Beneficial Effects of Estrogen in a Mouse Model of Cerebrovascular Insufficiency

BACKGROUND: The M(5) muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M(5) muscarinic acetylcholine knockout mice (M5R(-/-) mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain. METHODOLOGY/PRINCIPAL FINDINGS: We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R(-/-) mice. Here we show that female M5R(-/-) mice did not show the reduction in vascular area observed in male M5R(-/-) mice. However, ovariectomized female M5R(-/-) mice displayed phenotypic changes similar to male M5R(-/-) mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17beta-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R(-/-) mice treated with E2. Treatment with E2 also improved the performance of male M5R(-/-) mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R(-/-) mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that M5R(-/-) mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction

Distribution and Structure of Synapses on Medial Vestibular Nuclear Neurons Targeted by Cerebellar Flocculus Purkinje Cells and Vestibular Nerve in Mice: Light and Electron Microscopy Studies

<div><p>Adaptations of vestibulo-ocular and optokinetic response eye movements have been studied as an experimental model of cerebellum-dependent motor learning. Several previous physiological and pharmacological studies have consistently suggested that the cerebellar flocculus (FL) Purkinje cells (P-cells) and the medial vestibular nucleus (MVN) neurons targeted by FL (FL-targeted MVN neurons) may respectively maintain the memory traces of short- and long-term adaptation. To study the basic structures of the FL-MVN synapses by light microscopy (LM) and electron microscopy (EM), we injected green florescence protein (GFP)-expressing lentivirus into FL to anterogradely label the FL P-cell axons in C57BL/6J mice. The FL P-cell axonal boutons were distributed in the magnocellular MVN and in the border region of parvocellular MVN and prepositus hypoglossi (PrH). In the magnocellular MVN, the FL-P cell axons mainly terminated on somata and proximal dendrites. On the other hand, in the parvocellular MVN/PrH, the FL P-cell axonal synaptic boutons mainly terminated on the relatively small-diameter (< 1 μm) distal dendrites of MVN neurons, forming symmetrical synapses. The majority of such parvocellular MVN/PrH neurons were determined to be glutamatergic by immunocytochemistry and in-situ hybridization of GFP expressing transgenic mice. To further examine the spatial relationship between the synapses of FL P-cells and those of vestibular nerve on the neurons of the parvocellular MVN/PrH, we added injections of biotinylated dextran amine into the semicircular canal and anterogradely labeled vestibular nerve axons in some mice. The MVN dendrites receiving the FL P-cell axonal synaptic boutons often closely apposed vestibular nerve synaptic boutons in both LM and EM studies. Such a partial overlap of synaptic boutons of FL P-cell axons with those of vestibular nerve axons in the distal dendrites of MVN neurons suggests that inhibitory synapses of FL P-cells may influence the function of neighboring excitatory synapses of vestibular nerve in the parvocellular MVN/PrH neurons.</p></div

Axodendritic synapses between FL P-cells and parvocellular MVN/PrH neurons.

<p><b>A1</b>–<b>A4</b>, <b>B1</b>–<b>B4</b>, Photographs of two examples of EM serial sections (interval, 0.14 μm) for FL P-cell axonal boutons forming symmetrical synapses on parvocellular MVN/PrH neurons. Edges of synapses were shown by arrowheads. <b>C</b>, Distribution of the mean diameters for post-synaptic dendrites. Note that FL P-cell axonal boutons formed synapses on relatively small diameter dendrites (mean, 0.73 μm; n = 40 dendrites from three mice). den, dendrite of MVN/PrH neuron; Pb, P-cell axonal bouton. Scale bars, 0.2 μm.</p

Labeled Purkinje cell axonal boutons after FL lentivirus injections.

<p>Labeled Purkinje cell axonal boutons after FL lentivirus injections.</p

Neurons that receive FL P-cell projections on their distal dendrites in the parvocellular MVN/PrH are predominantly glutamatergic.

<p><b>A</b> and <b>B</b>, Representative images of MVN/PrH neurons of Thy1-GFP M-line transgenic mouse that were stained with anti-GFP (green) and anti-PKCγ (red) antibodies. <b>C</b>, <b>D</b>, and <b>E</b>, Images of coronal sections of MVN/PrH stained with either one of three antibodies against neurotransmitters (anti-Glu, anti-Gly, and anti-GABA). The majority of GFP-labeled neurons were stained positive for anti-Glu antibody (<b>C</b>), and some of the remaining neurons were positive for anti-Gly antibody (<b>D</b>), but not positive for anti-GABA antibody (<b>E</b>). Scale bars, 200 μm (<b>A</b>), 100 μm (<b>B</b>), and 50 μm (<b>C</b>–<b>E</b>). GABA, γ-aminobutyric acid; Glu, glutamate; Gly, glycine.</p

Anterogradely labeled FL P-cell axons in the medial vestibular nucleus (MVN) and prepositus hypoglossi (PrH) in case (#) 94R.

<p><b>A</b>, GFP expression in FL at 3 weeks after lentiviral injection. Sections were stained with anti-GFP (green) and neuronal marker anti-NeuN (red) antibodies. <b>B</b>, GFP was preferentially expressed in FL P-cells. <b>C</b>, GFP-labeled axons of FL P-cells in the ventromedial MVN. FL, flocculus; icp, inferior cerebellar peduncle; MVMC, magnocellular MVN; MVPC, parvocellular MVN; P-cell, Purkinje cell; PFL, paraflocculus; PrH, prepositus hypoglossi nucleus; 4V, fourth ventricle. Scale bars, 200 μm (<b>A</b> and <b>C</b>) and 50 μm (<b>B</b>).</p

Distribution of vestibular nerve axonal boutons and FL P-cell axonal boutons in the parvocellular MVN/PrH.

<p><b>A</b>, Low magnification image of MVN/PrH showing two distinct types of synaptic inputs. BDA was injected into the horizontal semicircular canal after the lentivirus injection into FL. FL P-cell axons and vestibular nerve axons were visualized by anti-GFP antibody (green) and Alexa 594 streptavidine (red), respectively. Note that the distribution of FL P-cell axons overlapped partially with that of the vestibular nerve axons in MVN/PrH. <b>B</b>, High magnification confocal image of the region marked by square in A. Some of the FL P-cell axonal boutons were closely located to those of the vestibular nerve axonal boutons (arrowheads). <b>C1</b>–<b>C3</b>, <b>D1</b>–<b>D3</b>, Two examples of serial EM photographs (interval, 0.14 μm in <b>C1</b>–<b>C3</b>, 0.21 μm in <b>D1</b>–<b>D3</b>) of MVN/PrH neuronal dendrites. GFP (FL P-cell axons) was detected by DAB reaction products, and BDA (vestibular nerve axons) was detected by silver-enhanced gold particles. Note that the FL P-cell axons formed symmetrical synapses (arrowheads), whereas the vestibular nerve axons formed asymmetrical synapses (arrows) on the same MVN neuronal dendrites. den, dendrite of MVN/PrH neuron; MVN, medial vestibular nucleus; Pb, P-cell axonal bouton; PrH, prepositus hypoglossi nucleus; Vb, vestibular nerve axonal bouton; 4V, fourth ventricle. Scale bars, 200 μm (<b>A</b>), 20 μm (<b>B</b>), and 0.2 μm (<b>C1</b>–<b>C3</b>, <b>D1</b>–<b>D3</b>).</p

A putative scheme for the two different types of FL-targeted HVOR/HOKR-relaying neurons in mouse MVN.

<p>In magnocellular MVN, FL P-cell axons make synaptic contacts on the proximal dendrites and somata of neurons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.ref034" target="_blank">34</a>], Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.g003" target="_blank">3</a> in the present study), which are presumably inhibitory glycinergic neurons projecting to the ipsilateral abducens nucleus [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.ref034" target="_blank">34</a>]. By contrast, in parvocelliar MVN/PrH, FL P-cells make synaptic contacts on the distal dendrites of neurons (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.g005" target="_blank">5</a>), which are presumably excitatory glutamatergic (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.g004" target="_blank">Fig 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.s002" target="_blank">S2 Fig</a>) neurons projecting to the ipsilateral oculmotor nucleus [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.ref053" target="_blank">53</a>]. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164037#pone.0164037.s004" target="_blank">S1 Table</a>. ABN, abducens nucleus; Glu, glutamatergic (excitatory) neuron; Gly, glycinergic (inhibitory) neuron; OMN, oculomotor nucleus.</p

Two distinct types of FL P-cell axonal boutons observed in MVN/PrH (case (#) 94R).

<p><b>A</b>–<b>C</b>, Merged images of GFP-fluorescent FL P-cell axonal boutons (green) and anti-NeuN-labelled somata (red) of MVN neurons. In <b>A</b>, anterogradely labeled FL P-cell axonal boutons were observed in the magnocellular MVN, in which the somata and proximal dendrites of MVN neurons were surrounded by a small group of axons, showing basket like structures (arrowheads in <b>B</b> and <b>C</b>). <b>D</b>–<b>F</b>, Similar to <b>A</b>–<b>C</b>, but for those observed in the parvocellular MVN/PrH, in which axonal boutons showing <i>en passant</i> structures were dominantly observed (arrows in <b>E</b> and <b>F</b>). MVMC, mgnocellular NVN; MVPC, parvocellular MVN; PrH, prepositus hypoglossi nucleus; 4V, fourth ventricle. Scale bars, 200 μm (<b>A</b> and <b>D</b>) and 20 μm (<b>B</b>, <b>C</b>, <b>E</b> and <b>F</b>).</p