Holger Rosenbrock

Effect of chronic intermittent restraint stress on hippocampal expression of marker proteins for synaptic plasticity and progenitor cell proliferation in rats.

Chronic restraint stress may change hippocampal mRNA levels of markers for synaptic plasticity such as synaptophysin, growth-associated protein 43 (GAP-43), and brain-derived neurotrophic factor (BDNF). In order to examine the relation between that stressor and those biochemical markers on protein level as well as the Ki-67 protein, a marker of progenitor cell proliferation, we subjected rats to chronic intermittent restraint stress for 6 h per day for 14 days excluding the weekends. This stress intensity caused a significant increase in adrenal gland weight and decrease in body weight gain. However, we did not find significant alteration of protein expression levels for synaptophysin, GAP-43, and BDNF by using Western blot analysis. Unlike these findings, the hippocampal protein expression of Ki-67 was significantly reduced by using both Western blot and immunohistochemical analyses. This reduction of Ki-67 expression in chronically stressed rats was correlated with increased adrenal gland weight and decreased body weight gain. All marker proteins used did not show any changes of hippocampal expression level after a single restraint stress session of 3 h. In conclusion, chronic intermittent restraint stress caused changes in the physiological stress response in rats, and a decrease of hippocampal progenitor cells using the Ki-67 protein as marker which indicates a suppression of adult neurogenesis. The results might contribute to understand the relationship between stress and cellular neurobiology of depression, since chronic antidepressant treatment have been shown to increase adult neurogenesis in the rat hippocampus.

The Selective mGlu5 Receptor Antagonist MTEP, Similar to NMDA Receptor Antagonists, Induces Social Isolation in Rats

It has repeatedly been shown that uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists can mimic certain aspects of positive and negative symptoms of schizophrenia in human volunteers and laboratory animals. The purpose of the present study was to expand these findings and to determine whether the selective metabotropic glutamate receptor subtype 5 (mGluR5) antagonist, MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine), could induce similar effects in Wistar rats. First, MTEP (1.0–10.0 mg/kg; intraperitoneally) after acute and subchronic (daily for 5 days) administration as well as the uncompetitive antagonists of the NMDA receptor of either high affinity, phencyclidine (0.5–4.0 mg/kg; subcutaneously (s.c.)) and (+)-MK-801 (0.03–0.25 mg/kg; s.c.), or low–moderate affinity, ketamine (2.0–16.0 mg/kg; s.c.) and memantine (0.15–20.0 mg/kg; s.c.), following daily administration for 3 days were tested in the social interaction test to determine their ability to reproduce the negative and positive symptoms measured by social isolation and stereotyped behavior, respectively. Second, the compounds were tested in the motility test following acute administration to determine their ability to induce locomotor hyperactivity reflecting the positive symptoms. In line with previous findings, all examined NMDA receptor antagonists produced social interaction deficits, locomotor hyperactivity, and stereotypy except memantine. Notably, this study found that MTEP following both acute and subchronic administration dose-dependently induced social isolation, but did not cause either locomotor hyperactivity or stereotypy. These data demonstrate that social behavior deficits in rats can be caused by both the blockade of the NMDA receptor and the inhibition of mGluR5, whereas mGluR5 antagonists may not independently be able to mimic the positive symptoms.

Functional interaction of metabotropic glutamate receptor 5 and NMDA-receptor by a metabotropic glutamate receptor 5 positive allosteric modulator.

The NMDA (N-methyl-D-aspartate)-receptor is fundamentally involved in cognitive functions. Recent studies demonstrated a functional interaction between the metabotropic glutamate receptor 5 (mGlu(5) receptor) and the NMDA-receptor in neurons. In rat hippocampal slices, it was shown that activation of mGlu(5) receptor by a positive modulator in the presence of a subthreshold agonist concentration potentiated NMDA-receptor mediated currents and phosphorylation of intracellular signalling proteins. In the present study, we investigated the functional interaction of mGlu(5) receptor and NMDA-receptor by the selective mGlu(5) receptor positive modulator ADX-47273 in-vitro and in-vivo. In rat primary neurons, this compound potentiated Ca(2+) mobilization in the presence of a subthreshold concentration of the mGluR(1/5) agonist DHPG (0.3 microM) with an EC(50) of 0.28+/-0.05 microM. NMDA-induced Ca(2+)-mobilization in primary neurons could be potentiated when neurons were pre-stimulated with 1 microM ADX-47273 in the presence of 0.3 microM DHPG. The specific mGlu(5) receptor antagonist MPEP and the Src-family kinase inhibitor PP2 blocked this potentiation demonstrating the functional interaction of the NMDA-receptor and mGlu(5) receptor in neurons. Furthermore, ADX-47273 elicited an enhancement of NMDA-receptor dependent long-term potentiation in rat hippocampal slices that could be reversed by MPEP. After intraperitoneal administration to rats, ADX-47273 showed a dose-dependent reduction of NMDA-receptor antagonist (ketamine) induced hyperlocomotion, supporting the mechanistic interaction of the NMDA-receptor and mGlu(5) receptor in-vivo. In conclusion, these findings further support the idea of a functional interaction between the mGlu(5) receptor and NMDA-receptor, which may provide a pharmacological strategy for addressing CNS diseases with cognitive impairments linked to NMDA-receptor hypofunction.

Inhibition of acetylcholinesterase and phosphodiesterase-9A has differential effects on hippocampal early and late LTP.

Donepezil is the current standard symptomatic treatment of mild-to-moderate Alzheimers disease (AD) patients. It aims to compensate for the deficit in cholinergic neurotransmission by blocking acetylcholinesterase (AChE) and thus increases the concentration of extracellular acetylcholine. However, experience from clinical practice demonstrated that AChE inhibitors only have moderate treatment effects. As a potential new approach for memory enhancement, inhibition of specific phosphodiesterases (PDEs) has gained attention. Among those are PDE9A inhibitors which increase the levels of the second messenger cyclic guanosine monophosphate (cGMP) intracellularly. In order to gain more insight into the potential impact of extracellularly acting AChEs and intracellularly acting PDE9A inhibitors on synaptic plasticity, we analyzed the effects of the AChE inhibitor donepezil and the PDE9A inhibitor BAY 73-6691 on long-term potentiation (LTP) in rat hippocampal slices, a widely accepted cellular experimental model of memory formation. Generally, LTP can be differentiated into an early and a late form, being protein-synthesis independent and protein-synthesis dependent, respectively. Donepezil was found to increase early LTP, but did not affect late LTP. In contrast, BAY 73-6691 demonstrated enhancing effects on both early and late LTP and even transformed early into late LTP. Furthermore, it was shown that this transformation into late LTP was dependent on the NO-cGMP-PKG pathway. In conclusion, this study demonstrates that BAY 73-6691 exhibits a stronger effect in enhancing and prolonging LTP than donepezil suggesting that PDE9 inhibition might be more efficacious in enhancing learning and memory.

PDE9A inhibition rescues amyloid beta-induced deficits in synaptic plasticity and cognition.

The cyclic nucleotide cGMP is an important intracellular messenger for synaptic plasticity and memory function in rodents. Therefore, inhibition of cGMP degrading phosphodiesterases, like PDE9A, has gained interest as potential target for treatment of cognition deficits in indications like Alzheimers disease (AD). In fact, PDE9A inhibition results in increased hippocampal long-term potentiation and exhibits procognitive effects in rodents. To date, however, no evidence has been published linking PDE9A inhibition to the pathologic hallmarks of AD such as amyloid beta (Aβ) deposition. Therefore, we investigated the role of PDE9A inhibition in an AD relevant context by testing its effects on Aβ-related deficits in synaptic plasticity and cognition. The PDE9A inhibitor BAY 73-6691 was found to restore long-term potentiation impaired by Aβ42 oligomers. Furthermore, we demonstrated that BAY 73-6691 enhanced cGMP levels in the hippocampus of APP transgenic tg2576 mice and improved memory performance of these mice. Altogether, our results support the hypothesis that inhibition of PDE9A could be a beneficial approach for the treatment of memory impairment in AD patients.

Sub-chronic rolipram treatment leads to a persistent improvement in long-term object memory in rats

In the present study the effects of sub-chronic rolipram treatment in an object recognition task in 3-month-old male rats were investigated. Rats remember which object they have explored in a previous trial (T1) when they are tested 1 h later (T2). However, when tested 24 h later, they do not remember which object was presented to them in the first trial. Drug treatments may improve discrimination performance after 24 h, i.e., improve memory for the familiar object. Rats were sub-chronically treated with 0.5 mg/kg rolipram (p.o.) for five consecutive days and tested with a 24 h delay between T1 and T2. Memory performance in the object recognition task was assessed before, during and after sub-chronic treatment. In addition, we investigated whether the timing of the final dose, i.e., 24, 1, or 6 h before training, had an effect on memory performance. During sub-chronic treatment, i.e., after 2-3 days of rolipram treatment, moderate effects on memory performance were observed. Regardless of when the final administration was given, sub-chronic rolipram treatment improved long-term memory performance. Since plasma and brain rolipram levels were undetectable at 24 h before the test, and acute treatment with rolipram 24 h before training had no effects, the observed memory enhancement cannot be attributed to acute rolipram effects. The long-term memory enhancing effects of rolipram might be explained by long-lasting neuronal changes by the chronic treatment due to recurring activation of the cAMP/PKA/CREB pathway leading to CREB phosphorylation.

Globular and Protofibrillar Aβ Aggregates Impair Neurotransmission by Different Mechanisms

In Alzheimers disease, substantial evidence indicates the causative role of soluble amyloid β (Aβ) aggregates. Although a variety of Aβ assemblies have been described, the debate about their individual relevance is still ongoing. One critical issue hampering this debate is the use of different methods for the characterization of endogenous and synthetic peptide and their intrinsic limitations for distinguishing Aβ aggregates. Here, we used different protocols for the establishment of prefibrillar Aβ assemblies with varying morphologies and sizes and compared them in a head-to-head fashion. Aggregation was characterized via the monomeric peptide over time until spheroidal, protofibrillar, or fibrillar Aβ aggregates were predominant. It could be shown that a change in the ionic environment induced a structural rearrangement, which consequently confounds the delineation of a measured neurotoxicity toward a distinct Aβ assembly. Here, neuronal binding and hippocampal neurotransmission were found to be suitable to account for the synaptotoxicity to different Aβ assemblies, based on the stability of the applied Aβ aggregates in these settings. In contrast to monomeric or fibrillar Aβ, different prefibrillar Aβ aggregates targeted neurons and impaired hippocampal neurotransmission with nanomolar potency, albeit by different modalities. Spheroidal Aβ aggregates inhibited NMDAR-dependent long-term potentiation, as opposed to protofibrillar Aβ aggregates, which inhibited AMPAR-dominated basal neurotransmission. In addition, a provoked structural conversion of spheroidal to protofibrillar Aβ assemblies resulted in a time-dependent suppression of basal neurotransmission, indicative of a mechanistic switch in synaptic impairment. Thus, we emphasize the importance of addressing the metastability of prefacto characterized Aβ aggregates in assigning a biological effect.

A multi-slice recording system for stable late phase hippocampal long-term potentiation experiments

A major challenge in neuroscience is identifying the cellular and molecular processes underlying learning and memory formation. In the past decades, significant progress has been made in understanding cellular and synaptic mechanisms underlying hippocampal learning and memory using long-term potentiation (LTP) experiments in brain slices as a model system. To expedite LTP measurements it is helpful to further optimize such recording systems. Here, we describe a modification of a multi-slice recording system (SliceMaster, Scientifica Limited, East Sussex, UK) that allows absolutely stable measurements of field excitatory postsynaptic potentials (fEPSPs) for up to 8 h in up to eight slices simultaneously. The software Notocord(®) was used for on-line data acquisition and to control the digital pattern generator which can generate different patterns for slice stimulation, inducing different types of LTP. Moreover, in contrast to common gravity-driven perfusion systems, a Pumped Perfusion System was employed to recycle drug solutions applied to the hippocampal slice. In addition, slices were positioned on two stacked grids for optimal recording of fEPSPs. These two stacked grids were placed in the measuring chambers allowing recordings for several hours without any perturbances. In summary, this modified slice-recording system improves throughput and allows for better statistical design, increases number of used slices per animal and enables very robust LTP measurements for up to 7 h. Hence, this system is suitable not only to investigate molecular mechanisms underlying the late phase of LTP, but also to screen candidate compounds in the context of drug discovery.

Differential effect of the mGlu5 receptor positive allosteric modulator ADX-47273 on early and late hippocampal LTP.

Conflicting findings are reported in the literature about the involvement of the mGlu5 receptor in hippocampal long-term potentiation (LTP), which might be a consequence of different sub-types of LTP induced by the investigators due to the specific experimental conditions used. A comparable controversy came up in the past concerning the influence of different experimental conditions on the involvement of L-type voltage dependent calcium channels (L-VDCCs) and NMDA receptors in hippocampal LTP. In this study, two stimulation protocols with otherwise identical conditions were used to probe modulatory effects of mGlu5 receptor activation in NMDA receptor and L-VDCCs dependent CA1 LTP: weak high frequency stimulation (20 stimuli at 100 Hz) to induce early LTP and repeated strong high frequency stimulation (3 times 100 stimuli at 100 Hz with 5 min interval) to induce late LTP, which - in contrast to early LTP - was shown to be protein-synthesis dependent. Using the NMDA receptor antagonist MK-801 and the L-type calcium channel blocker nifedipine, early LTP was shown to be dependent on NMDA receptors only, whereas late LTP was demonstrated to be dependent on NMDA receptors and L-VDCCs in about equal parts. Moreover, late LTP, but not early LTP, was increased by the mGlu5 receptor positive allosteric modulator ADX-47273, indicating that artificial augmentation of mGlu5 receptor activation by endogenous glutamate may boost the protein-synthesis dependent form of LTP but not the protein-synthesis independent form.

Automated classification of the behavior of rats in the forced swimming test with support vector machines

The forced swimming test of rats or mice is a frequently used behavioral test to evaluate compounds for antidepressant activity in vivo. The aim of this study was to replace the human observer, needed to score and analyze the behavior of animals, by a fully automated method. For this purpose, in a first step from a video recording of each rat, an activity profile was calculated, from which subsequently a set of meaningful properties was extracted. This set was finally used to train a Support Vector Machine (SVM). Furthermore, specialized kernel functions, namely the so-called time resolved p-spectrum and modified optimal assignment kernels, were developed to calculate the similarity of activity profiles. Our method allows for a very reliable discrimination of animals treated with antidepressants of different classes (tricyclics imipramine and desipramine as well as selective serotonin reuptake inhibitor, SSRI, fluoxetine) versus a vehicle-treated group. Moreover, our technique is able to classify between tricyclic antidepressants and SSRIs. The results of this work enabled the development of an automated and highly accurate behavior classification system.