Guido C. Koopmans

Phosphodiesterase type 5 inhibition improves early memory consolidation of object information

The nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway is assumed to play an important role in processes underlying learning and memory. We used phosphodiesterase type 5 (PDE5) inhibitors to study the role of cGMP in object- and spatial memory. Our results and those reported in other studies indicate that elevated hippocampal cGMP levels are required to improve the memory performance of rodents in object recognition and passive avoidance learning, but not in spatial learning. The timing of treatment modulates the effects on memory and strongly supports a role for cGMP in early stages of memory formation. Alternative explanations for the improved memory performance of PDE5 inhibitors are also discussed. Immunocytochemical studies showed that in vitro slice incubations with PDE5 inhibitors increase NO-stimulated cGMP levels mainly in hippocampal varicose fibers. Reviewing the available data on the localization of the different components of the NO-cGMP signaling pathway, indicates a complex interaction between NO and cGMP, which may be independent of each other. It is discussed that further studies are needed, immunocytochemical and behavioral, to better understand the cGMP-mediated molecular mechanisms underlying memory formation.

Olfactory ensheathing cells, olfactory nerve fibroblasts and biomatrices to promote long-distance axon regrowth and functional recovery in the dorsally hemisected adult rat spinal cord.

Cellular transplantation, including olfactory ensheathing cells (OEC) and olfactory nerve fibroblasts (ONF), after experimental spinal cord injury in the rat has previously resulted in regrowth of severed corticospinal (CS) axons across small lesion gaps and partial functional recovery. In order to stimulate CS axon regrowth across large lesion gaps, we used a multifactorial transplantation strategy to create an OEC/ONF continuum in spinal cords with a 2-mm-long dorsal hemisection lesion gap. This strategy involved the use of aligned OEC/ONF-poly(D,L)-lactide biomatrix bridges within the lesion gap and OEC/ONF injections at 1 mm rostral and caudal to the lesion gap. In order to test the effects of this complete strategy, control animals only received injections with culture medium rostral and caudal to the lesion gap. Anatomically, our multifactorial intervention resulted in an enhanced presence of injured CS axons directly rostral to the lesion gap (65.0 +/- 12.8% in transplanted animals versus 13.1 +/- 3.9% in control animals). No regrowth of these axons was observed through the lesion site, which may be related to a lack of OEC/ONF survival on the biomatrices. Furthermore, a 10-fold increase of neurofilament-positive axon ingrowth into the lesion site as compared to untreated control animals was observed. With the use of quantitative gait analysis, a modest recovery in stride length and swing speed of the hind limbs was observed. Although multifactorial strategies may be needed to stimulate repair of large spinal lesion gaps, we conclude that the combined use of OEC/ONF and poly(D,L)-lactide biomatrices is rather limited.

Assessment of spatial learning abilities of mice in a new circular maze

In the present study, we tested the spatial learning behavior of four different mouse strains (129/Sv, BALB/c, C57BL and Swiss) in a newly developed circular maze. The maze was based on the circular Barnes maze, which was initially developed for rats. Since mice do not readily enter holes in floor, additional reinforcers (positive and negative) or pretraining procedures have been used to train the animals. Because these methods are not always desirable, we examined whether mice are more willing to enter escape holes (12), which were located in the rim of the apparatus. C57BL mice appeared to improve their performance on three different measures of spatial learning: latency to find escape hole, distance to escape hole and errors (visit to other holes). The other strains also improved their performance although this was only seen for one parameter (i.e. 129/Sv and BALB/c on latency, and Swiss on distance). When the animals were trained to find another location, it was found that only the performance of the C57BL mice was transiently impaired. The C57BL mice were also very efficient in improving their performance in a repeated acquisition paradigm (six trials per day on four successive days). Applying a probe trial procedure, a clear preference for the goal location was found. These findings indicate that these mice used a spatial search strategy. Although this circular maze can be used as an additional tool to assess spatial learning in (genetically modified) mice, it is noted that strain differences in spatial learning seem to be independent of task. Further, our data with different strains indicate that different measures of behavior should be evaluated to assess the spatial learning performance of mice.

High frequency stimulation of the subthalamic nucleus improves speed of locomotion but impairs forelimb movement in parkinsonian rats

The subthalamic nucleus (STN) plays an important role in motor and non-motor behavior in Parkinsons disease, but its involvement in gait functions is largely unknown. In this study, we investigated the role of the STN on gait in a rat model of PD using the CatWalk method. Parkinsonian rats received bilateral high frequency stimulation (HFS) with different stimulation amplitudes of the STN. Rats were rendered parkinsonian by bilateral injections of 6-hydroxydopamine (6-OHDA) into the striatum. One group of 6-OHDA animals was implanted bilaterally with stimulation electrodes at the level of the STN. Stimulations were performed at 130 Hz (frequency), 60 micros (pulse width) and varying amplitudes of 0, 3, 30 and 150 microA. Rats were evaluated in an automated quantitative gait analysis method (CatWalk method). After behavioral evaluations, rats were killed and the brains processed for histological stainings to determine the impact of the dopaminergic lesion (tyrosine hydroxylase immunohistochemistry) and the localization of the electrode tip (hematoxylin-eosin histochemistry). Results show that bilateral 6-OHDA infusion significantly decreased (70%) the number of dopaminergic cells in the substantia nigra pars compacta (SNc). Due to 6-OHDA treatment, the gait parameters changed considerably. There was a general slowness. The most pronounced effects were seen at the level of the hind paws. Due to implantation of STN electrodes the step pattern changed. STN electrical stimulation improved the general slowness but induced slowing of the forelimb movement. Furthermore, we found that HFS with a medium amplitude significantly changed speed, the so-called dynamic aspect of gait. The static features of gait were only significantly influenced with low amplitude. Remarkably, STN stimulation affected predominantly the forepaws/limbs.

LDL receptor deficiency results in decreased cell proliferation and presynaptic bouton density in the murine hippocampus

An aberrant cholesterol metabolism in the brain may contribute to the pathogenesis of Alzheimers disease (AD). The LDL receptor (LDLR) regulates plasma cholesterol levels and recently we and others obtained evidence that it is also involved in regulating brain cholesterol homeostasis. Moreover, we found that LDLR-deficient mice display impaired spatial memory. Because cholesterol, in part derived from cellular uptake via LDLR, is required for peripheral cell proliferation and growth, we examined the effect of absence of the LDLR on hippocampal proliferation and the density of synaptic connections. Mice deficient for the LDLR displayed a reduced number of proliferating (BrdU-labeled) cells in the hippocampus as compared to wild type control mice. In addition, the number of synaptophysin-immunoreactive presynaptic boutons in the hippocampal CA1 and the dentate gyrus (DG) areas, but not in cortical areas, was lower in the LDLR-knockout mice than in the control mice. In vitro experiments showed that LDLR activity is increased when cell growth is enhanced by the addition of N2 supplement. This further supports a role for the LDLR in the outgrowth of neurites. These findings support the notion that, similar to its role in the periphery, the LDLR is important for the cellular uptake of cholesterol in the brain and that disturbance of this process affects neuronal plasticity.

Characterization of retinaldehyde dehydrogenase-2 induction in NG2-positive glia after spinal cord contusion injury

The transcriptional activator retinoic acid (RA) supports axonal regeneration of several neuronal cell populations in vitro, and it has been suggested that its receptor RARβ2 may be used to support axonal regeneration in the adult mammalian spinal cord. We have previously shown that spinal cord injury induces activity of the RA synthesizing enzyme retinaldehyde dehydrogenase (RALDH)2 in NG2‐positive cells. This report quantifies the increase of RALDH2 protein in the injured spinal cord and characterizes the RALDH2/NG2 expressing cells probably as a unique RA synthesizing subpopulation of activated oligodendrocyte precursors or “polydendrocytes”. In the uninjured spinal cord low levels of RALDH2 are present in oligodendrocytes as well as in the meninges and in blood vessels. Following injury there is a significant increase in RALDH2 in these latter two tissues and, given that the RALDH2/NG2 positive cells are clustered in the same area, this implies that these are specific foci of RA synthesis. It is presumed that these cells release RA in a paracrine fashion in the region of the wound; however, the RALDH2/NG2‐immunoreactive cells expressed the retinoid receptors RARα, RARβ, RXRα and RXRβ, suggesting that RA also serves an autocrine function.

Limitations in transplantation of astroglia-biomatrix bridges to stimulate corticospinal axon regrowth across large spinal lesion gaps.

Regrowth of injured axons across rather small spinal cord lesion gaps and subsequent functional recovery has been obtained after many interventions. Long-distance regeneration of injured axons across clinically relevant large spinal lesion gaps is relatively unexplored. Here, we aimed at stimulating long-distance regrowth of the injured corticospinal (CS) tract. During development, an oriented framework of immature astrocytes is important for correct CS axon outgrowth. Furthermore, a continuous growth promoting substrate may be needed to maintain a CS axon regrowth response across relatively large spinal lesion gaps. Hence, we acutely transplanted poly(D,L)-lactide matrices, which after seeded with immature astrocytes render aligned astrocyte-biomatrix complexes (R. Deumens, et al. Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro. Neuroscience 125 (3) (2004) 591-604), into 2-mm long dorsal hemisection lesion gaps. In order to create a growth promoting continuum, astrocyte suspensions were also injected rostral and caudal to the lesion gap. During 2 months, locomotion was continuously monitored. Histological analysis showed that astrocytes injected into host spinal tissue survived, but did not migrate. None of the astrocytes on the biomatrices survived within the lesion gap. BDA-labeled CS axons did not penetrate the graft. However, directly rostral to the lesion gap, 120.9+/-38.5% of the BDA-labeled CS axons were present in contrast to 12.8+/-3.9% in untreated control animals. The observed anatomical changes were not accompanied by locomotor improvements as analyzed with the BBB and CatWalk. We conclude that although multifactorial strategies may be needed to stimulate long-distance CS axon regrowth, future studies should focus on enhancing the viability of cell/biomatrix complexes within large spinal lesion gaps.

Stimulation of neurite outgrowth on neonatal cerebral astrocytes is enhanced in the presence of BDNF.

An area of increasing interest in spinal cord injury (SCI) research is the development of multi-factorial strategies to promote repair. In this respect, a prominent role is played by cell transplantation, the reparative effect of which can be enhanced by additional use of neurotrophic factors. Immature astrocytes have shown their merit in stimulating axon regeneration upon transplantation into the injured spinal cord. Brain-derived neurotrophic factor (BDNF) influences a wide range of descending axon tracts in the injured spinal cord. In the present study, we hypothesized that the neurite outgrowth of neonatal cortical neurons on immature astrocytes is enhanced in the presence of BDNF. To test this hypothesis, neonatal cortical neurons were cultured on neonatal astrocytes for 2 days in absence or presence of BDNF. The length of the longest neurite and the number of primary neurites per neuron were taken as measures to study neurite outgrowth. We show that BDNF dose-dependently enhanced neurite outgrowth of neonatal cerebral cortical neurons grown on immature astrocytes. Compared to conditions without BDNF, the length of the longest neurite increased by 25.5 and 28.8% in presence of 10 and 100 pg/ml BDNF, respectively. BDNF did not alter the density of the immature astrocytes. We conclude that the presence of BDNF enhances the neurite outgrowth on immature astrocytes. A multi-factorial strategy based on transplantation of neonatal astrocytes in the presence of additional BDNF is recommended and may stimulate axon regrowth after experimental injury to the central nervous system.

Functional recovery, serotonergic sprouting, and endogenous progenitor fates in response to delayed environmental enrichment after spinal cord injury.

Environmental enrichment (EE) is a way to induce voluntary locomotor training that positively affects locomotor recovery after acute spinal cord injury (SCI). The beneficial effect on SCI outcome is thought to be based on enhanced plasticity in motor pathways, triggered by locomotor-specific sensory feedback to the spinal cord circuitry for locomotion (central pattern generators [CPGs]). In view of chronic SCI, we tested the hypothesis that EE improves motor outcome after SCI in the rat when started after a clinically relevant delay of 3 weeks. At the CPG level (i.e., the spinal L1-L2 level), where EE-related sensory feedback is processed, two key mechanisms of anatomical plasticity were examined: (1) serotonergic innervation, and (2) survival and differentiation of spinal cord progenitor cells. Delayed EE improved interlimb coordination, which was associated with an increased serotonergic innervation of the ventro-lateral grey matter within the L1-L2 segments. Although spinal cord progenitor cells were found to differentiate into both neurons and glial cells, EE did not affect their survival. These results show that EE induces a substantial improvement of motor outcome after SCI when commenced after a clinically-relevant delay. Increased serotonergic innervation of the lumbar CPG area is therefore suggested to play an important role in the EE-induced recovery of interlimb coordination.