Equilibrium studies on neutral red-DNA binding.
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Animals↗
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Photodynamic inactivation of verrucae vulgares. II.
Photodynamic inactivation therapy, consisting of a double-blind, paired comparison treatment schedule, was used in treating 56 patients for recalcitrant, symmetrical verrucae vulgares. 0.1% proflavine in 100% dimethylsulphoxide (DMSO) and 0.1% neutral red in 100% DMSO were used as active dyes, and 1% picric acid in 100% DMSO and 1% color ruber in 100% DMSO and 1% color ruber in 100% DMSO served as corresponding placebos. A Westinghouse sunlamp and black light were used to irradiate the warts dyed with proflavine and its placebo, and the warts dyed with neutral red and its placebo were irradiated with an ordinary light bulb (Osram 588597). 50 patients completed the treatment. 10 of the 27 patients treated with proflavine and 10 of the 23 patients treated with neutral red were cured by the end of an 8 week period, with the warts disappearing simultaneously from the actively as well as the placebo-treated side. Complement fixing antibodies against wart virus were detected in one of the cured patients and 2 who were treatment failures.
Acridines↗
Relevance and applicability of a simple earthworm biomarker of copper exposure. I. Links to ecological effects in a laboratory study with Eisenia andrei.
A simple earthworm biomarker, neutral-red retention by coelomocyte lysosomes, was measured concurrently with ecological parameters in order to link effects at different levels of biological organization in a laboratory study. Exposure of the earthworm Eisenia andrei to an increasing range of soil copper concentrations in the laboratory indicated a threshold range for the neutral-red assay at soil copper concentrations between 40 and 80 mg Cu kg-1. This threshold coincided with the soil to worm copper bioconcentration factor decreasing from 1 to 0.3 and thus an apparent onset of a copper regulatory mechanism. Effects at the individual and population levels only occurred at soil copper concentrations beyond that of the biomarker threshold. Thus, it was possible to differentiate between exposure and toxicity. It was therefore possible to conclude that the neutral-red assay has great potential and relevance in earthworm ecotoxicity studies. The assay provides an early warning that can be linked directly to important physiological changes that were observed to precede adverse effects on individuals or populations in this laboratory study.
Analysis of Variance↗
Cytotoxicity of absorption enhancers in Caco-2 cell monolayers.
This study was performed to evaluate the utility of absorption enhancers with reference to mucosal cell cytotoxicity. Overall assessment of the damage to plasma, lysosomal and nuclear membranes by three absorption enhancers, sodium deoxycholate, sodium caprate and dipotassium glycyrrhizinate, was performed on Caco-2 cell monolayers. The cytotoxicities of sodium deoxycholate (0.02-0.1% w/v), sodium caprate (0.1-0.5% w/v) and dipotassium glycyrrhizinate (0.5-2% w/v) were evaluated by the trypan blue-exclusion test, the protein-release test, the neutral-red assay, the DNA--propidium iodide staining test and the test for recovery of transepithelial electrical resistance (TEER) up to 24 h after treatment with each enhancer. Sodium dodecyl sulphate (SDS; 0.1% w/v), a potent surfactant, was used as positive control. SDS at this level was significantly cytotoxic whereas dipotassium glycyrrhizinate was not cytotoxic in any tests. Results from the trypan blue-exclusion and protein-release tests showed that high concentrations of sodium caprate (0.5% w/v) and sodium deoxycholate (0.1% w/v) were significantly cytotoxic to the plasma membrane. The neutral-red assay, an indicator of damage to lysosomal membranes, revealed that 0.5% (w/v) sodium caprate had no effect whereas the uptake of neutral red was slightly increased by treatment with 0.1% (w/v) sodium deoxycholate, implying that the compound had cell-growth-enhancing activity. Nuclear-membrane damage, as evaluated by the DNA--propidium iodide staining test, was severe in cell monolayers treated with 0.5% (w/v) sodium caprate compared with that induced by 0.1% (w/v) sodium deoxycholate. In the TEER recovery test, TEER failed to recover 24 h after treatment with 0.5% (w/v) sodium caprate and 0.1% (w/v) SDS, but recovered after treatment with 0.1% (w/v) sodium deoxycholate. The recovery of TEER might be related to nuclear membrane damage and cell-growth-enhancing activity. These results indicate that of the three classes of enhancer, dipotassium glycyrrhizinate was not cytotoxic and that high concentrations of sodium caprate and sodium deoxycholate could damage plasma and nuclear membranes.
Caco-2 Cells↗
Modification of the agar overlay assay: assessment of the influence of acrylics used in orthodontics on proliferation and differentiation of primary and transformed fibroblasts.
In orthodontics, removable acrylic appliances are preferably produced from methylmetacrylates. However, in the adjacent oral mucosal tissue, cell damage may be caused by the evaporation of residual monomer. The aim of this study was to modify the classic agar overlay assay in order to apply histochemical methods by comparing conventionally used transformed mouse fibroblasts (L-929) with primary human gingival fibroblasts to further elucidate the term toxicity. While proliferation was assessed via the incorporation of the base analogon bromdesoxyuridine, differentiation was investigated by detection of the fibroblast-specific intermediate filament vimentin. After the monomers had taken effect, reduced proliferation extending over the inhibition area was observed by indirect immunofluorescence, independent of cell type. In contrast to this observation, a few cells within the inhibition area which could not be clearly detected by neutral red staining still exhibited mitotic activity. Detection of the differentiation-specific intermediate filament vimentin was comparable with the degree of neutral red fading visible in the classic agar overlay assay. The study showed that the inhibition areas with primary gingival fibroblasts were smaller (approximately 1/3) after monomer action compared with conventionally applied transformed fibroblasts. The results indicate that the modified assay is comparable with the classic method. Evaluation of neutral red staining with respect to toxic material influence can moreover be supplemented by histochemical studies of typical cell properties.
Acrylic Resins↗
Rapid ecotoxicological testing using transformed BF-2 cells incorporating a luminescent reporter gene.
Since there is an ethical need to minimise the experimental use of higher organisms such as fish, especially those used in acute toxicity testing, fish cells are considered to be useful surrogates for fish in toxicity screening. The use of fish cell lines in conventional bioassays such as neutral red retention assays is however labour intensive, lengthy and costly. The use of luminescent reporter genes has been explored in our laboratory. In this study, a transfected BF-2 cell line (BF-2/luc1) was used for rapid toxicity testing on selected chemicals and results were compared with those obtained with in vivo fish testing and in vitro fish cell neutral red retention assays. The effect of temperature on the sensitivity of BF-2/luc1 was also investigated. BF-2/luc1 cells were harvested and suspended in PBS at 2.5-3.0x10(6)cells/ml. Individual aliquots of the suspended cells (40 microl each) were incubated for either 0.5 or 6 h at room temperature (22 degrees C) in the presence or absence of the toxicants. Bioluminescence was assayed using 17.5 microl Brightglo luciferase reagent which lysed the cells and provided the substrate luciferin. Luminescence was measured in a luminometer (Turner TD 20/20). The EC50 values obtained from BF-2/luc1 cells (0.5-6 h) generally compared well with the LC50 values (24-96 h) obtained from the in vivo fish tests on a range of species. The present study also showed that BF-2/luc1 cell sensitivity increased significantly when incubation temperature during toxicant exposure increased from 15 to 35 degrees C. The use of luminescent reporter genes in monitoring fish cells offers the possible advantages of increased sensitivity over the neutral red retention assay and a more rapid test to replace stain based bioassays, and provides a rapid screening method that could reduce the need for acute fish toxicity testing.
Animals↗
Effect of biflavones of Ginkgo biloba against UVB-induced cytotoxicity in vitro.
The effect of Ginkgo biloba extract on Ultraviolet B (UVB) irradiated fibroblasts was examined by using a neutral red dye uptake assay and a lactic dehydrogenase (LDH) release assay. Crude extract along with individual components, including flavone-glycosides and biflavones, were applied to cultured normal human skin fibroblasts for 12 hours, and 0, 20, 40 and 80 mJ/cm2 of UVB were irradiated. Two synthetic flavonoids, quercetin and rutin, which have polyphenol structures close to the flavonoids in Ginkgo biloba extract, were used to compare any structure-related activity under the same conditions. At the concentrations (from 0.25 to 2 mg/ml) treated with biflavone components (isoginkgetin/ginkgetin, sciadopitysin) and quercetin, high neutral red dye uptake was detected with gradual increases in UVB irradiation. The time-course release of LDH was determined as the cytotoxicity index (%) during 24 hours following a high dose UVB irradiation (200 mJ/cm2), and the pattern of this cytotoxicity index was similar to that of the neutral red dye uptake results. Sciadopitysin, isoginkgetin/ginkgetin and quercetin treatments lowered cytotoxicity indices to 50.81, 67.81 and 62.19%, respectively, compared to 95.38% for the untreated control. The antioxidant potential of biflavones of Ginkgo biloba could be explained on the basis of structure-related activity; hydroxy- and methyl-substitutions on the basic structure of these flavonoids played a role, as other reports have suggested.
Cell Death↗
Antifusion activity in sera from persons infected with human immunodeficiency virus type 1.
Cell-to-cell fusion plays an important role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infections. An assay to measure the antifusion activity of serum has been developed by using the fusion event that occurs between H9 cells chronically infected with HIV-1 (H9IIIB) and fusion-susceptible MT-2 cells. The endpoint is determined by measuring neutral red uptake in cells after syncytium formation is allowed to occur in the presence of various serum dilutions. The assessment of antifusion activity in serum by neutral red uptake has been shown to correlate with syncytium reduction as determined by direct counting. The optimal number and ratio of cells in the suspension for efficiency and speed of the assay have been determined. With this assay it was shown that 50% of 36 serum specimens capable of neutralizing cell-free virions failed to inhibit syncytium formation. The assay can thus measure a distinct activity in HIV-1-immune human sera which is a subset of neutralization activity. Because of the potential role of this activity in the rate of disease progression and protective immune responses, the antifusion assay will be an important tool for the investigation of disease pathogenesis and for acquired immunodeficiency syndrome vaccine development. The assay can also be applied to the investigation of the pathogenesis of the fusion event at the cellular level. The ability to use absorbance measurements rather than syncytium counts as the endpoint facilitates direct computer-assisted data analysis, which expedites the performance of the assay.
Cell Fusion↗
Comparison of the streak-plate and pour-plate techniques for determination of coliform counts of mild samples using crystal violet-neutral red-lactose-bile (VRBL) medium.
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Animals↗
[Biofilm disclosing agents in complete denture: clinical and antimicrobial evaluation].
This study evaluated the disclosing ability, removal facility and antimicrobial effect of biofilm disclosing agents applied on complete dentures. Disclosing ability was evaluated by means of the visual method. The solutions were applied on the internal surface of dentures. After being photographed, the dentures were brushed with denture-specific brush and dentifrice and photographed again. The obtained slides were projected on paper (10 X amplification) and the total and stained surfaces were outlined with graphite, cut off and weighed, in order to assess removal facility. The evaluation of antimicrobial effects was carried out by means of the method of diffusion in agar, and the results were obtained by measuring the length of the halos and rings. In terms of disclosing ability, the best solutions were 0.05% methylene blue, 5% erythrosin, 1% sodic fluorescein, Replak and 1% neutral red. One percent eosin, 1% sodic fluorescein and 5% erythrosin were the most easily removed solutions. One percent eosin, 5% erythrosin, 1% sodic fluorescein, 0.3% proflavine, Replak and 1% neutral red presented no antimicrobial effect. The solutions which presented the greatest disclosing ability and removal facility as well as absence of antimicrobial effect - which are essential requirements in the assessment of hygiene methods and guidance on oral health - were 1% eosin, 1% neutral red and 5% erythrosin.
Adult↗
Species- and sex-specific renal cytotoxicity of ochratoxin A and B in vitro.
Four different cell models were chosen for comparison of OTA and OTB toxicity: primary porcine (PKC), rat (RPTC) and human renal proximal epithelial cells (HKC) from both sexes and a porcine renal cell line: LLC-PK1. Culture conditions were tested and optimized for each respective cell type (species/sex and origin). All cell types were characterized for epithelial origin and growth patterns and following optimization of dosing strategies and assay procedures, a strict study design was implemented to avoid systemic variations. Due to possible sensitivity differences, three simple endpoints were chosen to provide basic data for interspecies comparison: neutral red uptake, MTT reduction and cell number. Of the endpoints tested neutral red appeared the most sensitive, although all three parameters yielded comparable EC50's. Sex-differences were observed between male and female HKC cells following 96 h exposure to OTA, with HKC(m) being more sensitive than HKC(f). No sex-difference was observed in PKC cells, however, the PKC were approximately 3 and 10 times more sensitive than HKC(m) and HKC(f), respectively, to OTA and OTB. Interestingly, the CI95 of the EC50 values obtained for OTA (15.5-16.5 microM) and OTB (17.0-2 1.0 microM) were comparable in the PKC cells. In contrast, OTB had lower cytotoxicity than OTA in HKC and LLC-PK1 (approx. 2-fold) and no effects in RPTC. Overall, HKC(m) were nearly as sensitive as PKC towards OTA, followed by RPTC, LLC-PK1 and HKC(f), thus suggesting a sex specific sensitivity in humans towards OTA induced cytotoxicity.
Animals↗
In vitro effect of Pt and Pd mercaptopyridine complexes.
In previous research we studied the cytotoxic effect of new Pt mercaptopyridine (MP) complexes on several tumoral cell lines (F10, Föhn, LoVo and HeLa) as well as on the fibroblast cell line (3T3). The more interesting Pt compounds are compared here to Pd mercaptopyridine analogs, in order to evaluate the metals influence on activity. Earlier, the complexes C/2 = [Pt(MP)3Cl]Cl; C/5 = [Pt(MP)3Br]Br; C/8 = [Pd(MP)3Cl]Cl and C/11 = [Pd(MP)3Br]Br and cis-DDP as reference were tested on 3T3 and LoVo cells, by Sauter's multiwells technique and neutral red uptake. The results obtained using lysosomal neutral red uptake to confirm those by the Sauter's multiwells techniques, showing that C/2 and C/11 are the most active complexes. In particular, C/2 shows a significantly higher cytotoxic activity than cis-DDP on LoVo cells, and equivalent on 3T3. C/5 complex also induces an interesting cell growth reduction, but only on LoVo, while C/8 is completely inactive on all cell lines. Because the major limitation to the successful treatment of platinum-based chemotherapeutic regimens is the emergence of drug resistance, the activity of the four complexes on cis-DDP sensitive (M5076) and cis-DDP resistant cancer cells (M5/DDPc) has been tested. The data reported in this work make devident that the presence of ligands with sulfur donor atoms may be of particular importance in confirming the antitumor properties of Pt complexes. In fact, Pt mercaptopyridine C/2 is also more active than cis-DDP against cells made resistant to cis-DDP. Moreover, the results obtained with Pd complex C/11, especially on LoVo cells, showed that this metal could be considered interesting in the design of potential new antitumor drugs.
Antineoplastic Agents↗
Measurement of intracellular pH in hamster diaphragm by absorption spectrophotometry.
The regulation of intracellular pH (pHi) is important in controlling muscle contraction. In these experiments, a spectrophotometric method of determining pHi was developed, and the method was then used to study muscle pHi regulation during CO2-induced changes in extracellular pH (pHb). Studies were performed in vitro on 27 diaphragm muscle strips obtained from adult hamsters. pHi was measured from the ratio of the absorbances of the acid (lambda = 530 nm) and alkaline (lambda = 460 nm) forms of a vital dye, neutral red, using the unstained diaphragm spectrum as a reference blank. A standard neutral red calibration curve constructed from eight diaphragm muscle homogenates indicated that the absorbance ratio was highly linear, with pH over the range 6.00-8.00. In intact muscle strips gassed with 95% O2-5% CO2, pHb was 7.45 +/- 0.03 (SE) and pHi was 7.00 +/- 0.01 (SE). When the muscle was aerated with CO2 concentrations from 3 to 30%, pHb and pHi changed rapidly and reached a steady state in 10-15 min. However, when pHb ranged from 6.80-7.80, pHi changed little from the value observed when pHb was 7.40. When pHb was less than 6.80 or greater than 7.80, changes in pHi and pHb were quantitatively similar. The results suggest that, in the isolated diaphragm, overall pHi is stable and effectively buffered over a wide range of CO2-induced changes in buffer solution pH.
Animals↗
A new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress. Application to dystrophin-deficient DMD skeletal muscles.
This is the first report on the development of an immunohistochemical technique, combined with quantitative image analysis, for the assessment of oxidative stress quantitatively in nuclear DNA in situ, and its application to measure DNA damage in Duchenne muscular dystrophic (DMD) muscles. Three sequential staining procedures for cell nuclei, a cell marker, and a product of oxidative DNA damage, 8-oxoguanine (8-oxoG), were performed. First, the nuclei in muscle sections were stained with Neutral Red followed by the capture of their images with an image analysis system used for absorbance measurements. Second, the same sections were then immunostained for laminin in basement membranes as the cell marker. Next, the sections were treated with 2 N HCl to remove the bound Neutral Red and to denature tissue DNA. Third, the sections were immunostained for 8-oxoG in DNA, using diaminobenzidine (DAB) to reveal the antibody complex. This was followed by capture of the images of the immunostained sections as previously. The absorbances at 451.2 nm of bound Neutral Red and DAB polymer oxides, the final product of 8-oxoG immunostaining, were measured in the same myonuclei in the sections. Analysis of these absorbances permitted indices of the 8-oxoG content, independent of the nuclear densities, to be determined in nuclear DNA in single myofibres and myosatellite cells surrounded by basement membranes. We found that the mean index for the myonuclei in biceps brachii muscles of 2- to 7-year-old patients was 14% higher than that in age-matched normal controls. This finding of the increased oxidative stress in the myonuclei in young DMD muscles agrees with the previous reports of increased oxidative stress in the cytoplasm in the DMD myofibres and myosatellite cells. The present technique for the quantitative assessment of oxidative stress in nuclear DNA in situ is applicable not only in biomedical research but also in the development of effective drugs for degenerative diseases related to oxidative stress.
Animals↗
Measurement of protection afforded by ultraviolet-absorbing window film using an in vitro model of photodamage.
BACKGROUND AND OBJECTIVES: The effects of chronic sun damage including telangiectasias, solar lentigos, rhytides, enlarged pores, sagging skin, and pre-cancerous and cancerous growths are among the most common presenting complaints in a dermatologist's office. These changes are often worse on the driver's side of the face, emphasizing the role of UVA exposure received while driving in producing these changes. This study was undertaken to measure the ability of car window glass alone and in combination with ultraviolet (UV)-absorbing film to reduce UV-damage as measured using an established in vitro model of photoprotection. STUDY DESIGN MATERIALS AND METHODS: Using the 3T3 neutral red uptake photoprotection assay with solar simulating radiation (SSR) administered by a xenon arc solar simulator, we measured the photoprotection ability of auto glass, window film that filters UV radiation, and the combination of window film and auto glass. RESULTS: As measured by the 3T3 neutral red uptake photoprotection assay, auto glass reduced cell death from SSR by 29%, while window film reduced it 90%, and the combination of auto glass and film reduced cell death by 93%, when compared to unfiltered SSR. CONCLUSIONS: Window film that filters UV radiation results in dramatic reductions in cytotoxicity when measured by the neutral red uptake photoprotection assay. Widespread use of window film provides an ever-present barrier to ultraviolet A (UVA) exposure and could potentially reduce the detrimental effects of UVA, including photoaging, skin cancer, and ocular damage, such as cataracts. In addition, such film is essential for patients suffering from conditions sensitive to UV radiation, such as lupus erythematosis.
Animals↗
Validity of eucaryote inhibitors for assessing production and grazing mortality of marine bacterioplankton.
Application of eucaryote inhibitors to the estimation of production and grazing mortality of bacterioplankton was evaluated. Exposure to a range of concentrations of thiram, cycloheximide, and neutral red (0.4 to 210, 36 to 1,777, 4 to 346 muM, respectively) was 98 to 100% effective at inhibiting growth of a chrysomonad in culture. Exposure to colchicine and griseofulvin (50 to 1,000 muM for both) yielded only 24 to 94 and 53 to 79% inhibition, respectively. Exposures to thiram, neutral red, and griseofulvin were 90 to 100% effective at inhibiting growth in culture of a ciliate, Cyclidium sp., and the responses to colchicine and cycloheximide were variable (64 to 100 and 0 to 100% inhibition, respectively). Thiram and neutral red inhibited field populations of nanozooplankton more effectively than cycloheximide and colchicine. Direct effects of eucaryote inhibitors on growing cultures of bacterioplankton varied with parameters measured and duration of exposure. After 3-day exposures, specific growth rates and "instantaneous" heterotrophic potential ([C]glucose uptake) were not consistently affected, but biosynthetic activity (RNA and DNA syntheses) was depressed. The degree of inhibition of isolates and field populations of phytoplankton depended upon type of inhibitor and phytoplankton species. In field experiments, it was possible to calculate rates of bacterioplankton production and grazing mortality for only 16 of 29 inhibitor experiments and for 4 of 10 size fractionation experiments. Bacterioplankton production and mortality estimates varied greatly with the eucaryote inhibitor used, and those derived from inhibition techniques were substantially different from those derived from fractionation techniques. The poor performances of both techniques are attributed to the following: (i) effects of inhibitors on phytoplankton, (ii) indirect effects of the inhibitors on bacterioplankton, and (iii) insufficient separation of grazers from prey by filtration techniques. Because of the inconsistent results obtained in this investigation, we strongly recommend exercising caution in the application of inhibitor techniques to ecological problems, especially in phototrophically dominated systems.
Journal Article↗
In vitro evaluation of the cytotoxic potential of alkylphenols from Ginkgo biloba L.
Extracts from the leaves of Ginkgo biloba L. belong to the most widely used phytopharmaceuticals. In crude Ginkgo extracts, ginkgolic acids (GA) and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardous compounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties. To further assess the cytotoxic potential of GA, their effect on the human keratinocyte cell line HaCaT and the rhesus monkey kidney tubular epithelial cell line LLC-MK(2) was investigated. The action of a defined mixture of GA on cell growth, viability and integrity was evaluated by the neutral red uptake assay as well as the release of lactate dehydrogenase (LDH) and acid phosphatase (ACP). Cell morphology was examined by electron microscopy. For comparison, the effect of the standardized Ginkgo extract EGb 761, which contains less than 5 ppm GA, was also investigated. Following incubation of cells with EGb 761, neutral red uptake was half-maximally inhibited at concentrations of 900 mg/l (HaCaT) and 1480 mg/ml (LLC-MK(2)). The corresponding IC(50)-values for the mixture of GA ranged between 22 mg/l (HaCaT) and 4.6 mg/l (LLC-MK(2)), respectively. In parallel to the inhibition of neutral red uptake, a concentration dependent release of LDH was observed when cells were incubated in the presence of GA (1-100 mg/l). In contrast, even at a concentration of 1800 mg/l EGb 761 did not cause release of LDH above controls. Since GA interacted with the assay for ACP, no index of lysosomal damage could be established by this method. Incubation of HaCaT cells with GA for 18 h increased the proportion of apoptotic cells from about 6% (control) to nearly 80% at concentrations of >or=30 mg/l. Electron microscopic analysis of HaCaT cells revealed a drug induced formation of myelinosomes possibly due to the inhibition of lysosomal enzymes, while morphological evaluation of LLC-MK(2) cells indicated that the cytotoxic activity of GA in these cells is primarily mediated by transformation of mitochondria, which is probably induced by uncoupling of oxidative phosphorylation.
Animals↗
Role of climbing fibers in determining the spatial patterns of activation in the cerebellar cortex to peripheral stimulation: an optical imaging study.
The spatial patterns of activation in the rat cerebellar cortex evoked by ipsilateral face stimulation were mapped using optical imaging based on the pH sensitive dye, Neutral Red. The aims of the study were to characterize the optical responses evoked by peripheral stimulation and test the hypothesis that the resultant parasagittal banding is due to climbing fiber activation. In the anesthetized rat Crus I and II of the cerebellar cortex were stained with Neutral Red. Epi-fluorescent changes produced by a train of stimuli (5-10s and 4-20 Hz) to the ipsilateral face were monitored in time using a fast, high resolution charge-coupled device camera. The patterns of activation were quantified using a two-dimensional fast Fourier transform analysis that removed signals with high spatial frequencies and minimized the contribution of horizontal structural elements (i.e. blood vessels). The dominant spatial pattern of activation evoked by face stimulation was that of parasagittal bands. The bands were highly frequency-dependent and were elicited most strongly by stimulus frequencies in the range of 6-8 Hz. There was a large fall-off in the response for frequencies above and below. The optical signal evoked by face stimulation built up over a period of 10s and then gradually decayed. Within a folium the individual parasagittal bands exhibited some frequency and temporal specificity. Stimulation of the contralateral inferior olive also resulted in the activation of parasagittal bands with characteristics similar to the bands evoked by face stimulation, including a preferred stimulus frequency which peaked at 10 Hz. Injection of lidocaine into the contralateral inferior olive blocked the parasagittal bands evoked by ipsilateral face stimulation, while control injections of saline had no effect. The results confirm that a parasagittal banding pattern is a dominant feature of the functional architecture of the cerebellar cortex. The parasagittal banding pattern observed with Neutral Red is due primarily to the activation of climbing fiber afferents. The frequency tuning of the responses, with the preference for peripheral stimuli of 6-8 Hz, is in agreement with previous findings that the inferior olive is inherently rhythmic. These observations support the hypothesis that inferior olivary neurons are dynamically coupled into groups that activate parasagittal bands of Purkinje cells in the cerebellar cortex. The frequency tuning also supports the hypothesis that the climbing fiber system is involved with timing. Activation of this afferent system may require stimuli with appropriate frequency content and stimuli synchronized to the rhythmicity of the inferior olive.
Action Potentials↗