Reinhard Jork

Polymyxin B, an inhibitor of protein kinase C, prevents the maintenance of synaptic long-term potentiation in hippocampal CA1 neurons.

The involvement of protein kinase C (PKC)-mediated processes in mechanisms of long-term potentiation (LTP) was suggested by recent studies which have demonstrated a correlation between PKC activation and LTP. However, it was not possible to tell whether there is a causal relationship between the two events. Therefore, we have examined the induction and maintenance of LTP in rat hippocampal slices in the presence of a relatively selective PKC inhibitor, using extracellular electrophysiological techniques. Bath application of 0.1-100 microM polymyxin B did not influence the occurrence of post-tetanic and long-term potentiation usually seen in test responses 1 and 10 min after a 100-Hz/1 s tetanic stimulation of stratum radiatum fibers. However, 20 microM polymyxin B significantly depressed the increase in population spike amplitude and population excitatory postsynaptic potential (EPSP) slope from 30 to 120 min onwards, following repeated tetanization. Immediately after the drug application only weak and reversible effects were seen by the same parameters in test responses of a non-tetanized control input. A late (greater than 6 h) heterosynaptic potentiation of the population spike in the control input was blocked by polymyxin B treatment. Whereas the EPSP-LTP was fully blocked, some potentiation of the population spike still remained, suggesting the independence of PKC of the additional spike (E/S) potentiation for the first 6 h. These results provide direct evidence that the PKC activation is not essential for the initial phase of LTP, but is a necessary condition for a medium and a late, protein synthesis-dependent phase in this monosynaptic pathway, i.e. for the maintenance of synaptic LTP.

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).

Inhibition of evoked glutamate release by the neuroprotective 5-HT1A receptor agonist BAY x 3702 in vitro and in vivo

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT(1A) receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC(50)=1 microM). This effect was blocked by the selective 5-HT(1A) receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT(1A) receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 microg/kg or 10 microg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.

Upregulation of ICAM-1 and MCP-1 but not of MIP-2 and sensorimotor deficit in response to traumatic axonal injury in rats.

The pathophysiology of traumatic axonal injury (TAI) is only partially understood. In this study, we investigated the inflammatory response as well as the extent of neurological deficit in a rat model of traumatic brain injury (TBI). Forty‐two adult rats were subjected to moderate impact‐acceleration brain injury and their brains were analyzed immunohistochemically for ICAM‐1 expression and neutrophil infiltration from 1 hr up to 14 days after trauma. In addition, the chemotactic factors MIP‐2 and MCP‐1 were measured in brain homogenates by ELISA. For evaluating the neurological deficit, three sensorimotor tests were applied for the first time in this model. In the first 24 hr after trauma, the number of ICAM‐1 positive vessels increased up to 4‐fold in cortical and subcortical regions compared with sham operated controls (P < 0.05). Maximal ICAM‐1 expression (up to 8‐fold increase) was detected after 4 days (P < 0.001 vs. 24 hr), returning to control levels in all brain regions by 7 days after trauma. MCP‐1 was elevated between 4 hr and 16 hr post‐injury as compared with controls. In contrast, neither neutrophil infiltration nor elevation of MIP‐2, both events relevant in focal brain injury, could be detected. In all neurological tests, a significant deficit was observed in traumatized rats as compared with sham operated animals from Day 1 post‐injury (grasping reflex of the hindpaws: P < 0.001, vibrissae‐evoked forelimb placing: P = 0.002, lateral stepping: P = 0.037). In conclusion, after moderate impact acceleration brain injury ICAM‐1 upregulation has been demonstrated in the absence of neutrophil infiltration and is paralleled by a selective induction of chemokines, pointing out that individual and distinct inflammatory events occur after diffuse vs. focal TBI. J. Neurosci. Res. 63:438–446, 2001.

5-HT1A receptor agonist-mediated protection from MPTP toxicity in mouse and macaque models of Parkinson's disease

Excitotoxicity-mediated cell death is involved in Parkinsons disease (PD). 5-HT1A receptor agonists can protect from such mechanisms. The current study demonstrates that the 5-HT1A agonists BAY 639044 and repinotan have neuroprotective effects in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In addition, we also show that both compounds delay the appearance of parkinsonian motor abnormalities in a MPTP monkey model that recapitulates the progressive nature of PD. Thus, BAY 639044 or repinotan treatment was initiated when there was 30% neuronal death in the substantia nigra pars compacta, and nerve terminal loss in the striatum was 40%, i.e., compatible with the clinical situation where early symptomatic patients would receive such a treatment. The delay in appearance of parkinsonian motor abnormalities is a consequence of partial neuroprotection of nigrostriatal dopamine neurons, both at neuronal and terminal levels as shown for BAY 639044. These results suggest that 5-HT1A agonists, such as BAY 639044, may protect from neurodegeneration and delay the worsening of motor symptoms in Parkinson patients.

Dopamine induced changes in protein phosphorylation and polyphosphoinositide metabolism in rat hippocampus

Effects of dopamine (DA) on endogenous phosphorylation of hippocampal proteins and polyphosphoinositides were studied in subcellular fractions from a crude mitocondrial/synaptosomal preparation. DA induced a concentration-dependent decrease in the in vitro phosphorylation of the protein B-50 (-22.1% at 10(-5) M DA), whereas no changes were found in phosphoproteins in other subcellular fractions. Treatment of hippocampal slices with 5 X 10(-4) M DA resulted in a 45.8% increase in post hoc phosphorylation of B-50 in SPM and it affected post hoc phosphorylation of several proteins in a cytosolic fraction. In vitro phosphorylation of SPM with DA (5 X 10(-4) M) increased endogenous TPI phosphorylation (+51.6%), whereas treatment of slices with DA (5 X 10(-4) M) resulted in a 39.4% decrease in post hoc TPI phosphorylation. This decrease could be blocked by haloperidol. Significant changes induced by DA (5 X 10(-4) M) were also found in 32P-incorporation into PA (in vitro: -32.4% and post hoc: +39.3%), but were not found in DPI labeling. The data provide evidence for DA-induced changes in phosphorylation of proteins and polyphosphoinositides in rat hippocampal SPM.

Effects of subdural haematoma on sensorimotor functioning and spatial learning in rats

Twenty per cent of all strokes are haemorrhagic in character and are associated with severe disturbances in sensorimotor behaviour and cognition. Although spontaneous recovery of pre-stroke functioning occurs in some cases, the process is demanding, slow, and often incomplete. A first step in the preclinical testing of new putative, neuroprotective and recovery-supporting therapeutics is to validate animal models of brain injury. In a series of four experiments we evaluated the behavioural impairments and the time course of recovery of functional deficits in rats with an experimentally induced subdural haematoma. We found that unilateral subdural haematoma resulted in dysfunction in both simple reflexive (experiment 1) and skilled sensorimotor behaviour (experiment 2). Reflexive behaviour did not recover, or recovered only marginally, and neither did the deficits in skilled forepaw use. Bilateral subdural haematoma impaired the learning and memory performance of adult (experiment 3) and old rats (experiment 4) in the Morris water escape task. Considering the diversity of the deficits found in our experiments, we conclude that different models are needed to cover the broad range of deficits seen in stroke patients.

4-Aminopyridine affects synaptosomal protein phosphorylation in rat hippocampal slices

Rat brain hippocampal slices were incubated with or without the convulsant 4-aminopyridine (4-AP). From these slices a crude mitochondrial/synaptosomal membrane fraction was prepared and analyzed for endogenous protein phosphorylation. 4-AP (10(-5) M) stimulated the phosphorylation of a 50 kDa protein by 86%. The phosphorylation of this 50 kDa protein is Ca2+/calmodulin-dependent and we suggest that this protein is the lower molecular weight subunit of Ca2+/calmodulin-dependent protein kinase II (CaMK II).