Additions to the knowledge of the genus Helvella in Europe. New records and de novo description of five species from the Nordic region.

Helvella is a species-rich genus, forming a large variation of astounding ascocarps in many different habitats. During the last decade, molecular markers and morphological characters have been combined to delimit and identify cryptic species in this genus. We report on a list of 54 species of Helvella s.s. in the Nordic region and describe five new species, i.e. H. bresadolae, H. convexa, H. japonica, H. nordlandica and H. oroarctica. The morphological and molecular characteristics of the new species and the emended / hypocrateriformis, / fibrosa-macropus, and / fallax-pezizoides lineages of Helvella s.s. are shortly commented upon. Further we include a discussion of the distribution of species in the Nordic region based on a large set of studied collections. The ecological versatility and variable geographic patterns of these species indicate that cryptic species may have contrasting ecology in their local habitats. Citation: Skrede I, Løken SB, Mathiesen C, Schumacher T (2023). Additions to the knowledge of the genus Helvella in Europe. New records and de novo description of five species from the Nordic region. Fungal Systematics and Evolution 11: 71-84. doi: 10.3114/fuse.2023.11.06.

The genera Helvella and Dissingia (Ascomycota: Pezizomycetes) in Europe - Notes on species from Spain.

Phylogenetic analyses of 115 newly collected Helvella specimens from Spain using three genetic markers [heat shock protein 90 (hsp), RNA polymerase II second largest subunit (rpb2) and the nuclear large subunit ribosomal DNA (LSU)] confirm the assignment of the Spanish collections to one Dissingia and 30 Helvella species. The analyses were supplemented with an additional sample of 65 Spanish and extralimital Helvella specimens from the fungaria of Oslo (O), Trondheim (TRH), Copenhagen (C), Uppsala (UPS), Stockholm (S) and Venice (MCVE). Nine species are described as new, i.e. Helvella fuscolacunosa, H. hispanica, H. iberica, H. inexpectata, H. neopallescens, H. phlebophoroides, H. poculiformis, H. retinervis, and H. terricola. We present photographs of a selection of fresh specimens and provide descriptions of all species of this diverse South European Mediterranean element of the genera in Europe.

Temporal coupling between neuronal activity and blood flow in rat cerebellar cortex as indicated by field potential analysis.

1. Laser-Doppler flowmetry and extracellular recordings of field potentials were used to examine the temporal coupling between neuronal activity and increases in cerebellar blood flow (CeBF). 2. Climbing fibre-evoked increases in CeBF were dependent on stimulus duration, indicating that increases in CeBF reflected a time integral in neuronal activity. The simplest way to represent neuronal activity over time was to obtain a running summation of evoked field potential amplitudes (runSigmaFP). RunSigmaFP was calculated for each stimulus protocol and compared with the time course of the CeBF responses to demonstrate coupling between nerve cell activity and CeBF. 3. In the climbing fibre system, the amplitude and time course of CeBF were in agreement with the calculated postsynaptic runSigmaFP (2-20 Hz for 60 s). This suggested coupling between CeBF and neuronal activity in this excitatory, monosynaptic, afferent-input system under these conditions. There was no correlation between runSigmaFP and CeBF during prolonged stimulation. 4. Parallel fibre-evoked increases in CeBF correlated with runSigmaFP of pre- and postsynaptic potentials (2-15 Hz for 60 s). At higher stimulation frequencies and during longer-lasting stimulation the time course and amplitudes of CeBF responses correlated with runSigmaFP of presynaptic, but not postsynaptic potentials. This suggested a more complex relationship in this mixed inhibitory-excitatory, disynaptic, afferent-input system. 5. This study has demonstrated temporal coupling between neuronal activity and CeBF in the monosynaptic, excitatory climbing-fibre system. In the mixed mono- and disynaptic parallel fibre system, temporal coupling was most clearly observed at low stimulation frequencies. We propose that appropriate modelling of electrophysiological data is needed to document functional coupling of neuronal activity and blood flow.

Modification of activity-dependent increases in cerebellar blood flow by extracellular potassium in anaesthetized rats.

1. The hypothesis that potassium ions mediate activity-dependent increases of cerebral blood flow was examined in rat cerebellar cortex using ion-selective microelectrodes and laser-Doppler flowmetry. Increases of cerebellar blood flow (CeBF) and extracellular potassium concentration ([K+]o) were evoked by stimulation of parallel fibres and climbing fibres, and by microinjection of KCl into the cortex. 2. For parallel fibre stimulation, there was a maximal increase in [K+]o to 6.3 +/- 0.5 mM and in CeBF of 122 +/- 11 %. Climbing fibre stimulation gave a maximal increase in [K+]o to 4.4 +/- 0.2 mM and in CeBF of 157 +/- 20 %. This indicates different maxima for [K+]o and CeBF, dependent on the afferent system activated. 3. [K+]o and CeBF responses evoked by parallel or climbing fibre stimulation increased rapidly at the onset of stimulation, but exhibited different time courses during the remainder of the stimulation period and during return to baseline. 4. Microinjections of KCl into the cortex increased [K+]o to levels comparable to those evoked by parallel fibre stimulation. The corresponding CeBF increases were the same as, or smaller than, for parallel fibre stimulation, and much smaller than for climbing fibre stimulation. This suggests that mediators other than [K+]o are important for activity-dependent cerebral blood flow increases. 5. The present study showed that increased [K+]o is involved in CeBF regulation in the parallel fibre system, but is of limited importance for CeBF regulation in the climbing fibre system. The hypothesis that K+ is a major mediator of activity-dependent blood flow increases is probably not generally applicable to all brain regions and all types of neuronal stimulation.

SPD 502: a water-soluble and in vivo long-lasting AMPA antagonist with neuroprotective activity.

Accumulating preclinical data suggest that compounds that block the excitatory effect of glutamate on excitatory amino acid receptors may have neuroprotective effects and utility for the treatment of neurodegeneration after brain ischemia. In the present study, the in vitro and in vivo pharmacological properties of the novel glutamate antagonist SPD 502 [8-methyl-5(4-(N,N-dimethylsulfamoyl)phenyl)-6,7, 8,9,-tetrahydro-1H-pyrrolo[3,2-h]-isoquinoline-2, 3-dione-3-O-(4-hydroxybutyric acid-2-yl)oxime] are described. In binding studies, SPD 502 was shown to display selectivity for the [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-binding site (IC50 = 0.043 microM) compared with the [3H]kainate- (IC50 = 81 microM), [3H]cis-4-phosphonomethyl-2-piperidine carboxylic acid-(CGS 19755), and [3H]glycine-binding sites (IC50 > 30 microM) in rat cortical membranes. In an in vitro functional assay, SPD 502 blocked the AMPA-induced release of [3H]gamma-aminobutyric acid from cultured mouse cortical neurons in a competitive manner with an IC50 value of 0.23 microM. Furthermore, SPD 502 potently and selectively inhibited AMPA-induced currents in cortical neurons with an IC50 value of 0.15 microM. In in vivo electrophysiology, SPD 502 blocked AMPA-evoked spike activity in rat hippocampus after i.v. administration with an ED50 value of 6.1 mg/kg and with a duration of action of more than 1 h. Furthermore, SPD 502 increased the seizure threshold for electroshock-induced tonic seizures in mice at i.v doses of 40 mg/kg and higher. In the two-vessel occlusion model of transient forebrain ischemia in gerbils, SPD 502 (10 mg/kg bolus injection followed by a 10 mg/kg/h infusion for 2 h) resulted in a highly significant protection against the ischemia-induced damage in the hippocampal CA1 pyramidal neurons.

Modification of activity-dependent increases of cerebral blood flow by excitatory synaptic activity and spikes in rat cerebellar cortex.

1. Mechanisms of activity-dependent increases in cerebral blood flow (CBF) were examined in rat cerebellar cortex using the laser Doppler flow technique and extracellular recordings of single unit activity and field potentials. 2. Stimulation of the monosynaptic climbing fibre system evoked long-lasting complex spikes in Purkinje cells, and extracellular field potentials with a characteristic profile that indicated contributions from both passive and active membrane mechanisms. The concomitant CBF increases were reproducible at fairly short intervals, and suggest that both synaptic activity and spikes may contribute to increased CBF. 3. Stimulation of the disynaptic parallel fibre system inhibited the spiking activity in Purkinje cells, while the postsynaptic activity increased as indicated by the simultaneously recorded field potential. Nevertheless, CBF always increased. The inhibition of spike firing activity was partly dependent on GABAergic transmission, but may also relate to the intrinsic membrane properties of Purkinje cells. 4. The CBF increases evoked by parallel or climbing fibre stimulation were highly correlated to the sum of neural activities, i.e. the negativity of field potentials multiplied by the stimulus frequency. This suggests a robust link between extracellular current flow and activity-dependent increases in CBF. 5. AMPA receptor blockade attenuated CBF increases and field potential amplitudes, while NMDA receptor antagonism did not. This is consistent with the idea that the CBF responses are of neuronal origin. 6. This study has shown that activity-dependent CBF increases evoked by stimulation of cerebellar parallel fibres are dependent on synaptic excitation, including excitation of inhibitory interneurones, whereas the net activity of Purkinje cells, the principal neurones of the cerebellar cortex, is unimportant for the vascular response. For the climbing fibre system, not only synaptic activity but also the generation of complex spikes from Purkinje cells contribute to the increases in CBF. The strong correlation between CBF and field potential amplitudes suggests that extracellular ion fluxes contribute to the coupling of brain activity to blood flow.

In vivo and in vitro evaluation of AMPA receptor antagonists in rat hippocampal neurones and cultured mouse cortical neurones.

The effects of four glutamate receptor antagonists on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-responses were evaluated using both in vitro and in vivo electrophysiological techniques: whole cell patch-clamp recordings from cultured mouse cortical neurones and microiontophoresis in the rat hippocampus. The compounds tested were NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), GYKI 52466 (1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine), PNQX (pyrido[3, 4-f]quinoxaline-2,3-dione, 1,4,7,8,9,10-hexahydro-9-methyl-6-nitro-, methanesulfonate), NS377 (7-ethyl-5-phenyl-1,6,7,8-tetrahydro-1,7-diaza-as-indacene-2 ,3-dione), and MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenz(a,d)cycloheptene-5,10-imine hydrogen maleate). In vitro, the IC50 values (in microM) for inhibition of AMPA-evoked inward currents were approximately 0.4 for NBQX, approximately 7.5 for GYKI 52466, approximately 1 for PNQX and approximately 15 for NS377. PNQX and NS377 also inhibited NMDA-induced currents with IC50 values at approximately 5 and approximately 18 microM, respectively, while NBQX at 60 microM and GYKI 52466 at 100 microM had only weak effects. The ED50 values in micromol/kg i.v. for inhibition of AMPA-evoked hippocampal neuronal spike activity in vivo were approximately 32 for NBQX, approximately 19 for GYKI 52466, approximately 17 for PNQX and approximately 11 for NS377 with efficacy values (maximal inhibition) between 71% and 81%. The ED50 values (in [Lmol/kg i.v.) and efficacy values for inhibition of NMDA-evoked hippocampal neuronal spike activity were approximately 28 with an efficacy of 61% for NBQX, approximately 16 with 35% for PNQX and approximately 6 with 61% for NS377. GYKI 52466 did not significantly affect NMDA responses, whereas MK-801 showed NMDA specificity in vivo.

Laminar analysis of activity-dependent increases of CBF in rat cerebellar cortex: dependence on synaptic strength.

The purpose of the present study was to examine mechanisms of activity-dependent changes of cerebral blood flow (CBF) in rat cerebellar cortex by laser-Doppler flowmetry, using two synaptic inputs that excite different regions of the same target cell and with different synaptic strength. The apical part of Purkinje cells was activated by electrical stimulation of parallel fibers, whereas the cell soma and the proximal part of the dendritic tree were activated by climbing fibers using harmaline (40 mg/kg ip) or electrical stimulation of the inferior olive. Glass microelectrodes were used for recordings of field potentials and single-unit activity of Purkinje cells. CBF increases evoked by parallel fibers were most pronounced in the upper cortical layers. In contrast, climbing fiber stimulation increased CBF in the entire cortex. Inhibition of nitric oxide (NO) synthase activity by NG-nitro-L-arginine (L-NNA) or guanylate cyclase activity by 1H-[1,2,4(oxadiazolo)4,3-a]quinoxaline-1-one did not affect basal or harmaline-induced Purkinje cell activity but attenuated harmaline- and parallel fiber-evoked CBF increases by approximately 40-50%. Application of 8-(p-sulfophenyl)theophylline and adenosine deaminase reduced the harmaline-evoked CBF increase without any effect on the parallel fiber-evoked CBF response. The results suggest that CBF increases elicited by activation of Purkinje cells are partially mediated by the NO-guanosine 3',5'-cyclic monophosphate system independent of the input function but that adenosine contributes as well when climbing fibers are activated. This is the first demonstration of variations of coupling as a function of postsynaptic activity in the same cell.

The ultrastructure and innervation of the ear of the gar, Lepisosteus osseus.

The endorgans of the inner ear of the gar were examined using transmission and scanning electron microscopy as well as nerve staining. The ultrastructure of the sensory hair cells and supporting cells of the gar ear are similar to cells in other bony fishes, whereas there are significant differences between the gar and other bony fishes in the orientations patterns of the sensory hair cells on the saccular and lagenar sensory epithelia. The saccular sensory epithelium has two regions, a main region and a secondary region ventral to the main region. The ciliary bundles on the main region are divided into two groups, one oriented dorsally and the other ventrally. Furthermore, as a result of curvature of the saccular sensory epithelium, the dorsal and ventral ciliary bundles on the rostral portion of the epithelium are rotated ninety degrees and are thus oriented on the animal's rostro-caudal axis. Hair cells on the secondary region are generally oriented ventrally. The lagenar epithelium has three groups of sensory hair cells. The groups on the rostral and caudal ends of the macula are oriented dorsally, whereas the middle group is oriented ventrally. Hair cell orientations on the utricular epithelium and macula neglecta are similar to those in other bony fishes. Nerve fiber diameters can be divided into three size classes, 1-8 microns, 9-13 microns, and 14 microns or more, with the smallest size class containing the majority of fibers. The distribution of the various classes of fiber diameters is not the same in nerve branches to each of the end organs. Similarly, the ratio of hair cells to axons differs in each end organ. The highest hair cell to axon ratio is in the utricle (23:1) and the smallest is in the macula neglecta (7:1). The number of sensory hair cells far exceed the number of eighth nerve axons in all sensory epithelia.

Hair cell orientation and a possible intraepithelial growth of the macula lagenae in the inner ear of the European eel.

The hair cell orientation of the macula lagenae in eels at three different stages of life was examined with the light microscope. Part of the posterior periphery of all three stages was occupied by a few rows of sensory cells having their kinocilium pointing in a direction opposite to the direction of the adjacent sensory cells. The width of this peripheral belt remained the same at all three stages of life, in spite of a considerable growth of the sensory epithelium, and the belt was always confined to the margin. This indicates an intraepithelial growth of at least part of the neuroepithelium of the lagenar macula of the European eel.