Neuronal repair. Asynchronous therapy restores motor control by rewiring of the rat corticospinal tract after stroke.

The brain exhibits limited capacity for spontaneous restoration of lost motor functions after stroke. Rehabilitation is the prevailing clinical approach to augment functional recovery, but the scientific basis is poorly understood. Here, we show nearly full recovery of skilled forelimb functions in rats with large strokes when a growth-promoting immunotherapy against a neurite growth-inhibitory protein was applied to boost the sprouting of new fibers, before stabilizing the newly formed circuits by intensive training. In contrast, early high-intensity training during the growth phase destroyed the effect and led to aberrant fiber patterns. Pharmacogenetic experiments identified a subset of corticospinal fibers originating in the intact half of the forebrain, side-switching in the spinal cord to newly innervate the impaired limb and restore skilled motor function.

Detection of anaphylatoxin receptors on CD83+ dendritic cells derived from human skin.

Dendritic cells (DC) are recruited to sites of inflammation for the initiation of immune responses. As the anaphylatoxins C5a and C3a are important mediators of inflammation, we investigated the expression of their receptors (C3aR and C5aR) on human DC. DC were isolated from human skin or generated from purified blood monocytes and were identified by their expression of CD1a or CD83. Freshly isolated or cultured dermal CD1a+ and CD83+ DC bound anti-C5aR and anti-C3aR monoclonal antibodies (mAbs), as detected by flow cytometry. C5a induced calcium fluxes in dermal CD1a+ and CD83+ DC, which could be inhibited by C17/5, an anti-C5a mAb. C3a did not induce calcium fluxes in these cells. Anaphylatoxin receptor expression was down-regulated on dermal DC by adding tumour necrosis factor-alpha (TNF-alpha) to the culture medium. On CD1a+ CD83- cells generated from isolated blood monocytes by culture with 6.25 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 125 U/ml of interleukin-4 (IL-4), expression of both C5aR and C3aR was observed. In these cells, both C5a and C3a induced calcium fluxes. After addition of TNF-alpha to the culture medium, the majority of the CD1a+ cells expressed CD83+. These cells - expressing a phenotype of 'mature DC' - down-regulated the expression of the anaphylatoxin receptors and lost their reactivity to the respective ligands. Our results demonstrate the expression of the anaphylatoxin receptors C5aR and C3aR on human skin-derived DC and blood-derived cells expressing the DC-associated membrane molecule, CD1a. Furthermore, the expression of anaphylatoxin receptors on CD83+ dermal DC is indicative of an intermediate stage of maturation of these cells, which was not observed on in vitro-differentiated CD83+ cells.

Neuronal plasticity and formation of new synaptic contacts follow pyramidal lesions and neutralization of Nogo-A: a light and electron microscopic study in the pontine nuclei of adult rats.

Regeneration and compensatory sprouting are limited after lesions in the mature mammalian central nervous system in contrast to the developing central nervous system (CNS). After neutralization of the growth inhibitor Nogo-A, however, massive sprouting and rearrangements of fiber connections occurred after unilateral pyramidal tract lesions in adult rats: Corticofugal fibers from the lesioned side crossed the midline of the brainstem and innervated the contralateral basilar pontine nuclei. To determine whether these newly sprouted fibers formed synaptic contacts, we analyzed the corticofugal fibers in the basilar pontine nuclei contralateral to the lesion by light and electron microscopy 2 weeks after pyramidotomy and treatment with the Nogo-A-inhibiting monoclonal antibody IN-1 (mAb IN-1). The mAb IN-1, but not a control antibody, led to structural changes in the basilar pons ipsilateral and contralateral to the lesion site. Fibers sprouted across the pontine midline and terminated topographically. They established asymmetric synaptic contacts with the characteristics of normal corticopontine terminals. These results show that adult CNS fibers are able to sprout and to form new synaptic contacts after a lesion when a growth-permissive microenvironment is provided.

GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization.

GABAB (gamma-aminobutyric acid)-receptors have been implicated in central nervous system (CNS) functions, e.g. cognition and pain perception, and dysfunctions including spasticity and absence epilepsy. To permit an analysis of the two known GABAB-receptor splice variants GABAB-R1a (GB1a) and GABAB-R1b (GB1b), their distribution pattern has been differentiated in the rat brain, using Western blotting and immunohistochemistry with isoform-specific antisera. During postnatal maturation, the expression of the two splice variants was differentially regulated with GB1a being preponderant at birth. In adult brain, GB1b-immunoreactivity (-IR) was predominant, and the two isoforms largely accounted for the pattern of GABAB-receptor binding sites in the brain. Receptor heterogeneity was pronounced in the hippocampus, where both isoforms occurred in CA1, but only GB1b in CA3. Similarly, in the cerebellum, GB1b was exclusively found in Purkinje cells in a zebrin-like pattern. The staining was most pronounced in Purkinje cell dendrites and spines. Using electron microscopy, over 80% of the spine profiles in which a synaptic contact with a parallel fibre was visible contained GB1b-IR at extrasynaptic sites. This subcellular localization is unrelated to GABAergic inputs, indicating that the role of GABAB-receptors in vivo extends beyond synaptic GABAergic neurotransmission and may, in the cerebellum, involve taurine as a ligand.

A highly sensitive immunofluorescence procedure for analyzing the subcellular distribution of GABAA receptor subunits in the human brain.

We designed a protocol to improve the immunohistochemical analysis of human brain structures, which overcomes the limited detection sensitivity, high background, and intense autofluorescence commonly associated with human tissue. This procedure was evaluated by using antibodies against major GABAA receptor subunits (alpha1, alpha2, alpha3, gamma2) in autopsy and surgical specimens. Tissue blocks were briefly fixed by immersion and pretreated with microwave irradiation in sodium citrate buffer. Immunoperoxidase staining revealed a marked enhancement of cell surface immunoreactivity and reduction of background in microwave-irradiated tissue, irrespective of its origin. For confocal laser scanning microscopy, immunofluorescence staining was optimized with the tyramide signal amplification (TSA) technique. This procedure not only dramatically increased the sensitivity for antigen detection but also totally suppressed autofluorescence, thus revealing the cellular and subcellular distribution of GABAA receptor subunits. A distinct neuron-specific expression pattern of the alpha-subunit variants was observed in cerebral cortex and hippocampal formation, along with widespread expression of the gamma2-subunit. Of particular interest was the prominent alpha2- and alpha3-subunit staining on the initial axon segment of pyramidal neurons. This protocol represents a major improvement for high-resolution studies of human brain tissue aimed at investigating morphological alterations underlying neurological diseases.

Synapse-specific localization of NMDA and GABA(A) receptor subunits revealed by antigen-retrieval immunohistochemistry.

Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of gamma-aminobutyric acid type A (GABA[A]) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABA(A) receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABAA receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive ofsynaptic receptors. In hippocampus CA1-CA3 fields, the NR2Aand NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABAA receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The alpha2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell-surface-associated staining is therefore strongly suggestive of an 'unmasking' of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABAA receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses.

Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: an immunohistochemical study.

To assess the significance of GABAA-receptor heterogeneity, which is based on a family of at least 15 subunits, the cellular localization and subunit composition of GABAA-receptor subtypes were analyzed immunohistochemically in the rat spinal cord. The distribution of subunits alpha 1, alpha 2, alpha 3, alpha 5, beta 2,3, and gamma 2 was investigated with subunit-specific antibodies, and their colocalization within individual neurons was visualized by double-immunofluorescence staining. The results reveal a widespread expression of the subunits, alpha 3, beta 2,3, and gamma 2 in the spinal cord, whereas the three other alpha subunits displayed a more restricted, lamina-specific distribution. The alpha 1 and alpha 5 subunits were most abundant in the intermediate zone, whereas the alpha 2 subunit was predominant in the superficial layers of the dorsal horn and in somatic and preganglionic motoneurons. From colocalization studies, seven subunit combinations could be identified (alpha 3/beta 2,3/gamma 2; alpha 2/beta 2,3/gamma 2; alpha 1/beta 2,3/gamma 2; alpha 5/beta 2,3/gamma 2; alpha 1/alpha 5/beta 2,3/gamma 2; alpha 2/gamma 2; alpha 2/alpha 5/gamma 2) that correspond presumably to distinct receptor subtypes. Although most neurons expressed the subunit triplet alpha x/beta 2,3/gamma 2, the beta 2,3 subunits could not be detected in motoneurons that may thus possess "atypical" receptor subtypes (alpha 2/gamma 2 and alpha 2/alpha 5/gamma 2). ON the subcellular level, aggregates of immunoreactivity, suggestive of postsynaptic GABAA receptors, typically were seen on the surface of neuronal somata and proximal dendrites. In addition, an intense diffuse staining was observed in laminae I--III for the subunits alpha 2, alpha 3, beta 2,3, and gamma 2, presumably localized on primary afferent terminals. The localization of GABAA-receptor subtypes in distinct laminar compartments of the spinal cord suggests that GABAA-receptor heterogeneity is of relevance for the modulation of sensory inputs, nociception, and motor control at segmental levels.