The gut microbiota and metabolome are associated with diminished COVID-19 vaccine-induced antibody responses in immunosuppressed inflammatory bowel disease patients.

BACKGROUND: Patients with inflammatory bowel disease (IBD) treated with anti-TNF therapy exhibit attenuated humoral immune responses to vaccination against SARS-CoV-2. The gut microbiota and its functional metabolic output, which are perturbed in IBD, play an important role in shaping host immune responses. We explored whether the gut microbiota and metabolome could explain variation in anti-SARS-CoV-2 vaccination responses in immunosuppressed IBD patients. METHODS: Faecal and serum samples were prospectively collected from infliximab-treated patients with IBD in the CLARITY-IBD study undergoing vaccination against SARS-CoV-2. Antibody responses were measured following two doses of either ChAdOx1 nCoV-19 or BNT162b2 vaccine. Patients were classified as having responses above or below the geometric mean of the wider CLARITY-IBD cohort. 16S rRNA gene amplicon sequencing, nuclear magnetic resonance (NMR) spectroscopy and bile acid profiling with ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS) were performed on faecal samples. Univariate, multivariable and correlation analyses were performed to determine gut microbial and metabolomic predictors of response to vaccination. FINDINGS: Forty-three infliximab-treated patients with IBD were recruited (30 Crohn's disease, 12 ulcerative colitis, 1 IBD-unclassified; 26 with concomitant thiopurine therapy). Eight patients had evidence of prior SARS-CoV-2 infection. Seventeen patients (39.5%) had a serological response below the geometric mean. Gut microbiota diversity was lower in below average responders (p = 0.037). Bilophila abundance was associated with better serological response, while Streptococcus was associated with poorer response. The faecal metabolome was distinct between above and below average responders (OPLS-DA R2X 0.25, R2Y 0.26, Q2 0.15; CV-ANOVA p = 0.038). Trimethylamine, isobutyrate and omega-muricholic acid were associated with better response, while succinate, phenylalanine, taurolithocholate and taurodeoxycholate were associated with poorer response. INTERPRETATION: Our data suggest that there is an association between the gut microbiota and variable serological response to vaccination against SARS-CoV-2 in immunocompromised patients. Microbial metabolites including trimethylamine may be important in mitigating anti-TNF-induced attenuation of the immune response. FUNDING: JLA is the recipient of an NIHR Academic Clinical Lectureship (CL-2019-21-502), funded by Imperial College London and The Joyce and Norman Freed Charitable Trust. BHM is the recipient of an NIHR Academic Clinical Lectureship (CL-2019-21-002). The Division of Digestive Diseases at Imperial College London receives financial and infrastructure support from the NIHR Imperial Biomedical Research Centre (BRC) based at Imperial College Healthcare NHS Trust and Imperial College London. Metabolomics studies were performed at the MRC-NIHR National Phenome Centre at Imperial College London; this work was supported by the Medical Research Council (MRC), the National Institute of Health Research (NIHR) (grant number MC_PC_12025) and infrastructure support was provided by the NIHR Imperial Biomedical Research Centre (BRC). The NIHR Exeter Clinical Research Facility is a partnership between the University of Exeter Medical School College of Medicine and Health, and Royal Devon and Exeter NHS Foundation Trust. This project is supported by the National Institute for Health Research (NIHR) Exeter Clinical Research Facility. The views expressed are those of the authors and not necessarily those of the NIHR or the UK Department of Health and Social Care.

CytokineLink: a cytokine communication map to analyse immune responses in inflammatory and infectious diseases

Intercellular communication mediated by cytokines is critical to the development of immune responses, particularly in the context of infectious and inflammatory diseases. By releasing these small molecular weight peptides, the source cells can influence numerous intracellular processes in the target cells, including the secretion of other cytokines downstream. However, there are no readily available bioinformatic resources that can model cytokine - cytokine interactions. In this effort, we built a communication map between major tissues and blood cells that reveals how cytokine-mediated intercellular networks form during homeostatic conditions. We collated the most prevalent cytokines from literature, and assigned the proteins and their corresponding receptors to source tissue and blood cell types based on enriched consensus RNA-Seq data from the Human Protein Atlas database. To assign more confidence to the interactions, we integrated literature information on cell - cytokine interactions from two systems immunology databases, immuneXpresso and ImmunoGlobe. From the collated information, we defined two metanetworks: a cell-cell communication network connected by cytokines; and a cytokine-cytokine interaction network depicting the potential ways in which cytokines can affect the activity of each other. Using expression data from disease states, we then applied this resource to reveal perturbations in cytokine-mediated intercellular signalling in inflammatory and infectious diseases (ulcerative colitis and COVID-19, respectively). For ulcerative colitis, with CytokineLink we demonstrated a significant rewiring of cytokine-mediated intercellular communication between non-inflamed and inflamed colonic tissues. For COVID-19, we were able to identify inactive cell types and cytokine interactions that may be important following SARS-CoV-2 infection when comparing the cytokine response with other viruses capable of initiating a cytokine storm. Such findings have potential to inform the development of novel, cytokine-targeted therapeutic strategies. CytokineLink is freely available for the scientific community through the NDEx platform and the project github repository (https://github.com/korcsmarosgroup/CytokineLink).

Inhibition of the neuromuscular acetylcholine receptor with atracurium activates FOXO/DAF-16-induced longevity.

Transcriptome-based drug screening is emerging as a powerful tool to identify geroprotective compounds to intervene in age-related disease. We hypothesized that, by mimicking the transcriptional signature of the highly conserved longevity intervention of FOXO3 (daf-16 in worms) overexpression, we could identify and repurpose compounds with similar downstream effects to increase longevity. Our in silico screen, utilizing the LINCS transcriptome database of genetic and compound interventions, identified several FDA-approved compounds that activate FOXO downstream targets in mammalian cells. These included the neuromuscular blocker atracurium, which also robustly extends both lifespan and healthspan in Caenorhabditis elegans. This longevity is dependent on both daf-16 signaling and inhibition of the neuromuscular acetylcholine receptor subunit unc-38. We found unc-38 RNAi to improve healthspan, lifespan, and stimulate DAF-16 nuclear localization, similar to atracurium treatment. Finally, using RNA-seq transcriptomics, we identify atracurium activation of DAF-16 downstream effectors. Together, these data demonstrate the capacity to mimic genetic lifespan interventions with drugs, and in doing so, reveal that the neuromuscular acetylcholine receptor regulates the highly conserved FOXO/DAF-16 longevity pathway.

Effect of Cannabidiol on the Long-Term Toxicity and Lifespan in the Preclinical Model Caenorhabditis elegans.

Introduction: Despite widespread use of cannabidiol (CBD), no lifelong toxicity study has been published to date. Caenorhabditis elegans is often used in preclinical lifelong toxicity studies, due to an estimated 60-80% of their genes having a human ortholog, and their short lifespan of ∼2-3 weeks. In this study, we examined both acute and long-term exposure studies of CBD at physiologically relevant concentrations. Materials and Methods: Acute toxicity was determined by treating day 1 adults with a wide range of CBD concentrations (0.4 µM to 4 mM) and assessing mortality and motility compared to control animals. Thermotolerance was examined by treating adult animals with CBD (0.4 µM to 4 mM) and exposing them to 37°C for 4 h, and then scoring for the number of alive animals treated with CBD compared to controls. Long-term toxicity was assessed by exposing day 1 adults to 10, 40, and 100 µM CBD until all animals perished. Control animals had no active drug exposure. Results: We report both acute and long-term exposure studies of CBD to adult C. elegans at physiologically relevant concentrations. Acute toxicity results showed that no animal died when exposed to 0.4-4000 µM CBD. The thermotolerance study showed that 40 µM CBD, but not other treatment levels, significantly increased resistance to heat stress by 141% compared to the untreated controls. Notably, whole-life exposure of C. elegans to 10-100 µM CBD revealed a maximum life extension of 18% observed at 40 µM CBD. In addition, motility analysis of the same groups revealed an increase in late-stage life activity by up to 206% compared to controls. Conclusion: These results serve as the only CBD lifelong exposure data in an in vivo model to date. While further research into the lifelong use of CBD should be carried out in mammalian models, the C. elegans model indicates a lack of long-term toxicity at physiologically relevant concentrations.

SARS-CoV-2 causes a different cytokine response compared to other cytokine storm-causing respiratory viruses in severely ill patients

Hyper-induction of pro-inflammatory cytokines, also known as a cytokine storm or cytokine release syndrome (CRS) is one of the key aspects of the currently ongoing SARS-CoV-2 pandemic. This process occurs when a large number of innate and adaptive immune cells are activated, and start producing pro-inflammatory cytokines, establishing an exacerbated feedback loop of inflammation. It is one of the factors contributing to the mortality observed with COVID-19 for a subgroup of patients. CRS is not unique to SARS-CoV-2 infection; it was prevalent in most of the major human coronavirus and influenza A subtype outbreaks of the past two decades (H5N1, SARS-CoV, MERS-CoV, H7N9). Here, we collected changing cytokine levels upon infection with the aforementioned viral pathogens through a comprehensive literature search. We analysed published patient data to highlight the conserved and unique cytokine responses caused by these viruses. A map of such responses could help specialists identify interventions that successfully alleviated CRS in different diseases and evaluate whether they could be used in COVID-19 cases.

C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress.

The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 µm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neurons that did not produce exophers. The extruded exopher transits through surrounding tissue in which some contents appear degraded, but some non-degradable materials can subsequently be found in more remote cells, suggesting secondary release. Our observations suggest that exopher-genesis is a potential response to rid cells of neurotoxic components when proteostasis and organelle function are challenged. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.

Copper Uptake Efficiency and Its Distribution Within Bioenergy Grass Giant Reed.

To evaluate copper uptake and its toxicity on bioenergy grass giant reed (Arundo donax L.), experiments were carried out using two epigenetic clonal lines - American (BL) and Hungarian (20SZ) ecotypes - grown on elevated Cu concentrations up to 26.8 mg L(-1). Neither ecotype showed any noticeable foliar symptoms of Cu toxicity at concentrations tested up to 10 mg L(-1). Dry mass of plants of both ecotypes significantly increased at the highest Cu treatment compared to control. Although the BL ecotype had greater capacity to uptake Cu than 20SZ, the dry mass and shoot length of BL was higher than that of 20SZ. Values of bioconcentration and transportation factors were higher in the BL than in the 20SZ ecotype. Almost 45 % of total Cu content within the whole plant was found in the plant root of both ecotypes. This demonstrated both ecotypes can be utilized for Cu phytoremediation alongside with significant biomass production.

Laboratory and Clinical features of EIA Toxin-positive and EIA Toxin-negative Community-acquired Clostridium difficile Infection.

Studies have described the clinical course of patients with Clostridium difficile infection (CDI) with positive enzyme immunoassay (EIA) for toxins A and B. Limited information is available for the patients with negative EIA but positive for the toxin B gene (TcdB) by the PCR. The aim of our study is to determine if there are any differences that exist among the clinical and laboratory parameters in the patients tested to be positive by EIA for toxin and those who were negative. This is a retrospective cohort study conducted in a 700-bed teaching hospital. We reviewed charts of the patients with presumptive CDI between January 2006 and July 2013. We divided these patients into two groups, EIA-positive and EIA-negative, based on result of EIA for toxins A and B and the requirement for a positive PCR analysis of the TcdB gene. The EIA-positive group had significantly higher white blood cell counts (p<0.001), with a significantly greater percentage of bands (p<0.0001). Albumin and total protein both exhibit significantly (p<0.0001, both comparisons) lower values in the EIA-positive group. Among clinical findings, the EIA-positive group had significantly longer length of hospital stay (p=0.010). These data suggest that an infection with an EIA-negative strain of C. difficile presents laboratory markers closer to those of healthy subjects and clinical features suggesting considerably less severe than infection with EIA-positive C. difficile.

Flow-scanning optical tomography.

We present a 3D tomography technique for in vivo observation of microscopic samples. The method combines flow in a microfluidic channel, illumination through a slit aperture, and a Fourier lens for simultaneous acquisition of multiple perspective angles in the phase-space domain. The technique is non-invasive and naturally robust to parasitic sample motion. 3D absorption is retrieved using standard back-projection algorithms, here a limited-domain inverse radon transform. Simultaneously, 3D differential phase contrast images are obtained by computational refocusing and comparison of complementary illumination angles. We implement the technique on a modified glass slide which can be mounted directly on existing optical microscopes. We demonstrate both amplitude and phase tomography on live, freely swimming C. elegans nematodes.

Phytoaccumulation potentials of two biotechnologically propagated ecotypes of Arundo donax in copper-contaminated synthetic wastewater.

An in vitro experiment was carried out to evaluate the phytoremediation potentials of two somatic embryo-derived ecotypes of Arundo donax-BL (American ecotype) and 20SZ (Hungarian ecotype)-of copper from synthetic wastewater. The two ecotypes were grown under sterile conditions in tubes containing a nutrient solution supplied with increasing doses of Cu (0, 1, 2, 3, 5, 10, and 26.8 mg L(-1)) for 6 weeks. The translocation and bioaccumulation factors and removal rate were estimated. In general, increasing Cu concentration in nutrient solution slightly decreased root, stem and leaf biomass without toxicity symptoms up to 26.8 mg L(-1). Moreover, both ecotypes showed high Cu removal efficiency from aqueous solution. However, Cu removal rate ranged between 96.6 to 98.8% for BL ecotype and 97 to 100% for 20SZ ecotype. Data illustrated that both BL and 20SZ ecotypes may be employed to treat Cu-contaminated water bodies up to 26.8 mg L(-1).

The polyamine analog PG11047 potentiates the antitumor activity of cisplatin and bevacizumab in preclinical models of lung and prostate cancer.

PURPOSE: PG11047 is a polyamine analog currently in Phase I trials for advanced cancer as a monotherapy and in combination with a number of approved anti-cancer agents. The use of polyamines as a target for antiproliferative therapy is based on findings that cells synthesize polyamines excessively when induced to grow and that polyamine metabolism is frequently dysregulated in cancer. A selective polyamine transport system provides access for PG11047 into rapidly dividing cells to inhibit polyamine biosynthetic enzymes, to induce the polyamine catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) which could subsequently induce reactive oxygen species that contribute to tumor cell responses to PG11047, and to function as a polyamine with altered function when it binds to natural polyamine binding sites. The objective of the present study was to assess the antitumor effects of PG11047 alone and in combination with approved anti-cancer agents. METHODS: The antitumor efficacy of PG11047 as a single agent, and in combination with cisplatin and bevacizumab, was tested in models of lung (A549) and prostate (DU-145) cancer, respectively. RESULTS: PG11047 significantly inhibited tumor development in both lung and prostate cancer models when administered as a single agent. In the lung cancer model, PG11047 potentiated the antitumor effect of cisplatin. Although potent activity was observed with PG11047 and bevacizumab when administered as single agents in the prostate cancer model, the combination arm significantly enhanced antitumor activity compared with either agent alone. In all experiments, PG11047 was well tolerated with no adverse effects on bodyweight gain. CONCLUSIONS: The preclinical data support the rationale for the current Phase I trials which are assessing PG11047 as a monotherapy and in combination with a number of approved anti-cancer agents including cisplatin and bevacizumab.

Regulation of cell growth by autophagy.

Cell growth-the primary determinant of cell size-has an intimate relationship with proliferation; cells divide only after they reach a critical size. Despite its developmental and medical significance, little is known about cellular pathways that mediate the growth of cells. Accumulating evidence demonstrates a role for autophagy-a mechanism of eukaryotic cells to digest their own constituents during development or starvation-in cell size control. Increasing autophagic activity by prolonged starvation, rapamycin treatment inhibiting TOR (target of rapamycin) signaling, or genetic intervention, causes cellular atrophy in worms, flies and mammalian cell cultures. In contrast, we have shown that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes, unc-51/Atg1 and bec-1/Atg6, confers reduced cell size. We argue that physiological levels of autophagy are required for normal cell size, whereas both insufficient and excessive levels of autophagy lead to retarded cell growth. Furthermore, we discuss data suggesting that the insulin/IGF-1 (insulin-like growth factor receptor-1) and TGF-beta (transforming growth factor-beta) signaling systems acting as major growth regulatory pathways converge on autophagy genes to control cell size. Thus, autophagy may act as a central regulatory mechanism of cell growth.

Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans.

Aging is a multifactorial process with many mechanisms contributing to the decline. Mutations decreasing insulin/IGF-1 (insulin-like growth factor-1) or TOR (target of rapamycin) kinase-mediated signaling, mitochondrial activity and food intake each extend life span in divergent animal phyla. Understanding how these genetically distinct mechanisms interact to control longevity is a fundamental and fascinating problem in biology. Here we show that mutational inactivation of autophagy genes, which are involved in the degradation of aberrant, damaged cytoplasmic constituents accumulating in all aging cells, accelerates the rate at which the tissues age in the nematode Caenorhabditis elegans. According to our results Drosophila flies deficient in autophagy are also short-lived. We further demonstrate that reduced activity of autophagy genes suppresses life span extension in mutant nematodes with inherent dietary restriction, aberrant insulin/IGF-1 or TOR signaling, and lowered mitochondrial respiration. These findings suggest that the autophagy gene cascade functions downstream of and is inhibited by different longevity pathways in C. elegans, therefore, their effects converge on autophagy genes to slow down aging and lengthen life span. Thus, autophagy may act as a central regulatory mechanism of animal aging.

Autophagy in Caenorhabditis elegans.

Autophagy (cellular self-eating) is a highly regulated, lysosome-mediated catabolic process of eukaryotic cells to segregate by a special membrane and subsequently degrade their own constituents during development or starvation. Electron microscopy analysis reveals autophagic elements in various cell types of the nematode Caenorhabditis elegans, whose genome contains counterparts of several yeast genes involved in autophagy. Genetic manipulation inactivating autophagy-related genes in C. elegans causes defects in development, affects dauer larval morphogenesis, accelerates aging thereby shortening life span, reduces cell size, decreases survival during starvation, promotes apoptotic cell death, and protects neurons from undergoing hyperactive ion channel- or neurotoxin-induced degeneration. These results implicate autophagy in various developmental and cellular functions such as reproductive growth, aging, and cell growth, as well as cell survival and loss. This chapter discusses methods of inactivating C. elegans autophagy genes by RNA interference, testing the resistance of autophagy-deficient nematodes to starvation-induced stress, handling mutants carrying a deletion in the autophagy pathway, and monitoring autophagic activity by using LysoTracker Red dye or reporters labeled with green fluorescent protein. Such methods may be adaptable to identify additional roles of autophagy in development and cellular function, and may also help to detect the intracellular accumulation of autophagy proteins and monitor autophagosome formation.

Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans.

Here we show that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes unc-51/atg1 and bec-1/atg6/beclin1 results in small body size without affecting cell number. Furthermore, loss-of-function mutations in unc-51 and bec-1 suppress the giant phenotype of mutant animals with aberrant insulin-like growth factor-1 (insulin/IGF-1) or transforming growth factor-beta (TGF-beta) signaling. This function for unc-51 and bec-1 in cell size control and their interaction with these two growth modulatory pathways may represent a link between the hormonal and nutritional regulation of cell growth.

Janus-faced autophagy: a dual role of cellular self-eating in neurodegeneration?

Autophagy is a highly regulated cellular pathway used by eukaryotic cells to consume parts of their constituents during development or starvation. It is associated with extensive rearrangements of intracellular membranes, and involves the cooperation of many gene products in the regulation and execution phase by largely unknown mechanisms. Recent results strongly indicate the role of autophagy in the degradation of damaged macromolecules, in particular misfolded, aberrant proteins, and in organelle turnover; in mutant mice with reduced autophagy, accumulation of abnormal cytosolic proteins as inclusion bodies and massive cell loss occur similarly to human neurodegenerative disorders. Thus, autophagy seems to prevent neurons from undergoing protein aggregation-induced degeneration. In contrast, we have shown that inactivation of genes involved in autophagosome formation suppresses neuronal demise induced by various hyperactivating ion channel mutations or by neurotoxins in the nematode Caenorhabditis elegans. These results raise the possibility that autophagy may also contribute to excitotoxic necrotic-like cell death. This way, autophagic degradation of cytoplasmic materials might have a dual role in the survival of neurons. Depending on the actual cellular milieu and insulting factor, it can act both as a protector and contributor to neuronal damage.

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs.

Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.

Influence of autophagy genes on ion-channel-dependent neuronal degeneration in Caenorhabditis elegans.

Necrotic cell death is a common feature in numerous human neurodegenerative disorders. In the nematode Caenorhabditis elegans, gain-of-function mutations in genes that encode specific ion channel subunits such as the degenerins DEG-1 and MEC-4, and the acetylcholine receptor subunit DEG-3 lead to necrotic-like degeneration of a subset of neurons. Neuronal demise caused by ion channel hyperactivity is accompanied by intense degradation of cytoplasmic contents, dramatic membrane infolding and vacuole formation; however, the cellular pathways underlying such processes remain largely unknown. Here we show that the function of three autophagy genes, whose yeast and mammalian orthologs are implicated in cytoplasmic self-degradation, membrane trafficking and the cellular response to starvation, contributes to ion-channel-dependent neurotoxicity in C. elegans. Inactivation of unc-51, bec-1 and lgg-1, the worm counterparts of the yeast autophagy genes Atg1, Atg6 and Atg8 respectively, partially suppresses degeneration of neurons with toxic ion channel variants. We also demonstrate that the TOR-kinase-mediated signaling pathway, a nutrient sensing system that downregulates the autophagy gene cascade, protects neurons from undergoing necrotic cell death, whereas nutrient deprivation promotes necrosis. Our findings reveal a role for autophagy genes in neuronal cell loss in C. elegans.

Molecular basis of inward rectification: structural features of the blocker defined by extended polyamine analogs.

Polyamines cause inward rectification of Kir K(+) channels by blocking deep within the channel pore. We investigated structural constraints of polyamine block of strongly rectifying mutant K(ATP) channels (Kir6.2[L164C,N160D,C166S] + SUR1). We studied three groups of polyamine analogs: 1) conformationally restricted linear tetra-amines with a cycloalkyl or alkene group between the second and third amines (CGC-11047, CGC-11093, CGC-11099, and CGC-11098), 2) conformationally restricted linear deca-amines with a cycloalkyl or alkene group between the fifth and sixth amines (CGC-11150, CGC-11179, and CGC-11241), and 3) cyclic tetra-amines (CGC-11174, CGC-11197, CGC-11199, and CGC-11254). All linear analogs cause a voltage-dependent block similar to that of spermine, but slightly weaker (at 1 microM, V(1/2) for spermine block = -10 +/- 1 mV, Z = 2.9 +/- 0.1, n = 19; V(1/2) for analogs varies from polyamine -7 to +10 mV, Z = 2.6-3.9). These data indicate tolerance for conformational restriction and an upper limit to the voltage dependence of the blocking process. There was no voltage-dependent block by the cyclic compounds; instead, they induce irreversible rundown of the current. Structural models of Kir channels suggest that a narrow entry at the top of the cytoplasmic pore may exclude cyclic analogs from the inner cavity, thereby explaining the structure-activity relationship that we observe.

Cholecystokinin-immunopositive basket and Schaffer collateral-associated interneurones target different domains of pyramidal cells in the CA1 area of the rat hippocampus.

Two types of GABAergic interneurone are known to express cholecystokinin-related peptides in the isocortex: basket cells, which preferentially innervate the somata and proximal dendrites of pyramidal cells; and double bouquet cells, which innervate distal dendrites and dendritic spines. In the hippocampus, cholecystokinin immunoreactivity has only been reported in basket cells. However, at least eight distinct GABAergic interneurone types terminate in the dendritic domain of CA1 pyramidal cells, some of them with as yet undetermined neurochemical characteristics. In order to establish whether more than one population of cholecystokinin-expressing interneurone exist in the hippocampus, we have performed whole-cell current clamp recordings from interneurones located in the stratum radiatum of the hippocampal CA1 region of developing rats. Recorded neurones were filled with biocytin to reveal their axonal targets, and were tested for the presence of pro-cholecystokinin immunoreactivity. The results show that two populations of cholecystokinin-immunoreactive interneurones exist in the CA1 area (n=15 positive cells). Cholecystokinin-positive basket cells (53%) preferentially innervate stratum pyramidale and adjacent strata oriens and radiatum. A second population of cholecystokinin-positive cells, previously described as Schaffer collateral-associated interneurones [Vida et al. (1998) J. Physiol. 506, 755-773], have axons that ramify almost exclusively in strata radiatum and oriens, overlapping with the Schaffer collateral/commissural pathway originating from CA3 pyramidal cells. Two of seven of the Schaffer collateral-associated cells were also immunopositive for calbindin. Soma position and orientation in stratum radiatum, the number and orientation of dendrites, and the passive and active membrane properties of the two cell populations are only slightly different. In addition, in stratum radiatum and its border with lacunosum of perfusion-fixed hippocampi, 31.6+/-3.8% (adult) or 26.8+/-2.9% (postnatal day 17-20) of cholecystokinin-positive cells were also immunoreactive for calbindin. Therefore, at least two populations of pro-cholecystokinin-immunopositive interneurones, basket and Schaffer collateral-associated cells, exist in the CA1 area of the hippocampus, and are probably homologous to cholecystokinin-immunopositive basket and double bouquet cells in the isocortex. It is not known if the GABAergic terminals of double bouquet cells are co-aligned with specific glutamatergic inputs. However, in the hippocampal CA1 area, it is clear that the terminals of Schaffer collateral-associated cells are co-stratified with the glutamatergic input from the CA3 area, with as yet unknown functional consequences. The division of the postsynaptic neuronal surface by two classes of GABAergic cell expressing cholecystokinin in both the hippocampus and isocortex provides further evidence for the uniform synaptic organisation of the cerebral cortex.