Oliver Weinmann

The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats

In contrast to peripheral nerves, central axons do not regenerate. Partial injuries to the spinal cord, however, are followed by functional recovery. We investigated the anatomical basis of this recovery and found that after incomplete spinal cord injury in rats, transected hindlimb corticospinal tract (CST) axons sprouted into the cervical gray matter to contact short and long propriospinal neurons (PSNs). Over 12 weeks, contacts with long PSNs that bridged the lesion were maintained, whereas contacts with short PSNs that did not bridge the lesion were lost. In turn, long PSNs arborize on lumbar motor neurons, creating a new intraspinal circuit relaying cortical input to its original spinal targets. We confirmed the functionality of this circuit by electrophysiological and behavioral testing before and after CST re-lesion. Retrograde transynaptic tracing confirmed its integrity, and revealed changes of cortical representation. Hence, after incomplete spinal cord injury, spontaneous extensive remodeling occurs, based on axonal sprout formation and removal. Such remodeling may be crucial for rehabilitation in humans.

Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury.

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion-regenerating and/or sprouting from spared intact fibers-were also found to be more frequent in Nogo-A-deficient animals.

Synapse‐specific localization of NMDA and GABAA 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 γ‐aminobutyric acid type A (GABAA) 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 GABAA 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 of synaptic receptors. In hippocampus CA1–CA3 fields, the NR2A and 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 α2 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. J. Comp. Neurol. 390:194–210, 1998.

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

GABAB (γ‐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.

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

Improving stroke recovery by timing treatment Patients recovering from strokes often fight a long uphill battle, with mixed results. Studying the effect of physical training on regeneration from damaged nerves in a model of stroke in rats, Wahl et al. show that timing matters. First, the researchers gave the rats a stroke, which damaged their ability to reach for food pellets with their forelimbs. The researchers then gave them physical training and treated them with an antibody to encourage neural regeneration. The rats improved more when the researchers waited until after the antibody treatment to start the training. Damaged circuits, it seems, need a little time to regrow before being called into action. Science, this issue p. 1250 A rat model of stroke shows that the rebuilding of spinal circuits in response to training is time-sensitive. 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.

Constraint-Induced Movement Therapy in the Adult Rat after Unilateral Corticospinal Tract Injury

Smaller spinal cord injuries often allow some degree of spontaneous behavioral improvements because of structural rearrangements within different descending fiber tracts or intraspinal circuits. In this study, we investigate whether rehabilitative training of the forelimb (forced limb use) influences behavioral recovery and plastic events after injury to a defined spinal tract, the corticospinal tract (CST). Female adult Lewis rats received a unilateral CST injury at the brainstem level. Use of the contralateral impaired forelimb was either restricted, by a cast, or forced, by casting the unimpaired forelimb immediately after injury for either 1 or 3 weeks. Forced use of the impaired forelimb was followed by full behavioral recovery on the irregular horizontal ladder, whereas animals that could not use their affected side remained impaired. BDA (biotinylated dextran amine) labeling of the intact CST showed lesion-induced growth across the midline where CST collaterals increased their innervation density and extended fibers toward the ventral and the dorsal horn in response to forced limb use. Gene chip analysis of the denervated ventral horn revealed changes in particular for growth factors, adhesion and guidance molecules, as well as components of synapse formation suggesting an important role for these factors in activity-dependent intraspinal reorganization after unilateral CST injury.

Red nucleus projections to distinct motor neuron pools in the rat spinal cord

Despite being one of the more extensively investigated descending pathways of the rat spinal cord, the termination pattern and postsynaptic targets of the rubrospinal tract (RST) still present some unresolved issues. In addition to locomotor functions, the RST is implicated in the control of limb movements such as reaching and grasping. Although a strong RST projection onto interneurons of intermediate Rexeds laminae V and VI have been described through the entire length of the rat spinal cord, the existence of direct rubro‐motoneuronal connections have not been demonstrated. In the present study, anterograde tracing of the rat RST with biotinylated dextran amine (BDA) was combined with injections of cholera toxin β‐subunit (CTβ) into selected groups of forelimb muscles to analyze in detail the rubral projections to the forelimb areas of the cervical spinal cord. The double‐staining procedure suggested a direct projection from the RST to specific populations of motoneurons. Three populations of forelimb muscles were distinguished, i.e., paw, “distal” muscles; forearm, “intermediate” muscles; and upper arm, “proximal” muscles. A somatotopic distribution of the corresponding motor neuron pools was present in the spinal cord segments C4‐Th1. Rubrospinal axons were seen in close apposition to the distal and intermediate muscle motoneurons, but were consistently absent in the most ventrally situated motor column projecting to proximal muscles. Microstimulation of the red nucleus resulted in electromyographic responses with shorter latency in the distal forelimb muscles than in proximal muscles. These experiments support a specific, preferential role of the RST in distal forelimb muscle control. J. Comp. Neurol. 448:349–359, 2002.

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Înjured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.

Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons

Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of γ-aminobutyric acid A (GABAA) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2–3 consecutive days for completion.

Intrathecally infused antibodies against Nogo-A penetrate the CNS and downregulate the endogenous neurite growth inhibitor Nogo-A

Neutralizing antibodies against the neurite growth inhibitory protein Nogo-A are known to induce regeneration, enhance compensatory growth, and enhance functional recovery. In intact adult rats and monkeys or spinal cord injured adult rats, antibodies reached the entire spinal cord and brain through the CSF circulation from intraventricular or intrathecal infusion sites. In the tissue, anti-Nogo antibodies were found inside Nogo-A expressing oligodendrocytes and neurons. Intracellularly, anti-Nogo-A antibodies were colocalized with endogenous Nogo-A in large organels, some of which containing the lysosomal marker cathepsin-D. This suggests antibody-induced internalization of cell surface Nogo-A. Total Nogo-A tissue levels in spinal cord were decreased in intact adult rats following 7 days of antibody infusion. This mechanism was confirmed in vitro; cultured oligodendrocytes and neurons had lower Nogo-A contents in the presence of anti-Nogo-A antibodies. These results demonstrate that antibodies against a CNS cell surface protein reach their antigen through the CSF and can induce its downregulation.