for the complete expression of the diet-induced obese phenotype and for the perpetuation of inflammation in the hypothalamus. In an obesity-prone mouse strain, the chemokine CX3CL1 (fractalkine) is rapidly induced in the neurons of the hypothalamus after the introduction of a high-fat diet. The inhibition of hypothalamic fractalkine reduces diet-induced hypothalamic inflammation and the recruitment of bone marrow-derived monocytic cells to the hypothalamus; in addition, this inhibition reduces obesity and protects against diet-induced glucose intolerance. Thus, fractalkine is an important player in the early induction of diet-induced hypothalamic inflammation, and its inhibition impairs the induction of the obese and glucose intolerance phenotypes.