Ein zerebraler Infarkt kann entweder durch eine Ischamie oder durch eine Blutung im Gehirn hervorgerufen werden. Verschiedene Mechanismen fuhren nach einer fokalen Ischamie zur sekundaren Ausbreitung des Infarktkernes, zur Schadigung in entfernten Hirnregionen sowie zur Bildung einer Glianarbe. Astrozyten gehoren zu den wichtigsten Komponenten der Glianarbe. Als Reaktion auf die Verletzung des Gehirns werden sie aktiviert, hypertrophieren, proliferieren und erhohen die Expression von Intermediarfilamenten wie dem sauren Gliafaserprotein (GFAP) und Nestin. Unter physiologischen Bedingungen zeigen Astrozyten ein hohes Mas an interzellularer Kopplung uber gap junctions. Dabei spielt Connexin 43 (Cx43) eine wichtige Rolle. Unter verschiedenen pahtophysiologischen Bedingungen und Erkrankungen des ZNS zeigen reaktive Astrozyten eine veranderte Expression von Cx43. Daher ergab sich die Frage, ob reaktive Astrozyten der Glianarbe nach einem fokalen ischamischen Infarkt auch eine solche Regulation erfahren und ob diese einer Dynamik unterliegt. Untersuchungen an Kulturen von neuronalen Progenitorzellen zeigten, dass Cx43 wahrend der Entwicklung die Proliferation, Differenzierung und Migration der Progenitorzellen beeinfluste. Es stellte sich daher die Frage, ob Cx43 auch die Proliferation und Migration der an der Glianarbe beteiligten Astrozyten beeinflusst. Nach einem Schlaganfall weisen auch entfernt vom Infarktkern liegende Gebiete funktionelle und strukturelle Veranderungen sowie eine variierte mRNA- und Proteinsynthese auf. Daher stellte sich die Frage, ob die Expression von Cx43 in entfernt vom Infarktkern liegenden Arealen Veranderungen zeigt. Um diesen Fragen nachzugehen, wurden fokale kortikale Infarkte im Hinterpfotenareal des somatosensorischen Kortex der Ratte mittels Photothrombose ausgelost. Die post-ischamische Expression von GFAP, Cx43 mRNA und Protein wurde am Tag 1, 3, 7, 14, 30, 60 und 120 in der Glianarbe, dem Hippokampus und dem somatosensorischen Kortex untersucht. In situ Hybridisierung und real time RT-PCR dienten zur Untersuchung der Cx43 mRNA Expression. Die Expression von GFAP und Cx43 Protein wurden mittels Immunhistochemie analysiert. Die Teilungsaktivitat der reaktiven Astrozyten wurde via Applikation von BrdU nach Induktion der Ischamie untersucht.
Der die ischamische Lasion flankierende, zerebrale Kortex zeigte eine gleichzeitige Heraufregulation von GFAP, Cx43 mRNA und Protein. Ab Tag 14 nach Induktion der Photothrombose war ein Band aus reaktiven Astrozyten am Boden der Lasion zu beobachten, in dem die Zellen eine Heraufregulation von Cx43 mRNA und Protein zeigten. Zur gleichen Zeit war ein Zellstrom zwischen dem lateralen Ventrikel und der Lasion sichtbar, in dem Zellen auch ein erhohtes Mas an GFAP, Cx43 mRNA und Protein Expression zeigten. Reaktive Astrozyten der Glianarbe, die die Lasion umhullten, besonders im Band am Boden der Lasion und im Strom zwischen dem lateralen Ventrikel und der Lasion, waren nachweislich proliferativ aktiv und exprimierten das Intermediarfilament Nestin. Im somatosensorischen Kortex der ipsilateralen Hemisphare wurde ein transienter Anstieg von Cx43 mRNA-positiven Zellen von einer Reduktion in der Immunreaktivitat des zugehorigen Proteins begleitet. Untersuchungen der einzelnen hippokampalen Strata zeigten ausschlieslich im Stratum Oriens direkt unter der Lasion eine erhohte Anzahl von Cx43 mRNA-positiven Astrozyten, die gleichzeitig eine Erhohung der Immunreaktivitat fur Cx43 Protein und GFAP aufwiesen. Die vorgelegte Arbeit zeigt, dass eine fokale kortikale Ischamie die Expression von Cx43 mRNA und Protein beeinflusst. Sie liefert erste Hinweise dafur, dass Cx43 an der Bildung der Glianarbe, besonders der Proliferation und Migration der reaktiven Astrozyten, beteiligt ist. Die erhohte Anzahl von Cx43 mRNA-positiven Zellen und die gesteigerte Expression von GFAP und Cx43 Protein im Stratum Oriens des Hippokampus lassen eine Beteiligung dieses Gebietes an der Glianarbenbildung vermuten. Die beobachtete Verringerung der Immunreaktivitat fur Cx43 im somatosensorischen Kortex lasst auf eine temporar gestorte interzellulare Kommunikation schliesen.
Key points Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d ‐tubocurarine and α‐bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium‐3 also enhanced stretch‐evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine‐tune proprioceptive afferent sensitivity. Abstract Muscle spindles are complex stretch‐sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ‐motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ‐motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp‐and‐hold stretches and sinusoidal vibrations before and after the addition of the competitive and non‐competitive AChR blockers d ‐tubocurarine and α‐bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp‐and‐hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high‐affinity choline transporter antagonist hemicholinium‐3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α‐bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine‐tuning muscle spindle sensitivity.
Animal models of focal ischemic infarcts reveal an impaired GABAergic (gamma-aminobutyric acid) neurotransmission. GABA, the main inhibitory neurotransmitter, is primarily taken up by specific sodium-dependent transporters. As these transporters play a crucial role in maintaining levels of GABA concentration, they may be functionally involved in ischemic processes. We investigated whether the mRNA and protein expression of GAT-1, the dominant neuronal GABA transporter, is altered after cortical infarct induced by photothrombosis in Wistar rats. In situ hybridization was performed to analyze GAT-1 mRNA-positive cells in cortical brain regions and the hippocampus. The lesion dramatically raised the number of GABA transporter mRNA-expressing cells in all investigated cortical regions. Double-labeling studies with a general neuronal marker and a marker for astrocytes revealed that cells expressing GAT-1 mRNA after photothrombosis are neurons. The mRNA expression pattern of all hippocampal subfields remained unchanged. In contrast, cortical GAT-1 protein density was only slightly affected and surprisingly in the opposite way. In the primary and secondary somatosensory cortex, density values were significantly reduced. Immunoreactivity was not altered in all investigated hippocampal areas. We found a marked discordance between the increased number of cells expressing GAT-1 mRNA in the cortex and the reduced tissue GAT-1 protein content. Focal brain ischemia obviously triggers mechanisms that interfere with GAT-1 transcriptional regulation and protein synthesis or turnover.
In early development, an excess of neurons is generated, of which later about half will be lost by cell death due to a limited supply of trophic support by their respective target areas. However, some of the neurons die when their axons have not yet reached their target, thus suggesting that additional causes of developmental cell death exist. Semaphorin 3A (Sema3A), in addition to its function as a guidance cue and mediator of timing and fasciculation of motor and sensory axon outgrowth, can also induce death of sensory neurons in vitro. However, it is unknown whether Neuropilin-1 (Npn-1), its binding receptor in axon guidance, also mediates the death-inducing activity. We show here that abolished Sema3A-Npn-1 signaling does not influence the cell death patterns of motor or sensory neurons in mouse during the developmental wave of programmed cell death. The number of motor and sensory neurons was unchanged at embryonic day 15.5 when this wave is concluded. Interestingly, the defasciculation of early motor and sensory projections that is observed in the absence of Sema3A or Npn-1 persists to postnatal stages. Thus, Sema3A-Npn-1 signaling plays an important role in the guidance and fasciculation of motor and sensory axons but does not contribute to the developmental elimination of these neurons.
Engrailed-1 (En1) is expressed in the ventral ectoderm of the developing limb where it plays an instructive role in the dorsal-ventral patterning of the forelimb. Besides its well-described role as a transcription factor in regulating gene expression through its DNA-binding domain, En1 may also be secreted to form an extracellular gradient, and directly impact on the formation of the retinotectal map. We show here that absence of En1 causes mispatterning of the forelimb and thus defects in the dorsal-ventral pathfinding choice of motor axons in vivo. In addition, En1 but not En2 also has a direct and specific repulsive effect on motor axons of the lateral aspect of the lateral motor column (LMC) but not on medial LMC projections. Moreover, an ectopic dorsal source of En1 pushes lateral LMC axons to the ventral limb in vivo. Thus, En1 controls the establishment of limb innervation through two distinct molecular mechanisms.
