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PubMed1983

Persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent.

Cell-free cytoplasmic extracts of the Syrian hamster cell lines C13/SV28 and BHK-21F were immunogenic in Syrian hamsters. The resulting antisera cross-reacted completely with antisera against lymphocytic choriomeningitis virus (LCMV) in an immunoradiometric assay employing BHK-21F antigen. Several other Syrian hamster cell lines not previously known to be infected with LCMV were also strongly positive when assayed for viral antigens. Also, several mouse sera and antisera raised in Syrian hamsters against cells transformed by papovaviruses had high titers of anti-LCMV activity. No cytopathic effect was evident in any of the persistently infected cell lines. Culture media from these cells were not infectious and showed no evidence of defective interfering particles. However, cell-free extracts of all the persistently infected cells contained material capable of transmitting the persistent infection to uninfected cells of Syrian hamsters, rats, mice, green monkeys, and humans. The onset of infection is much slower than when LCMV virions are used. When 2 X 10(6) uninfected BHK cells were treated with an extract from 100 persistently infected cells, the new infection was apparent within about 12 days. When an extract from 10(6) cells was used, the new infection was apparent within about 5 days, but not sooner. The intracellular infectious material was sensitive to treatment with deoxycholate, Nonidet P-40, or ether but resistant to treatment with RNase or trypsin. It was also large (5,000S) and heterodisperse on sucrose gradients. The infectious material was probably contained in large lipid vesicles and their integrity was probably essential for infection. When a few persistently infected cells were cocultivated with many uninfected cells, a few discrete colonies positive for LCMV antigens were observed after about 5 days. Since the culture media were not infectious, the infection probably spread by cell-cell contact. Several different experiments indicated that interferon did not play a major role in mediating persistence in this case. Persistent infections by LCMV can be maintained without expression of extracellular virus particles and without appearance of large amounts of viral antigens on the cell surface. Cell-cell contact could still allow transmission of intracellular infectious material. In an animal, these properties could circumvent immune surveillance.

Animals↗
PubMed1984

Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.

Animals↗
PubMed1986

Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type.

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.

Animals↗
PubMed1995

Canine distemper virus persistence in the nervous system is associated with noncytolytic selective virus spread.

Canine distemper virus (CDV), a negative-strand RNA morbillivirus, causes a progressive demyelinating disease in which virus persistence plays an essential role. The antiviral immune response leads to virus clearance in the inflammatory lesions. However, CDV can replicate and persist outside these inflammatory lesions within the brain. How CDV is capable of persisting in the presence of an effective antiviral immune response is poorly understood. In the present investigation, we studied several aspects of virus replication in primary dog brain cell cultures (DBCC), comparing an attenuated CDV strain and a virulent CDV strain. Confluent DBCC were infected with either virulent A75/17-CDV or attenuated Onderstepoort-CDV and monitored for 60 days. Persistence was not associated with defective virus production, because all mRNAs and corresponding proteins were continuously expressed in the noncytolytic infection. Quantitative measurements did not detect a difference between the two types of infection in the rate of virus transcription and protein synthesis at the level of the single cell. However, electron microscopy and virus titration experiments showed that in the persistent CDV infection virus budding is strongly limited compared with that of the attenuated virus. Morphometry and immunocytochemistry showed profound differences in the way the two viruses spread in the culture. The attenuated CDV spread randomly to immediately adjacent cells, whereas persistent CDV spread selectively to more-distant cells by way of cell processes. In conclusion, the present study supports a mechanism of CDV persistence through selective spread by way of cell processes, enabling virulent CDV to invade the central nervous system without the need of releasing much virus into the extracellular space.

Animals↗
PubMed1995

Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells.

Subacute sclerosing panencephalitis is a slowly progressing fatal human disease of the central nervous system which is a delayed sequel of measles virus (MV) infection. A typical pathological feature of this disease is the presence of viral ribonucleocapsid structures in the form of inclusion bodies and the absence of infectious virus or budding viral particles. The mechanisms governing the establishment and maintenance of a persistent MV infection in brain cells are still largely unknown. To understand the mechanisms underlying MV persistence in neuronal cells, a tissue culture model was studied. Clone NS20Y/MS of the murine neuroblastoma C1300 persistently infected with the wild-type Edmonston strain of MV secretes relatively high levels of alpha/beta interferon (IFN). As shown previously, treatment of the persistently infected cultures with anti-IFN serum converted the persistent state into a productive infection indicated by the appearance of multinucleated giant cells. In this study, we have investigated whether alpha/beta IFN produced by NS20Y/MS cells activates cellular protein tyrosine kinases which will induce tyrosine phosphorylating activity specific to virus-infected cells. We present data to show augmented protein tyrosine kinase activity in the persistently infected cells. We demonstrate that the MV N protein is phosphorylated on tyrosine in addition to serine and threonine in the persistent state but not in NS20Y cells acutely infected with MV.

2',5'-Oligoadenylate Synthetase↗
PubMed1996

Virulence as a positive trait in viral persistence.

A population replacement experiment has been devised to test the ability of a challenge virus to replace the resident virus in a persistently infected cell culture. BHK-21 cells persistently infected with foot-and-mouth disease virus of serotype C (clone C-S8c1) were challenged with a large excess of either the parental foot-and-mouth disease virus C-S8c1, genetically marked variants differing in their degree of virulence, or a mutant rescued after prolonged persistence in BHK-21 cells. After challenge, the composition of the resident virus population in the carrier culture was analyzed by reverse transcription-PCR amplification and nucleotide sequencing. The dominance of the initial persisting virus was seen in all cases, except when virulent viruses were used in the challenge. The experiments document that, paradoxically, virulence can be a positive factor in the reestablishment of a virus population in a persistently infected cell culture. A model based on the selection of virus-resistant cell variants during persistence is proposed to interpret these observations. Implications about the persistence of viruses in their host cells and organisms are discussed.

Animals↗
PubMed1997

Isolation of highly fusogenic variants of simian virus 5 from persistently infected cells that produce and respond to interferon.

A series of experiments were undertaken to examine how interferon and neutralizing antibodies influence the ability of simian virus 5 (SV5) (strain W3) to establish and maintain persistent infections in murine cells. In contrast to the rapid decline in SV5 protein synthesis observed in murine BALB/c fibroblasts (BF cells), which produce and respond to interferon, between 24 and 48 h postinfection there was no inhibition of virus protein synthesis in MSFI- cells, skin fibroblasts derived from alpha/beta-interferon receptor knockout BALB/c mice. Furthermore, the addition of anti-interferon antibodies to the culture medium of infected BF cells significantly reduced the observed decline in virus protein synthesis. Following infection of untreated BF cells, the majority replicated virus but survived the infection and eventually cleared the virus after 8 to 15 days. However, not all the cells were cured, and the cultures became persistently infected. Upon passage of persistently infected cultures, the virus fluxed between active and repressed states as a consequence of interferon production. This resulted in a balance being reached in which only 5 to 20% of the cells were infected at any one time. After 30 passages of the persistently infected cells, highly fusogenic virus variants arose (one of which was isolated and termed W3-f). W3-f remained as sensitive to interferon as the parental W3 isolate but, in the absence of interferon, spread much more rapidly than the parental W3 strain through BF cell monolayers. Sequence analysis revealed no deduced amino acid differences between the F proteins of W3 and W3-f. BF cell cultures persistently infected with W3-f were rapidly cleared of virus by the addition of virus-neutralizing antibodies to the culture medium. In contrast, neutralizing antibodies had little effect on the numbers of cells persistently infected with W3 over several passages. These results suggest that the ability of paramyxoviruses to cause cell-cell fusion may be selected for in vivo as a consequence of their adaptation to the interferon response rather than their need to escape from neutralizing antibodies. The significance of these observations with regard to persistent parainfluenza virus infections in vivo is further discussed.

Animals↗
PubMed1997

Evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection.

High-frequency RNA recombination has been proposed as an important mechanism for generating viral deletion variants of murine coronavirus. Indeed, a number of variants with deletions in the spike glycoprotein have been isolated from persistently infected animals. However, the significance of generating and potentially accumulating deletion variants in the persisting viral RNA population is unclear. To study this issue, we evaluated the evolution of spike variants by examining the population of spike RNA sequences detected in the brains and spinal cords of mice inoculated with coronavirus and sacrificed at 4, 42, or 100 days postinoculation. We focused on the S1 hypervariable region since previous investigators had shown that this region is subject to recombination and deletion. RNA isolated from the brains or spinal cords of infected mice was rescued by reverse transcription-PCR, and the amplified products were cloned and used in differential colony hybridizations to identify individual isolates with deletions. We found that 11 of 20 persistently infected mice harbored spike deletion variants (SDVs), indicating that deletions are common but not required for persistent infection. To determine if a specific type of SDV accumulated during persistence, we sequenced 106 of the deletion isolates. We identified 23 distinct patterns of SDVs, including 5 double-deletion variants. Furthermore, we found that each mouse harbored distinct variants in its central nervous system (CNS), suggesting that SDVs are generated during viral replication in the CNS. Interestingly, mice with the most severe and persisting neurological disease harbored the most prevalent and diverse quasispecies of SDVs. Overall, these findings illustrate the complexity of the population of persisting viral RNAs which may contribute to chronic disease.

Animals↗
PubMed1998

Viruses and cells with mutations affecting viral entry are selected during persistent rotavirus infections of MA104 cells.

To better understand mechanisms of persistent rotavirus infections of cultured cells, we established independent, persistently infected cultures of MA104 cells, using rotavirus strain SA11. The cultures were either passaged when the cells reached confluence or supplemented with fresh medium every 7 days. Viral titers in culture lysates varied from 10(4) to 10(7) PFU per ml during 350 days of culture maintenance. Trypan blue staining indicated that 72 to 100% of cells in the cultures were viable, and immunocytochemical staining using a monoclonal antibody directed against viral protein VP6 demonstrated that 38 to 63% of the cells contained rotavirus antigen. We tested the capacity of rotaviruses isolated from the persistently infected cultures (PI viruses) to infect cells cured of persistent infection. Although wild-type (wt) and PI viruses produced equivalent yields in parental MA104 cells, PI viruses produced greater yields than wt virus in cured cells, which indicates that viruses and cells coevolve during persistent rotavirus infections of MA104 cells. To determine whether mutations in viruses and cells selected during these persistent infections affect viral entry, we tested the effect of trypsin treatment of the viral inoculum on growth of wt and PI viruses. Trypsin pretreatment is required for postattachment penetration of rotavirus virions into cells. In contrast to the case with wt virus, PI viruses produced equivalent yields with and without trypsin pretreatment in parental MA104 cells. However, PI viruses required trypsin pretreatment for efficient growth in cured cells. These results indicate that mutant viruses and cells are selected during maintenance of persistent rotavirus infections of MA104 cells and suggest that mutations in each affect trypsin-dependent steps in rotavirus entry.

Biological Evolution↗
PubMed1999

Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy.

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.

Acute Disease↗
PubMed1999

Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E.

Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability.

Astrocytoma↗
PubMed1999

Acute and persistent infection of human neural cell lines by human coronavirus OC43.

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.

Cell Line↗
PubMed2000

Influence of the Theiler's virus L* protein on macrophage infection, viral persistence, and neurovirulence.

The genome of picornaviruses contains a large open reading frame (ORF) translated as a precursor polypeptide that is processed to yield all the proteins necessary for the viral life cycle. In persistent but not in neurovirulent strains of Theiler's virus, an overlapping ORF encodes an additional 18-kDa protein called L*. We confirmed previous work showing that the L* ORF of persistent strains facilitates the infection of macrophage cell lines, and we present evidence that this effect is due to the L* protein itself rather than to competition for the translation of the two overlapping ORFs. The introduction of an AUG codon to restore the L* ORF of the neurovirulent GDVII strain also enhanced the infection of macrophages, in spite of the divergent evolution of this protein. The presence or the absence of the L* AUG initiation codon had only a weak influence on the neurovirulence of the GDVII strain and on the persistence of the DA1 strain. The results obtained with DA1 in vivo contrast with the results reported previously for DAFL3, another molecular clone of the same virus strain, where the AUG-to-ACG mutation of the L* initiation codon totally blocked viral persistence (G. D. Ghadge, L. Ma, S. Sato, J. Kim, and R. P. Roos, J. Virol. 72:8605-8612, 1998). Thus, a factor that is critical for the persistence of a given clone of Theiler's virus is dispensable for the persistence of a closely related clone, indicating that different adjustments in the expression of persistence determinants occur in related viral strains.

Animals↗
PubMed2002

HLA-Cw*04 and hepatitis C virus persistence.

In studies of acute hepatitis C virus (HCV) infection, the early host immune response is one of the determinants of viral persistence. The class I human leukocyte antigens (HLA), which present foreign antigen to cytolytic T cells, are integral components of this response. We hypothesized that the highly polymorphic HLA genes affect the outcome of an HCV infection. To test this hypothesis, we molecularly typed 231 persons with well-documented clearance of an HCV infection and 444 matched persistently infected persons. HLA-A*1101 (odds ratio [OR], 0.49; 95% confidence interval [95% CI], 0.27 to 0.89), HLA-B*57 (OR, 0.62; 95% CI, 0.39 to 1.00), and HLA-Cw*0102 (OR, 0.43; 95% CI, 0.21 to 0.89) were associated with viral clearance, whereas HLA-A*2301 (OR, 1.78; 95% CI, 1.01 to 3.11) and HLA-Cw*04 (OR, 1.78; 95% CI, 1.21 to 2.59) were associated with viral persistence. HLA-Cw*04 is in strong linkage disequilibrium with HLA-B*53 and HLA-B*35, but only HLA-B*53 (OR, 1.70; 95% CI, 0.95 to 3.06) and the Cw*04-B*53 haplotype (OR, 1.76; 95% CI, 0.94 to 3.26) were weakly associated with viral persistence. HLA-B*53 has similar, but not necessarily identical, binding specificity to some HLA-B*35 subtypes (B*35-Px group). The association with the B*35-Px group was less strong than with HLA-B*53 alone. The association of HLA-Cw*04 with HCV persistence was codominant (two copies of the gene were more strongly associated with persistence than one copy). However, HLA-Cw*04 was not associated with HCV RNA levels among the persistently infected individuals. Since Cw*04 is a ligand for the killer immunoglobulin-like receptors on natural killer cells, these cells may be involved in recovery from HCV infection. Further investigation is needed to understand the relationship between class I alleles and HCV clearance.

Adult↗
PubMed2005

Limited sequence evolution within persistently targeted CD8 epitopes in chronic human immunodeficiency virus type 1 infection.

Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8 T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-gamma) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-gamma responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Association of antecedent malnutrition with persistent diarrhoea: a case-control study.

To determine the effect of nutritional state on persistent diarrhoea a case-control study was carried out on 756 children followed up prospectively for 18 months. Children who developed persistent diarrhoea were compared with population controls and controls with acute diarrhoea. The mean weight for age in the children with persistent diarrhoea (69.9%) was significantly lower than that in the population controls (77.0%) and the diarrhoeal controls (76.2%). Weight for age of less than or equal to 70% was associated with persistent diarrhoea in both case-control analyses (population controls, matched odds ratio 3.25; diarrhoeal controls, matched odds ratio 2.46). The corrected odds (multiple logistic regression) in the two analyses were 3.2 (95% confidence interval 1.3 to 8.1) and 3.4 (1.2 to 9.1). Weight for age of less than or equal to 70% increases the risk of persistent diarrhoea. In an underweight child there is a higher risk of diarrhoea becoming persistent. Prevention of malnutrition and intensive management of acute diarrhoea in malnourished children should help reduce the risk of the diarrhoea persisting.

Age Factors↗
PubMed2002

Mechanisms involved in persistent facilitation of neuromuscular synapses in aplysia.

Synaptic plasticity can last from a fraction of a second to weeks depending on how it was induced. The mechanisms that underlie short-, intermediate-, and long-term plasticity have been intensively studied at central synapses of both vertebrates and invertebrates; however, peripheral plasticity has not received as much attention. In this study, we investigated the mechanisms that contribute to a persistent form of plasticity at neuromuscular synapses in buccal muscle I3a of Aplysia. These synapses are reversibly facilitated by the small cardioactive peptide (SCP), a peptide cotransmitter that is intrinsic to the motor neurons, and persistently facilitated by serotonin (5HT) released from modulatory neurons that are extrinsic to the motor circuit. Many of the short-term effects of 5HT and SCP are mediated by the cAMP pathway, but little is known about the mechanisms that underlie persistent modulation. We were able to eliminate several possible mechanisms. One of these was the possibility that the apparent reversal of SCP's effects was due to desensitization of the SCP receptor. Superfusion for longer periods or with higher concentrations of SCP indicate that the SCP receptors do not desensitize. We also determined that new protein synthesis is not required for the persistent facilitation of EJPs. Another possibility was that 5HT was taken up and slowly re-released. Our results suggest that this mechanism is also unlikely. Activation of the cAMP pathway does not appear to mediate persistent effects; however, 5HT as well as SCP does cause persistent increases in cAMP levels that can prime I3a synapses and increase the effectiveness of activators of the cAMP pathway. Instead, the persistent effects of 5HT are mimicked by phorbol, suggesting that protein kinase C or an Aplysia homologue of unc13 may mediate these effects. These results, in combination with results from experiments on the sensory neurons that contribute to withdrawal reflexes in Aplysia, suggest that the mechanisms for intermediate- and long-term facilitation may reside in all of the synapses involved in the sensory to motor response reflex.

Animals↗
PubMed1996

Isolation and characterization of a persistent potassium current in neostriatal neurons.

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.

4-Aminopyridine↗