Manfred Krug

Anisomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CA1 region in vitro

Long-term potentiation (LTP) with its extremely long duration has been frequently regarded as an elementary mechanism of information storage in the nervous system or at least as a suitable model for the study of mechanisms underlying functional plasticity and processes of learning and memory formation. Considering the necessity of an increased protein synthesis for memory consolidation and for the maintenance of LTP in granular synapses in vivo it was of interest to determine whether the LTP of the CA1 region of the hippocampus depends on protein synthesis as well. For the solution of this question anisomycin (ANI), a reversible blocker of protein synthesis, was used at a concentration of 20 microM, which blocked the [3H]leucine incorporation in hippocampal slices by at least 85%. It has been shown that in the CA1 region in vitro the maintenance of LTP (i.e. a late phase greater than 5 h) depends on an ongoing protein synthesis. A 3-h treatment with ANI immediately following multiple tetanization resulted in gradually developing loss of field excitatory postsynaptic potential (EPSP) and population spike (PS) potentiation (15 +/- 19% increase of the PS instead of the 96 +/- 14% increase in non-treated control experiments at the 8th h after tetanization). Furthermore, a late PS potentiation (greater than 6 h) of a second non-tetanized pathway to CA1 pyramidal cells has been observed (increase by 64 +/- 18% at the 8th h) for the first time. This potentiation was ANI-sensitive as well and suggests that the maintenance of LTP is dependent on a postsynaptic mechanism.

Brevican-deficient mice display impaired hippocampal CA1 long- term potentiation but show no obvious deficits in learning and memory

ABSTRACT Brevican is a brain-specific proteoglycan which is found in specialized extracellular matrix structures called perineuronal nets. Brevican increases the invasiveness of glioma cells in vivo and has been suggested to play a role in central nervous system fiber tract development. To study the role of brevican in the development and function of the brain, we generated mice lacking a functional brevican gene. These mice are viable and fertile and have a normal life span. Brain anatomy was normal, although alterations in the expression of neurocan were detected. Perineuronal nets formed but appeared to be less prominent in mutant than in wild-type mice. Brevican-deficient mice showed significant deficits in the maintenance of hippocampal long-term potentiation (LTP). However, no obvious impairment of excitatory and inhibitory synaptic transmission was found, suggesting a complex cause for the LTP defect. Detailed behavioral analysis revealed no statistically significant deficits in learning and memory. These data indicate that brevican is not crucial for brain development but has restricted structural and functional roles.

Four different types of protease-activated receptors are widely expressed in the brain and are up-regulated in hippocampus by severe ischemia.

A variety of extracellular serine proteases are expressed in the central nervous system or might permeate the blood–brain barrier under pathological conditions. However, their intracerebral targets and physiological functions are largely unknown. Here, we show that four distinct subtypes of protease‐activated receptors (PARs) are abundantly expressed in the adult rat brain and in organotypic hippocampal slice cultures. PAR‐1 expression was significant in the hippocampus, cortex and amygdala. Highest densities of PAR‐2 and PAR‐3 were observed in hippocampus, cortex, amygdala, thalamus, hypothalamus and striatum. Apart from the striatum, a similar localization was found for PAR‐4. Within the hippocampal formation, each PAR subtype was predominantly localized in the pyramidal cell layers. Additionally, we identified PAR‐2 in mossy fibers between dentate gyrus and CA3, PAR‐3 in the subiculum and PAR‐4 in CA3 and in mossy fibres as well as in the stratum lacunosum moleculare. After exposing hippocampal slice cultures to a severe experimental ischemia (oxygen–glucose deprivation), the expression of PARs 1–3 was up‐regulated with subtype‐specific kinetics. The localization of PARs in brain regions particularly vulnerable to ischemic insults as well as distinct alterations in the expression pattern after experimental ischemia support the notion of an important role of extracellular serine proteases and PARs in cerebral ischemia.

Neurocan is dispensable for brain development.

ABSTRACT Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.

Different Mechanisms and Multiple Stages of LTP

On the basis of our own experimental data and results from other laboratories then available, we developed in 1972 a working hypothesis on neuronal mechanisms of memory. We suggested that the assumed stages of short-term, intermediate, and long-term memory, their different time course of origin and decay, their biochemical correlates as well as their sensitivity to interventions reflect the properties of the corresponding cellular mechanisms of a synaptic, synaptosomal and nuclear regulation of memory formation (Matthies, 1972).

Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long‐term potentiation

Neural recognition molecules such as the neural cell adhesion molecule (NCAM) have been implicated in synaptic plasticity, including long-term potentiation (LTP), sensitization, and learning and memory. The major isoform of NCAM carrying the longest cytoplasmic domain of all NCAM isoforms (NCAM180) is predominantly localized in postsynaptic membranes and postsynaptic densities of hippocampal neurons, with only a proportion of synapses carrying detectable levels of NCAM180. To investigate whether this differential expression of NCAM180 may correlate with distinct states of synaptic activity, LTP was induced by high-frequency stimulation of the perforant path and the percentage of NCAM180 immunopositive spine synapses determined in the outer third of the dentate molecular layer of the dentate gyrus by immunoelectron microscopy. Twenty-four hours following induction of LTP by high-frequency stimulation, the percentage of spine synapses expressing NCAM180 increases from 37% (passive control) to 70%. This increase was inhibited by the noncompetitive N-methyl-D-aspartate receptor antagonist MK801. Following repeated LTP induction at 10 consecutive days with one tetanization each day, 60% of all spine synapses were NCAM180 immunoreactive. Compared to passive control animals, the percentage of NCAM180 expressing synapses in low-frequency stimulated animals decreased from 37% to 28%. Spine synapses in the inner part of the dentate molecular layer not contacted by the afferents of the perforant path did not change the percentage of NCAM180-expressing synapses. The results obtained by the postembedding immunogold staining technique confirmed the difference in NCAM180 expression of spine synapses between passive control and potentiated animals. These observations suggest a role for NCAM180 in synaptic remodeling accompanying LTP.

Dopamine D1-deficient mutant mice do not express the late phase of hippocampal long-term potentiation.

THE possible involvement of the dopamine D1 receptor subtype in mechanisms of long-term potentiation (LTP) of the Schaffer collateral-commissural input of CA1 neurones was investigated using D1-deficient mutant mice. In transversal hippocampus slices from mice lacking the D1 receptor a normal post-tetanic and shortterm potentiation could be induced after applying a triple 100 Hz tetanization. However, the potentiated fEPSP in the mutant mice declined to control value about 140 min following tetanization, whereas in the wild type mice a normal, non-decremental LTP was observed. These data support the idea that besides the glutamatergic system, the synergistic activation of dopaminergic synapses is necessary for LTP maintenance.

Long-term potentiation induced in dendrites separated from rat's CA1 pyramidal somata does not establish a late phase

In 14 transversal hippocampal slices from rats the extracellular field-EPSP (excitatory postsynaptic potential) in the dendritic region of the CA1 subfield was recorded after isolating the apical dendrites from their somata by microsurgical cuts through the proximal stratum radiatum. Contrary to intact slices which, after tetanic stimulation of the Schaffer collaterals, showed long-term potentiation (LTP) until the end of registration after 8-10 h, LTP declined in the isolated dendrites to baseline values after 3 h. It is concluded that de-novo protein synthesis in postsynaptic cell bodies might be necessary for the maintenance of late phases of LTP.

Spinules in axospinous synapses of the rat dentate gyrus: changes in density following long-term potentiation

Following high frequency stimulation of the perforant path the density of axospinous synapses from the middle third of the molecular layer of the rat dentate gyrus did not change significantly. By contrast, within this synaptic population the density of axospinous synapses containing synaptic spinules increased markedly. We interpret these results in terms of a structural modification related to enhanced synaptic efficacy.

The role of superoxide dismutase and α-tocopherol in the development of seizures and kindling induced by pentylenetetrazol - influence of the radical scavenger α-phenyl-N-tert-butyl nitrone

Abstract Previous experiments have shown that the generation of free hydroxyl radicals in rat brain homogenates is increased following pentylenetetrazol (PTZ) kindling. The present study was performed in order to evaluate the involvement of endogeneous radical defence systems as the superoxide dismutase (SOD) and the level of α-tocopherol, an important lipid-soluble and membrane-bound antioxidant in brain homogenate of rats after acute seizure and kindling induced by PTZ. The activities of the total SOD were significantly reduced after acute seizure and tend towards an enhancement in kindled animals. Western blot analysis shows an upregulation of Mn-SOD in rat brain homogenates after kindling. The level of the chain-breaking antioxidant α-tocopherol was reduced in acutely convulsing rats and was not modified in kindled rats. Second, we studied the influence of exogeneously supplied radical scavenger α-phenyl-N-tert-butyl-nitrone (PBN) on seizure and kindling following PTZ treatment. After a single injection of PTZ at a dose evoking clonic–tonic seizures, PBN did not modify either the formation of free hydroxyl radicals measured by the levels of 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA or the susceptibility to PTZ. In the kindling group, subchronic treatment with PBN (over a period of 4 weeks) prevented the increase in the formation of free hydroxyl radicals, and the susceptibility to PTZ was transiently decreased during the development of kindling, but PBN did not influence the susceptibility to PTZ in fully kindled rats. Pretreatment with PBN increased the activities of total SOD and the protein content of Mn-SOD and decreased the level of α-tocopherol in comparison to saline controls. The results suggest that the formation of free hydroxyl radicals is not reflected by an enhanced susceptibility to PTZ classified according to the modified RACINE scale. Additionally, it may be assumed that the increased generation of hydroxyl radicals in kindled animals is not primary caused by an exhaustion of both the defence systems measured. Adaptive mechanisms, as the induction of Mn-SOD, may be taken into consideration to counteract oxidative stress-mediated free radical formation.