Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity

GABAergic (GABA = γ-aminobutyric acid) neurons from different brain regions contain high levels of parvalbumin, both in their soma and in their neurites. Parvalbumin is a slow Ca2+ buffer that may affect the amplitude and time course of intracellular Ca2+ transients in terminals after an action potential, and hence may regulate short-term synaptic plasticity. To test this possibility, we have applied paired-pulse stimulations (with 30- to 300-ms intervals) at GABAergic synapses between interneurons and Purkinje cells, both in wild-type (PV+/+) mice and in parvalbumin knockout (PV−/−) mice. We observed paired-pulse depression in PV+/+ mice, but paired-pulse facilitation in PV−/− mice. In paired recordings of connected interneuron-Purkinje cells, dialysis of the presynaptic interneuron with the slow Ca2+ buffer EGTA (1 mM) rescues paired-pulse depression in PV−/− mice. These data show that parvalbumin potently modulates short-term synaptic plasticity.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.

Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases δ and ɛ, is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases δ and ɛ. In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to γ-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40–45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after γ-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after γ-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumor-induced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.

An orchestrated gene expression component of neuronal programmed cell death revealed by cDNA array analysis

Programmed cell death (PCD) during neuronal development and disease has been shown to require de novo RNA synthesis. However, the time course and regulation of target genes is poorly understood. By using a brain-biased array of over 7,500 cDNAs, we profiled this gene expression component of PCD in cerebellar granule neurons challenged separately by potassium withdrawal, combined potassium and serum withdrawal, and kainic acid administration. We found that hundreds of genes were significantly regulated in discreet waves including known genes whose protein products are involved in PCD. A restricted set of genes was regulated by all models, providing evidence that signals inducing PCD can regulate large assemblages of genes (of which a restricted subset may be shared in multiple pathways).

Urocortin II: A member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)+ RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1–10 μg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Activation of Trk neurotrophin receptors in the absence of neurotrophins

Neurotrophins regulate neuronal cell survival and synaptic plasticity through activation of Trk receptor tyrosine kinases. Binding of neurotrophins to Trk receptors results in receptor autophosphorylation and downstream phosphorylation cascades. Here, we describe an approach to use small molecule agonists to transactivate Trk neurotrophin receptors. Activation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons was observed after treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors. These effects were reproduced by using the adenosine agonist CGS 21680 and were counteracted with the antagonist ZM 241385, indicating that this transactivation event by adenosine involves adenosine 2A receptors. The increase in Trk activity could be inhibited by the use of the Src family-specific inhibitor, PP1, or K252a, an inhibitor of Trk receptors. In contrast to other G protein-coupled receptor transactivation events, adenosine used Trk receptor signaling with a longer time course. Moreover, adenosine activated phosphatidylinositol 3-kinase/Akt through a Trk-dependent mechanism that resulted in increased cell survival after nerve growth factor or brain-derived neurotrophic factor withdrawal. Therefore, adenosine acting through the A2A receptors exerts a trophic effect through the engagement of Trk receptors. These results provide an explanation for neuroprotective actions of adenosine through a unique signaling mechanism and raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases.

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

Gamma oscillations synchronized between distant neuronal populations may be critical for binding together brain regions devoted to common processing tasks. Network modeling predicts that such synchrony depends in part on the fast time course of excitatory postsynaptic potentials (EPSPs) in interneurons, and that even moderate slowing of this time course will disrupt synchrony. We generated mice with slowed interneuron EPSPs by gene targeting, in which the gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was altered to drive expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit GluR-B. GluR-B is a determinant of the relatively slow EPSPs in excitatory neurons and is normally expressed at low levels in γ-aminobutyric acid (GABA)ergic interneurons, but at high levels in the GAD-GluR-B mice. In both wild-type and GAD-GluR-B mice, tetanic stimuli evoked gamma oscillations that were indistinguishable in local field potential recordings. Remarkably, however, oscillation synchrony between spatially separated sites was severely disrupted in the mutant, in association with changes in interneuron firing patterns. The congruence between mouse and model suggests that the rapid time course of AMPA receptor-mediated EPSPs in interneurons might serve to allow gamma oscillations to synchronize over distance.

The Mechanism of Rhythmic Ethylene Production in Sorghum. The Role of Phytochrome B and Simulated Shading1

Mutant sorghum (Sorghum bicolor [L.] Moench) deficient in functional phytochrome B exhibits reduced photoperiodic sensitivity and constitutively expresses a shade-avoidance phenotype. Under relatively bright, high red:far-red light, ethylene production by seedlings of wild-type and phytochrome B-mutant cultivars progresses through cycles in a circadian rhythm; however, the phytochrome B mutant produces ethylene peaks with approximately 10 times the amplitude of the wild type. Time-course northern blots show that the mutant's abundance of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase mRNA SbACO2 is cyclic and is commensurate with ethylene production, and that ACC oxidase activity follows the same pattern. Both SbACO2 abundance and ACC oxidase activity in the wild-type plant are very low under this regimen. ACC levels in the two cultivars did not demonstrate fluctuations coincident with the ethylene produced. Simulated shading caused the wild-type plant to mimic the phenotype of the mutant and to produce high amplitude rhythms of ethylene evolution. The circadian feature of the ethylene cycle is conditionally present in the mutant and absent in the wild-type plant under simulated shading. SbACO2 abundance in both cultivars demonstrates a high-amplitude diurnal cycle under these conditions; however, ACC oxidase activity, although elevated, does not exhibit a clear rhythm correlated with ethylene production. ACC levels in both cultivars show fluctuations corresponding to the ethylene rhythm previously observed. It appears that at least two separate mechanisms may be involved in generating high-amplitude ethylene rhythms in sorghum, one in response to the loss of phytochrome B function and another in response to shading.

Adventitious Root Growth and Cell-Cycle Induction in Deepwater Rice1

Deepwater rice (Oryza sativa) is adapted to survive conditions of severe flooding over extended periods of time. During such periods adventitious roots develop to provide water, nutrients, and anchorage. In the present study the growth of adventitious roots was induced by treatment with ethylene but not auxin, cytokinin, or gibberellin. Root elongation was enhanced between 8 and 10 h after submergence. The population of cells in the S phase and expression of the S-phase-specific histone H3 gene increased within 4 to 6 h. Within 6 to 8 h the G2-phase population increased. Cell-cycle activation was accompanied by sequential induction of a cdc2-activating kinase homolog, R2, of two cdc2 genes, cdc2Os-1 and cdc2Os-2, and of three cyclin genes, cycA1;3, cycB2;1, and cycB2;2, but only induction of the R2 gene expression preceded the induction of the S phase, possibly contributing to cell-cycle regulation in the G1 phase. Both cdc2 genes were expressed at slightly higher levels during DNA replication. Transcripts of the A-type cyclin accumulated during the S and G2 phases, and transcripts of the B-type cyclins accumulated during the G2 phase. Cyclin expression was induced at all nodes with a similar time course, suggesting that ethylene acts systemically and that root primordia respond to ethylene at an early developmental stage.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.

Causalgia, pathological pain, and adrenergic receptors

Control of expression of molecular receptors for chemical messengers and modulation of these receptors’ activity are now established as ways to alter cellular reaction. This paper extends these mechanisms to the arena of pathological pain by presenting the hypothesis that increased expression of α-adrenergic receptors in primary afferent neurons is part of the etiology of pain in classical causalgia. It is argued that partial denervation by lesion of peripheral nerve or by tissue destruction induces a change in peripheral nociceptors, making them excitable by sympathetic activity and adrenergic substances. This excitation is mediated by α-adrenergic receptors and has a time course reminiscent of experimental denervation supersensitivity. The change in neuronal phenotype is demonstrable after lesions of mixed nerves or of the sympathetic postganglionic supply. Similar partial denervations also produce a substantial increase in the number of dorsal root ganglion neurons evidencing the presence of α-adrenergic receptors. The hypothesis proposes the increased presence of α-adrenergic receptors in primary afferent neurons to result from an altered gene expression triggered by cytokines/growth factors produced by disconnection of peripheral nerve fibers from their cell bodies. These additional adrenergic receptors are suggested to make nociceptors and other primary afferent neurons excitable by local or circulating norepinephrine and epinephrine. For central pathways, the adrenergic excitation would be equivalent to that produced by noxious events and would consequently evoke pain. In support, evidence is cited for a form of denervation supersensitivity in causalgia and for increased expression of human α-adrenergic receptors after loss of sympathetic activity.

Event-related brain potentials in the study of visual selective attention

Event-related brain potentials (ERPs) provide high-resolution measures of the time course of neuronal activity patterns associated with perceptual and cognitive processes. New techniques for ERP source analysis and comparisons with data from blood-flow neuroimaging studies enable improved localization of cortical activity during visual selective attention. ERP modulations during spatial attention point toward a mechanism of gain control over information flow in extrastriate visual cortical pathways, starting about 80 ms after stimulus onset. Paying attention to nonspatial features such as color, motion, or shape is manifested by qualitatively different ERP patterns in multiple cortical areas that begin with latencies of 100–150 ms. The processing of nonspatial features seems to be contingent upon the prior selection of location, consistent with early selection theories of attention and with the hypothesis that spatial attention is “special.”

Spatially independent activity patterns in functional MRI data during the Stroop color-naming task

A method is given for determining the time course and spatial extent of consistently and transiently task-related activations from other physiological and artifactual components that contribute to functional MRI (fMRI) recordings. Independent component analysis (ICA) was used to analyze two fMRI data sets from a subject performing 6-min trials composed of alternating 40-sec Stroop color-naming and control task blocks. Each component consisted of a fixed three-dimensional spatial distribution of brain voxel values (a “map”) and an associated time course of activation. For each trial, the algorithm detected, without a priori knowledge of their spatial or temporal structure, one consistently task-related component activated during each Stroop task block, plus several transiently task-related components activated at the onset of one or two of the Stroop task blocks only. Activation patterns occurring during only part of the fMRI trial are not observed with other techniques, because their time courses cannot easily be known in advance. Other ICA components were related to physiological pulsations, head movements, or machine noise. By using higher-order statistics to specify stricter criteria for spatial independence between component maps, ICA produced improved estimates of the temporal and spatial extent of task-related activation in our data compared with principal component analysis (PCA). ICA appears to be a promising tool for exploratory analysis of fMRI data, particularly when the time courses of activation are not known in advance.