Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined, using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36 knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wildtype (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart brainstem preparation of the Cx36 KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36 KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters

Intravenously Injected Mesenchymal Stem Cells Penetrate the Brain and Treat Inflammation-Induced Brain Damage and Memory Impairment in Mice

Neuroinflammation is regarded as one of the pathogenic factors of Alzheimer disease (AD). Previously, we showed that mice regularly injected with bacterial lipopolysaccharide (LPS) possessed the AD-like symptoms like episodic memory decline, elevated amounts of amyloid beta (Aβ) peptide (1–42), and decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain. The use of mesenchymal stem cells (MSCs), which can differentiate into multiple cell types, including neurons, is an attractive idea of regenerative medicine, in particular, for neurodegenerative disorders like AD. In the present study, we aimed to investigate whether pathogenic effect of LPS on the brain and behavior of mice can be prevented or treated by injection of MSCs or MSC-produced soluble factors. Fluorescently-labeled MSCs, injected intravenously, were found in the brain blood vessels of LPS-treated mice. Mice co-injected with LPS and MSCs did not demonstrate episodic memory impairment, Aβ (1–42) accumulation, and nAChR decrease in the brain and brain mitochondria. Their mitochondria released less cytochrome c under the effect of Ca2+ compared to mitochondria of LPS-only-treated mice. Moreover, MSCs could reverse the pathogenic symptoms developed 3 weeks after LPS injection. Cultured MSCs produced IL-6 in response to LPS and MSCs effect in vivo was accompanied by additional stimulation of both micro- and macroglia. Xenogeneic (human) MSCs were almost as efficient as allogeneic (mouse) ones and regular injections of human MSC-conditioned medium also produced positive effect. These data allow suggesting MSCs as a potential therapeutic tool to cure neuroinflammation-related cognitive pathology

Neural Stem Cell Transplant-Induced Effect on Neurogenesis and Cognition in Alzheimer Tg2576 Mice Is Inhibited by Concomitant Treatment with Amyloid-Lowering or Cholinergic α7 Nicotinic Receptor Drugs.

Stimulating regeneration in the brain has the potential to rescue neuronal networks and counteract progressive pathological changes in Alzheimer's disease (AD). This study investigated whether drugs with different mechanisms of action could enhance neurogenesis and improve cognition in mice receiving human neural stem cell (hNSC) transplants. Six- to nine-month-old AD Tg2576 mice were treated for five weeks with the amyloid-modulatory and neurotrophic drug (+)-phenserine or with the partial α7 nicotinic receptor (nAChR) agonist JN403, combined with bilateral intrahippocampal hNSC transplantation. We observed improved spatial memory in hNSC-transplanted non-drug-treated Tg2576 mice but not in those receiving drugs, and this was accompanied by an increased number of Doublecortin- (DCX-) positive cells in the dentate gyrus, a surrogate marker for newly generated neurons. Treatment with (+)-phenserine did however improve graft survival in the hippocampus. An accumulation of α7 nAChR-expressing astrocytes was observed around the injection site, suggesting their involvement in repair and scarring processes. Interestingly, JN403 treatment decreased the number of α7 nAChR-expressing astrocytes, correlating with a reduction in the number of DCX-positive cells in the dentate gyrus. We conclude that transplanting hNSCs enhances endogenous neurogenesis and prevents further cognitive deterioration in Tg2576 mice, while simultaneous treatments with (+)-phenserine or JN403 result in countertherapeutic effects

α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid β42 accumulation in the mouse brain to impair memory.

Nicotinic acetylcholine receptors (nAChRs) expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ42), memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208) or injected with bacterial lipopolysaccharide (LPS) for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ40 and Aβ42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208) resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1) neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ42 accumulation and memory impairment in mice and (2) α7(1-208) nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease

Nicotinic Acetylcholine Receptor α9 and α10 Subunits Are Expressed in the Brain of Mice

The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are likely to be the evolutionary precursors to the entire cys-loop superfamily of ligand-gated ion channels, which includes acetylcholine, GABA, glycine and serotonin ionotropic receptors. nAChRs containing α9 and α10 subunits are found in the inner ear, dorsal root ganglia and many non-excitable tissues, but their expression in the central nervous system has not been definitely demonstrated. Here we show the presence of both α9 and α10 nAChR subunits in the mouse brain by RT-PCR and immunochemical approaches with a range of nAChR subunit-selective antibodies, which selectivity was demonstrated in the brain preparations of α7−/−, α9−/− and α10−/− mice. The α9 and α10 RNA transcripts were found in medulla oblongata (MO), cerebellum, midbrain (MB), thalamus and putamen (TP), somatosensory cortex (SC), frontal cortex (FC) and hippocampus. High α9-selective signal in ELISA was observed in the FC, SC, MO, TP and hippocampus and α10-selective signal was the highest in MO and FC. The α9 and α10 proteins were found in the brain mitochondria, while their presence on the plasma membrane has not been definitely confirmed The α7-, α9- and α10-selective antibodies stained mainly neurons and hypertrophied astrocytes, but not microglia. The α9- and α10-positive cells formed ordered structures or zones in cerebellum and superior olive (SO) and were randomly distributed among α7-positive cells in the FC; they were found in CA1, CA3 and CA4, but not in CA2 region of the hippocampus. The α9 and α10 subunits were up-regulated in α7−/− mice and both α7 and α9 subunits were down-regulated in α10−/− mice. We conclude that α9 and α10 nAChR subunits are expressed in distinct neurons of the mouse brain and in the brain mitochondria and are compensatory up-regulated in the absence of α7 subunits

The level of α7, β2 or β4 nAChR subunits in the brain sections of experimental mice studied by immunohistochemistry.

<p>Confocal microscopy images of the hippocampus CA1 and striatum of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with biotinylated α7-, β2- or β4-specific antibodies and developed with Extravidin-Cy3 (<i>red</i>). Cell nuclei are stained with DAPI (<i>blue</i>). Bar corresponds to 50μm, actual for each fragment of the panel.</p

The levels of different Aβ isoforms in the brain detergent lysates of experimental mice studied by Sandwich ELISA.

<p><b>A</b>—total Aβ₄₀ and Aβ₄₂, <b>B</b>—Aβ₄₀ and Aβ₄₂ bound to α7 nAChR in mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) compared to non-treated animals (Ctrl, n = 9) and to those “immunized” with complete Freund’s adjuvant (CFA, n = 5). The columns correspond to M±SE (n = 5); *—p<0.05; **—p<0.005; ***—p<0.0005 compared to Ctrl.</p

Episodic memory of experimental mice studied in the “Novel Object Recognition” test.

<p>Discrimination indexes calculated for mice immunized with 7(1–208) (n = 8) or injected with LPS (n = 5) compared to non-treated animals (n = 9) or those “immunized” with complete Freund’s adjuvant (CFA, n = 5). ***—p<0.0005 compared to non-treated mice (Ctrl).</p

The GFAP-positive astrocytes (A) and the number of nucleated cells (B) in the brain sections of experimental mice studied by immunohistochemistry.

<p><b>A</b>—Confocal microscopy images of the hippocampus CA1 (Hip), motor/somatosensory cortex (Crtx) or striatum (Str) of non-treated (Ctrl), α7(1–208)-immunized or LPS-injected mice stained with rabbit GFAP-specific antibody and developed with anti-rabbit Alexa 488 (<i>green</i>). Cell nuclei are stained with DAPI (<i>blue</i>). Bar corresponds to 50μm, actual for each fragment of the panel. <b>B</b>—The number of nucleated cells (DAPI-positive) in corresponding brain regions studied in all available sections (12 to 16 for each treatment for each region); *—p<0.05; **—p< 0.005.</p