Angela Kolodziej

Regional and cellular localization of the CXCl12/SDF-1 chemokine receptor CXCR7 in the developing and adult rat brain.

The chemokine stromal cell‐derived factor‐1 (SDF‐1) regulates neuronal development via the chemokine receptor CXCR4. In the adult brain the SDF‐1/CXCR4 system was implicated in neurogenesis, neuromodulation, brain inflammation, tumor growth, and HIV encephalopathy. Until the recent identification of RDC1/CXCR7 as the second SDF‐1 receptor, CXCR4 was considered to be the only receptor for SDF‐1. Here we provide the first map of CXCR7 mRNA expression in the embryonic and adult rat brain. At embryonic stages, CXCR7 and CXCR4 were codistributed in the germinative zone of the ganglionic eminences, caudate putamen, and along the routes of GABAergic precursors migrating toward the cortex. In the cortex, CXCR7 was identified in GABAergic precursors and in some reelin‐expressing Cajal‐Retzius cells. Unlike CXCR4, CXCR7 was abundant in neurons forming the cortical plate and sparse in the developing dentate gyrus and cerebellar external germinal layer. In the adult brain, CXCR7 was expressed by blood vessels, pyramidal cells in CA3, and mature dentate gyrus granule cells, which is reminiscent of the SDF‐1 pattern. CXCR7 and CXCR4 overlapped in the wall of the four ventricles. Further neuronal structures expressing CXCR7 comprised the olfactory bulb, accumbens shell, supraoptic and ventromedial hypothalamic nuclei, medial thalamus, and brain stem motor nuclei. Also, GLAST‐expressing astrocytes showed signals for CXCR7. Thus, CXCR4 and CXCR7 may cooperate or act independently in SDF‐1‐dependent neuronal development. In mature neurons and blood vessels CXCR7 appears to be the preponderant SDF‐1‐receptor. Indexing terms: J. Comp. Neurol. 510:207–220, 2008.

Reassessment of sst2 Somatostatin Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-1

OBJECTIVE The overexpression of somatostatin receptor 2 (sst2) in neuroendocrine tumors is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analog octreotide. Recent evidence has shown that the immunocytochemical evaluation of sst2A status is of value for predicting response to octreotide therapy and disease prognosis. However, due to the lack of monoclonal and limited availability of specific polyclonal anti-sst2A antibodies, only very few patients can currently benefit from in vitro sst2 evaluation. METHODS In the present study, we extensively characterized the novel rabbit monoclonal anti-sst2A antibody (clone UMB-1) using tissues from sst2-deficient mice and their wild-type littermates. UMB-1 was then subjected to a comparative study of immunohistochemistry on a series of histological specimens from formalin-fixed, paraffin-embedded human tumors and adjacent normal tissues. RESULTS Immunoprecipitation experiments unequivocally demonstrated that UMB-1 selectively detected its cognate sst2A and did not cross-react with other proteins present in crude tissue homogenates. The UMB-1 monoclonal antibody, when compared with currently available polyclonal antisera, yielded several times more effective immunohistochemical staining of fixed-embedded tissues with a predominance of plasma membrane staining and very low cytoplasmic signal even without heat-based antigen retrieval. In addition, dual immunofluorescence revealed for the first time that the sst2A is present on not only gastrin-containing but also ghrelin-containing cells in human gastric mucosa. CONCLUSION Thus, the rabbit monoclonal antibody UMB-1 may prove of great value in the assessment of sst2A status in human neuroendocrine tumors during routine histopathological examination.

Patterns of SDF-1α and SDF-1γ mRNAs, migration pathways, and phenotypes of CXCR4-expressing neurons in the developing rat telencephalon

Cortical GABAergic neurons originate in the ventral telencephalon, invade the cortex via tangential migration, and integrate into the cortical plate by surface‐directed and ventricle‐directed migration. In mice lacking CXCR4 or SDF‐1, GABAergic neurons fail to complete their migration. It is presently unknown which parts of the migration of CXCR4‐expressing GABAergic neurons are driven by SDF‐1. Here we compared patterns of SDF‐1 isoforms and CXCR4 in the developing rat telencephalon. In the ventral telencephalon, radial glia, striatal, and migratory GABAergic neurons expressed CXCR4. Tangentially migrating CXCR4‐expressing neurons populated the marginal zone and started to invade the lateral intermediate zone at embryonic day (E)14. Until E17 the spread of CXCR4‐expressing neurons in the dorsomedial direction was accompanied by progressive upregulation of SDF‐1α in the dorsomedial intermediate/subventricular zone. In the meninges, SDF‐1α and SDF‐1γ were expressed persistently. During invasion of the cortical plate the orientation of CXCR4‐immunoreactive neurons changed gradually from tangential (E17/E18) to radial (postnatal day [P] 0), which was paralleled by downregulation of SDF‐1α in the intermediate/subventricular zone. At E17, CXCR4‐immunoreactive cells were colabeled with markers for ventral forebrain‐derived neurons (Dlx) but not markers for glutamatergic (Tbr) or subplate (calretinin) neurons. Postnatally, calretinin‐ and somatostatin‐expressing but not parvalbumin‐expressing GABAergic neurons or pyramidal cells contained CXCR4. Pyramidal cells and few large blood vessels expressed SDF‐1α, while microvessels contained SDF‐1γ transcripts. In summary, SDF‐1α is expressed along cortical but not subcortical migration routes of GABAergic neurons. We propose that regulated expression of SDF‐1 in the intermediate/subventricular zone influences lateromedial tangential migration of CXCR4‐expressing GABAergic neurons. J. Comp. Neurol. 502:382–399, 2007.

Tonic Activation of CXC Chemokine Receptor 4 in Immature Granule Cells Supports Neurogenesis in the Adult Dentate Gyrus

Stromal-cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) play a well-established role during embryonic development of dentate gyrus granule cells. However, little is known about the regulation and function of CXCR4 in the postnatal dentate gyrus. Here, we identify a striking mismatch between intense CXCR4 mRNA and limited CXCR4 protein expression in adult rat subgranular layer (SGL) neurons. We demonstrate that CXCR4 protein expression in SGL neurons is progressively lost during postnatal day 15 (P15) to P21. This loss of CXCR4 protein expression was paralleled by a reduction in the number of SDF-1-responsive SGL neurons and a massive upregulation of SDF-1 mRNA in granule cells. Intraventricular infusion of the CXCR4-antagonist AMD3100 dramatically increased CXCR4 protein expression in SGL neurons, suggesting that CXCR4 is tonically activated and downregulated by endogenous SDF-1. Infusion of AMD3100 also facilitated detection of CXCR4 protein in bromodeoxyuridine-, nestin-, and doublecortin-labeled cells and showed that the vast majority of adult-born granule cells transiently expressed CXCR4. Chronic AMD3100 administration impaired formation of new granule cells as well as neurogenesis-dependent long-term recognition of novel objects. Therefore, our findings suggest that tonic activation of CXCR4 in newly formed granule cells by endogenous SDF-1 is essential for neurogenesis-dependent long-term memory in the adult hippocampus.

Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase‐activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP‐selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)‐dependent up‐regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2‐regulated Ser112 of pro‐apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short‐term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK‐dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt‐activating factors and conferred ischemic tolerance to neurons. Thus, ischemia‐induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.

Differential regulation of CXCL12 and PACAP mRNA expression after focal and global ischemia

Pituitary adenylate cyclase activating peptide (PACAP) and the chemokine stromal cell-derived factor (SDF-1) have been implicated in neuroprotection, neurogenesis, and regeneration. Focal ischemia is associated with rapid upregulation of PACAP in perifocal neurons and delayed induction of SDF-1 in hypoxic/ischemic tissues, the latter process being involved in the recruitment of stem cells and inflammatory cells. Here, we studied mRNA patterns of PACAP, SDF-1 and the cognate receptors PAC1 and CXCR4 by in situ hybridization in the rat hippocampus after transient global ischemia, a rat model for programmed death of CA1 pyramidal neurons. Cell death in CA1 was not associated with local induction of PACAP and SDF-1 expression or recruitment of CXCR4-expressing infiltrates. However, there was a transient, almost complete loss of SDF-1 expression in microvessels in all hippocampal regions. Granule cells transiently showed a decrease of SDF-1 and an increase of PACAP expression. While PAC1 mRNA was moderately decreased throughout the hippocampus, CXCR4 expression was selectively increased in the subgranular layer. We propose that altered PACAP and SDF-1 gene expression in granule cells plays a role in regulated neurogenesis after global ischemia. The finding that programmed neuronal death after global ischemia was not associated with SDF-1 upregulation or recruitment of CXCR4-expressing cells is in sharp contrast to SDF-1/CXCR4-mediated infiltration of infarct tissue after focal ischemia. Hence, the different modes of neuronal death after focal and global ischemia are associated with distinct SDF-1 and PACAP gene regulation patterns and distinct reorganization mechanisms.

SPECT-imaging of activity-dependent changes in regional cerebral blood flow induced by electrical and optogenetic self-stimulation in mice

Electrical and optogenetic methods for brain stimulation are widely used in rodents for manipulating behavior and analyzing functional connectivities in neuronal circuits. High-resolution in vivo imaging of the global, brain-wide, activation patterns induced by these stimulations has remained challenging, in particular in awake behaving mice. We here mapped brain activation patterns in awake, intracranially self-stimulating mice using a novel protocol for single-photon emission computed tomography (SPECT) imaging of regional cerebral blood flow (rCBF). Mice were implanted with either electrodes for electrical stimulation of the medial forebrain bundle (mfb-microstim) or with optical fibers for blue-light stimulation of channelrhodopsin-2 expressing neurons in the ventral tegmental area (vta-optostim). After training for self-stimulation by current or light application, respectively, mice were implanted with jugular vein catheters and intravenously injected with the flow tracer 99m-technetium hexamethylpropyleneamine oxime (99mTc-HMPAO) during seven to ten minutes of intracranial self-stimulation or ongoing behavior without stimulation. The 99mTc-brain distributions were mapped in anesthetized animals after stimulation using multipinhole SPECT. Upon self-stimulation rCBF strongly increased at the electrode tip in mfb-microstim mice. In vta-optostim mice peak activations were found outside the stimulation site. Partly overlapping brain-wide networks of activations and deactivations were found in both groups. When testing all self-stimulating mice against all controls highly significant activations were found in the rostromedial nucleus accumbens shell. SPECT-imaging of rCBF using intravenous tracer-injection during ongoing behavior is a new tool for imaging regional brain activation patterns in awake behaving rodents providing higher spatial and temporal resolutions than 18F-2-fluoro-2-dexoyglucose positron emission tomography.

Endogenous opioids inhibit ischemia‐induced generation of immature hippocampal neurons via the µ‐opioid receptor

It is established that hippocampal neurogenesis is dynamically regulated by physiological and pathological stimuli including learning, environmental complexity, mental disorders and brain lesion. Little is known about factors regulating adaptive changes in neurogenesis. Using µ‐opioid receptor (MOP)‐knockout mice we addressed whether endogenous opioids influence ischemia‐induced enhancement of hippocampal neurogenesis. Permanent middle cerebral artery occlusion (MCAO) produced similar corticostriatal infarcts in MOP‐knockout and wildtype mice. Analyses of BrdU/doublecortin‐colabelled cells in the granule cell layer 14 days after MCAO showed that ischemic knockouts contained more immature neurons generated during days 9–11 than wildtypes. After 29 days, similar quantities of BrdU/NeuN‐labelled cells were found in ischemic knockout and wildtype mice, suggesting that granule cells that were formed in excess during days 9–11 in the knockouts were eliminated by day 29. Neurogenesis was similar in knockout and wildtype mice subjected to sham operation. In addition to a transient increase in neurogenesis, MCAO caused a transient up‐regulation of preprodynorphin and preproenkephalin mRNA expression in the granule cell layer. Our findings suggest that activated signalling via endogenous opioids and the MOP limits the enhanced generation of neuronal cells after ischemic corticostriatal lesions.

A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis.

Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia.

Synaptic proteome changes in mouse brain regions upon auditory discrimination learning

Changes in synaptic efficacy underlying learning and memory processes are assumed to be associated with alterations of the protein composition of synapses. Here, we performed a quantitative proteomic screen to monitor changes in the synaptic proteome of four brain areas (auditory cortex, frontal cortex, hippocampus striatum) during auditory learning. Mice were trained in a shuttle box GO/NO‐GO paradigm to discriminate between rising and falling frequency modulated tones to avoid mild electric foot shock. Control‐treated mice received corresponding numbers of either the tones or the foot shocks. Six hours and 24 h later, the composition of a fraction enriched in synaptic cytomatrix‐associated proteins was compared to that obtained from naïve mice by quantitative mass spectrometry. In the synaptic protein fraction obtained from trained mice, the average percentage (±SEM) of downregulated proteins (59.9 ± 0.5%) exceeded that of upregulated proteins (23.5 ± 0.8%) in the brain regions studied. This effect was significantly smaller in foot shock (42.7 ± 0.6% down, 40.7 ± 1.0% up) and tone controls (43.9 ± 1.0% down, 39.7 ± 0.9% up). These data suggest that learning processes initially induce removal and/or degradation of proteins from presynaptic and postsynaptic cytoskeletal matrices before these structures can acquire a new, postlearning organisation. In silico analysis points to a general role of insulin‐like signalling in this process.