Fractionated brain X-irradiation profoundly reduces hippocampal immature neuron numbers without affecting spontaneous behavior in mice.

Whole brain radiotherapy (WBRT) is used to improve tumor control in patients with primary brain tumors, or brain metastasis from various primary tumors to improve tumor control. However, WBRT can lead to cognitive decline in patients. We assessed whether fractionated WBRT (fWBRT) affects spontaneous behavior of mice in automated home cages and cognition (spatial memory) using the Barnes maze. Male C57Bl/6j mice received bi-lateral fWBRT at a dosage of 4 Gy/day on 5 consecutive days. In line with previous reports, immunohistochemical analysis of doublecortin positive cells in the dentate gyrus showed a profound reduction in immature neurons 4 weeks after fWBRT. Surprisingly, spontaneous behavior as measured in automated home cages was not affected. Moreover, learning and memory measured with Barnes maze, was also not affected 4-6 weeks after fWBRT. At 10-11 weeks after fWBRT a significant difference in escape latency during the learning phase, but not in the probe test of the Barnes maze was observed. In conclusion, although we confirmed the serious adverse effect of fWBRT on neurogenesis 4 weeks after fWBRT, we did not find similar profound effects on spontaneous behavior in the automated home cage nor on learning abilities as measured by the Barnes maze. The relationship between the neurobiological effects of fWBRT and cognition seems more complex than often assumed and the choice of animal model, cognitive tasks, neurobiological parameters, and experimental set-up might be important factors in these types of experiments.

Neurobiological changes by cytotoxic agents in mice.

Cognitive deficit is a frequently reported side-effect of adjuvant chemotherapy. A large number of animal studies has been performed to examine the neurobiological mechanisms underlying this phenomenon, however, definite conclusions from these studies are restricted due to differences in experimental set-up. We systematically investigated the effects of 6 cytotoxic agents on various neurobiological parameters. C57Bl/6J mice were treated with cyclophosphamide, docetaxel, doxorubicin, 5-fluorouracil, methotrexate, or topotecan. The animals were sacrificed 3 or 15 weeks after treatment and the effect on neurogenesis, blood vessel density, and neuroinflammation was analyzed using immunohistochemistry. None of the cytostatic agents tested affected neurogenesis (cell survival or cell proliferation). Blood vessel density was increased in the hippocampus and prefrontal cortex 3 weeks after treatment with docetaxel and doxorubicin compared with control animals. A decrease in the number of microglial cells was observed in the prefrontal cortex after treatment with cyclophosphamide, docetaxel, 5-FU, and topotecan compared with control mice. The observed decrease in microglia cells is indicative of inflammation that occurred after treatment. Overall, the magnitude of the effects was relatively modest. Therefore, we conducted a similar study with topotecan in Abcg2;Abcb1a/b knock out and wildtype FVB mice. Animals were sacrificed 3 weeks after treatment and no notable effect was seen in hippocampal cell differentiation (DCX), microglia activation, or blood vessel density. Perhaps the FVB strain is more resistant to the neurotoxic effects of topotecan which makes this not the correct model to study the mechanism of chemotherapy-induced cognitive impairment.

Neurobeachin Regulates Glutamate- and GABA-Receptor Targeting to Synapses via Distinct Pathways.

Neurotransmission and synaptic strength depend on expression of post-synaptic receptors on the cell surface. Post-translational modification of receptors, trafficking to the synapse through the secretory pathway, and subsequent insertion into the synapse involves interaction of the receptor with A-kinase anchor proteins (AKAPs) and scaffolding proteins. Neurobeachin (Nbea), a brain specific AKAP, is required for synaptic surface expression of both glutamate and GABA receptors. Here, we investigated the role of Nbea-dependent targeting of postsynaptic receptors by studying Nbea interaction with synapse-associated protein 102 (SAP102/Dlg3) and protein kinase A subunit II (PKA II). A Nbea mutant lacking the PKA binding domain showed a similar distribution as wild-type Nbea in Nbea null neurons and partially restored GABA receptor surface expression. To understand the relevance of Nbea interaction with SAP102, we analysed SAP102 null mutant mice. Nbea levels were reduced by ~80% in SAP102 null mice, but glutamatergic receptor expression was normal. A single-point mutation in the pleckstrin homology domain of Nbea (E2218R) resulted in loss of binding with SAP102. When expressed in Nbea null neurons, this mutant fully restored GABA receptor surface expression, but not glutamate receptor expression. Our results suggest that the PKA-binding domain is not essential for Nbea's role in receptor targeting and that Nbea targets glutamate and GABA receptors to the synapse via distinct molecular pathways by interacting with specific effector proteins.

HCN channels are a novel therapeutic target for cognitive dysfunction in Neurofibromatosis type 1.

Cognitive impairments are a major clinical feature of the common neurogenetic disease neurofibromatosis type 1 (NF1). Previous studies have demonstrated that increased neuronal inhibition underlies the learning deficits in NF1, however, the molecular mechanism underlying this cell-type specificity has remained unknown. Here, we identify an interneuron-specific attenuation of hyperpolarization-activated cyclic nucleotide-gated (HCN) current as the cause for increased inhibition in Nf1 mutants. Mechanistically, we demonstrate that HCN1 is a novel NF1-interacting protein for which loss of NF1 results in a concomitant increase of interneuron excitability. Furthermore, the HCN channel agonist lamotrigine rescued the electrophysiological and cognitive deficits in two independent Nf1 mouse models, thereby establishing the importance of HCN channel dysfunction in NF1. Together, our results provide detailed mechanistic insights into the pathophysiology of NF1-associated cognitive defects, and identify a novel target for clinical drug development.

Cognitive impact of cytotoxic agents in mice.

RATIONALE AND OBJECTIVES: Adjuvant chemotherapy is associated with changes in cognition in a subgroup of cancer patients. Chemotherapy is generally given as a combination of cytotoxic agents, which makes it hard to define the agent responsible for these observed changes. Literature on animal experiments has been difficult to interpret due to variance in experimental setup. METHODS: We examined the effects of cytotoxic agents administered separately on various cognitive measures in a standardized animal model. Male C57Bl/6 mice received cyclophosphamide, docetaxel, doxorubicin, 5-fluorouracil, methotrexate, or topotecan. These agents represent different compound classes based on their working mechanism and are frequently prescribed in the clinic. A control group received saline. Behavioral testing started 2 or 15 weeks after treatment and included testing general measures of behavior and cognitive task performance: spontaneous behavior in an automated home cage, open field, novel location recognition (NLR), novel object recognition (NOR), Barnes maze, contextual fear conditioning, and a simple choice reaction time task (SCRTT). RESULTS: Cyclophosphamide, docetaxel, and doxorubicin administration affected spontaneous activity in the automated home cage. All cytotoxic agents affected memory (NLR and/or NOR). Spatial memory measured in the Barnes maze was affected after administration with doxorubicin, 5-fluorouracil, and topotecan. Decreased inhibition in the SCRTT was observed after treatment with cyclophosphamide, docetaxel, and topotecan. CONCLUSIONS: Our data show that, in mice, a single treatment with a cytotoxic agent causes cognitive impairment. Not all cytotoxic agents affected the same cognitive domains, which might be explained by differences in working mechanisms of the various agents.

Strain specificity and cholinergic modulation of visuospatial attention in three inbred mouse strains.

The tremendous increase in the use of mouse inbred strains and mutant mice to study the molecular basis of psychiatric disorders urges for a better understanding of attentional performance in mice. To this aim, we investigated possible strain differences in performance and cholinergic modulation of visuospatial attention in three widely used mouse inbred strains (129S2/SvHsd, C57BL/6JOlaHsd and DBA/2OlaHsd) in the five-choice serial reaction time task. Results indicated that after extended training, performance of 129S2/SvHsd mice was superior to that of C57BL/6JOlaHsd and DBA/2OlaHsd mice in terms of attention, omission errors, inhibitory control and latencies to correct choice. Increasing the attentional load resulted in comparable decrements in attention in all strains and inhibitory control impairments that were most pronounced in DBA/2OlaHsd mice. Further pharmacological evaluation indicated that all strains showed attentional impairments after treatment with the muscarinic and nicotinic antagonists scopolamine and mecamylamine, respectively. 129S2/SvHsd mice were less sensitive, whereas DBA/2OlaHsd mice appeared more sensitive to the detrimental effects of mecamylamine. In addition, subchronic, but not acute, nicotine treatment slightly improved attentional performance in all strains to the same extent. In conclusion, our data indicate strain specificity with particularly good performance of 129S2/SvHsd mice and strong cholinergic involvement in visuospatial attention in mice.

Spatio-temporal dynamics of the egg-laying-inducing peptides during an egg-laying cycle: a semiquantitative matrix-assisted laser desorption/ionization mass spectrometry approach.

The activity-dependent release of peptides from the neuro-endocrine caudodorsal cell (CDC) system of the freshwater snail Lymnaea stagnalis regulates egg laying and related behaviors. In this study, we optimized a mass spectrometry-based approach to study the spatio-temporal dynamics of peptides that are largely derived from the CDC hormone precursor during an egg-laying cycle and a CDC discharge in vitro. Semi-quantitative peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) indicated a massive depletion of peptides from the neurohemal area in the cerebral commissure (COM) during egg laying and the existence of a reserve pool of peptides in the CDC somata that were transported to the COM to restore peptide levels. The depletion of CDC peptides from the COM was correlated to their release during an induced electrical discharge in vitro. Moreover, MALDI-MS of the releasate revealed extensive truncation of the carboxyl terminal peptide. Finally, two novel peptides of 1788 and 5895 Da, not encoded by the CDC hormone precursor, also exhibited temporal quantitative changes similar to those of CDC peptides. Sequencing of the peptide of 1788 Da by tandem mass spectrometry yielded the novel sequence HF(FH)FYGPYDVFQRDVamide. Together, this implicates a more complex set of CDC peptides for the regulation of egg laying than previously anticipated.

Synapse formation between central neurons requires postsynaptic expression of the MEN1 tumor suppressor gene.

Synapse formation is a crucial step in the development of neuronal circuits and requires precise coordination of presynaptic and postsynaptic activities. However, molecular mechanisms that control the formation of functionally mature synaptic contacts, in particular between central neurons, remain poorly understood. To identify genes that are involved in the formation of central synapses, we made use of molluscan neurons that in culture form synaptic contacts between their somata (soma-soma synapses) in the absence of neurite outgrowth. Using single-cell mRNA differential display, we have identified a molluscan homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene encoding the transcription factor menin as a gene that is upregulated during synapse formation. In vitro antisense knock-down of MEN1 mRNA blocks the formation of mature synapses between different types of identified central neurons. Moreover, immunocytochemistry and cell-specific knock-down of MEN1 mRNA show that postsynaptic but not presynaptic expression is required for synapses to form. Together, our data demonstrate that menin is a synaptogenic factor that is critically involved in a general postsynaptic mechanism of synapse formation between central neurons.

Early evolutionary origin of the neurotrophin receptor family.

Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signalling system has been found in invertebrates. We describe a molluscan Trk receptor, designated Ltrk, from the snail Lymnaea stagnalis. The full-length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine-rich motifs flanked by cysteine clusters. In addition, Ltrk has a unique N-terminal extension and lacks immunoglobulin-like domains. Ltrk is expressed during development in a stage-specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT-3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signalling mechanisms may be highly conserved between vertebrates and invertebrates.

Identification of a molluscan homologue of the neuroendocrine polypeptide 7B2.

In vertebrates, interaction of prohormone convertase 2 (PC2) with the highly conserved polypeptide 7B2 is essential for transport and maturation of proPC2 in the regulated secretory pathway. In vitro, 7B2 displays a strong inhibitory activity toward PC2. Here, we characterize a cDNA encoding the first invertebrate 7B2-related protein (L7B2) from the brain of the mollusc Lymnaea stagnalis. The overall amino acid sequence identity between L7B2 and its vertebrate counterparts is surprisingly low (29%) and is restricted to a few small stretches of amino acid residues. Of particular interest are a conserved proline-rich region in the middle portion of the L7B2 sequence and a repeated conserved region in the carboxyl-terminal domain. Synthetic peptides corresponding to the carboxyl-terminal regions inhibit Lymnaea PC2 enzyme activity in extracts of insulin-producing neurons, in which both L7B2 and Lymnaea PC2 are abundantly expressed. Moreover, the peptides inhibit mouse PC2 enzyme activity. Our cloning of invertebrate 7B2 helps to delineate residues that are important for 7B2-PC2 interaction.

CRNF, a molluscan neurotrophic factor that interacts with the p75 neurotrophin receptor.

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis.

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not clear. To investigate this issue, we cloned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressin, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapeptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalian copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

The Light Green Cells of Lymnaea: a neuroendocrine model system for stimulus-induced expression of multiple peptide genes in a single cell type.

We review recent experiments showing that the cerebral neuroendocrine Light Green Cells (LGCs) of the freshwater snail, Lymnaea stagnalis, express a family of distinct though related molluscan insulin-related peptide (MIP) genes. The LGCs are involved in the regulation of a wide range of interrelated life processes associated with growth, (energy) metabolism and reproduction. We consider the mechanism of generation of diversity among MIPs, and present evidence that conditions with distinct effects on growth, metabolism and reproduction also can induce distinct patterns of expression of the MIP and schistosomin genes. The stimulus-dependent expression of multiple neuropeptide genes enormously increases the adaptive potential of a peptidergic neuron. We suggest that this contributes significantly to the information-handling capacity of the brain.

Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection.

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.