Behavioral effects associated with chronic ketamine or remacemide exposure in rats.

The effects of chronic exposure to ketamine or remacemide on the acquisition and performance of food-reinforced operant behaviors was assessed in female Sprague-Dawley rats. Ketamine is an anesthetic N-methyl-D-aspartate (NMDA) receptor antagonist, whereas remacemide is an active central nervous system compound with both NMDA receptor antagonist and sodium channel blocking properties. Learning, audio/visual discrimination and motivation were modeled using incremental repeated acquisition (IRA), audio/visual discrimination (AVD) and progressive ratio (PR) tasks, respectively. Ketamine (10 or 100 mg/kg/day), remacemide (100 or 150 mg/kg/day) or water was administered daily (7 days/week) via orogastric gavage beginning on postnatal day (PND) 23 and continuing until PND 257. Monday through Friday behavioral assessments began on PND 27 and continued until PND 383. Chronic treatment with the high dose of ketamine decreased response rate in all tasks suggesting decreased motivation or motoric capabilities. Chronic treatment with ketamine or remacemide had no effect on the acquisition of IRA task performance at any dose tested. While chronic treatment with either high-dose ketamine or low-dose remacemide only delayed the acquisition of AVD task performance for a brief period midway through treatment, chronic treatment with high-dose remacemide delayed the acquisition of AVD task performance until late in treatment. The findings for ketamine are quite different from those of MK-801 (the prototypic NMDA receptor antagonist) in a previous rat study in which MK-801 severely disrupted the acquisition of both IRA and AVD task performances. These observations suggest important differences in the mechanism of action between ketamine and MK-801. For example, ketamine has a much lower binding affinity than MK-801 for the NMDA receptor, the dopamine transporter and the dopamine D2 receptor. In addition, the findings for remacemide observed in rats are in marked contrast with those seen in monkeys where chronic remacemide had profound disruptive effects on the acquisition of both IRA and AVD task performances and suggest important species differences.

Effect of chronic MK-801 and/or phenytoin on the acquisition of complex behaviors in rats.

The purpose of the present experiment was to assess the effects of chronic MK-801 (an N-methyl-D-aspartate receptor antagonist) and/or phenytoin (a sodium channel blocker) treatment on behavioral acquisition and performance in rats. Learning, audio/visual discrimination and motivation were modeled using incremental repeated acquisition (IRA), audio/visual discrimination (AVD) and progressive ratio (PR) tasks, respectively. MK-801 and/or phenytoin were administered daily, 7 days/week by orogastric gavage beginning just after weaning on postnatal day (PND) 23 and continuing until PND 306. Monday through Friday behavioral assessments began on PND 27 and continued until PND 430. Throughout treatment, subjects in the high dose MK-801 (1.0 mg/kg/day) and the high dose drug combination (1.0 mg/kg/day MK-801+150 mg/kg/day phenytoin) groups exhibited decreased body weight gains compared to control subjects. For these two affected groups, response rates were also decreased in all tasks. Task acquisition, as evidenced by an increase in response accuracy, was decreased for both these groups in the AVD task, but only for the high dose MK-801 group in the IRA task. The data suggest that chronic MK-801 treatment adversely affects the acquisition of IRA and AVD task performance and that the inclusion of phenytoin in the MK-801 dosing regimen blocks some of the adverse effects of chronic MK-801 treatment on IRA task acquisition. These findings are in marked contrast with those observed in nonhuman primates and suggest important species differences associated with chronic exposure to compounds that block NMDA receptors and/or sodium channels.

The significance of ultrastructural features in acute myeloid leukemia: a study of 220 patients entered into the Medical Research Council's ninth acute myeloid leukemia trial.

Samples of peripheral blood or bone marrow obtained before treatment from 220 patients entered into the Medical Research Council's ninth acute myeloid leukemia (AML) trial were examined by electron microscopy. Several ultrastructural features showed strong correlations with each other, with variants of AML defined by the Hayhoe classification scheme, and with FAB type. However, no ultrastructural feature was found either uniquely in association with any other or specifically in any variant of AML. Each ultrastructural feature was tested for association with achievement of remission, remission duration, and survival time. Only Auer rods were associated with a high remission rate (p = 0.01), but even this association was not quite conventionally significant when the analysis was not quite conventionally significant when the analysis was stratified for age (p = 0.055). Shorter duration of remission was associated with cytoplasmic projections (p = 0.04 stratified for age). Overall survival was worse when convoluted or lobed nuclei were present but only when stratified for age and Hayhoe type (p = 0.04). The possibility of testing combinations of ultrastructural features for correlation with diagnosis or prognosis is discussed but would require data from more than the present 220 patients to be meaningful.

Paramagnetic microspheres as immunological markers for light and electron microscopy.

M-450 Dynabeads are magnetizable polystyrene microspheres 4.5 micron in diameter to which antibodies of IgM isotype can be physically adsorbed. Antibody-coated Dynabeads can be used to label cell surfaces and then to separate the rosetted cells by application of an external magnetic field. We demonstrate here, using cell lines K562 and U937 and the previously undescribed monoclonal antibodies CH-F42 and CH-E25, that Dynabeads can also be used to label cells at the ultrastructural level. Dynabeads can therefore provide a useful bridge between light and electron microscopy. The preservation of specific rosettes at the ultrastructural level without the formation of artifactual aggregates requires rapid but gentle fixation in dilute suspension. We have achieved this by fixing briefly in a large volume of buffered glutaraldehyde followed by neutralization of excess glutaraldehyde with ethanolamine.

Five new insulin-producing cell lines with differing secretory properties.

Five cell lines have been derived from a rat transplantable islet cell tumour using two different methods. The lines differ in morphology and contain and release different amounts of insulin and glucagon (insulin content, 1-90 pmol/10(6) cells; insulin release, 6-250 pmol/10(6) cells per 24 h; glucagon content, less than 0.005-35 pmol/10(6) cells; glucagon release, less than 0.05-10 pmol/10(6) cells per 24 h). All the lines responded to the presence of the secretagogues leucine (20 mmol/l) plus theophylline (5 mmol/l) by increasing the rate of release of insulin approximately twofold. A high extracellular concentration of potassium (40 mmol/l) caused a three- to tenfold calcium-dependent increase in release of insulin and a parallel release of glucagon. Increasing the concentration of glucose from 2.8 to 16.7 mmol/l did not alter the rate of insulin release by any of the cell lines.

Correlations between nuclear morphology and bundles of cytoplasmic fibrils in 50 cases of acute myeloid leukaemia.

An electron microscopic examination was carried out of peripheral blood or bone marrow samples, or both, from 50 patients entered into the Medical Research Council 9th Acute Myeloid Leukaemia Trial. The results showed a striking correlation between the presence of conspicuous bundles of fibrils within the cytoplasm of the leukaemic cells and the degree of convolution or lobulation of the nuclei. In none of the samples were predominantly convoluted or lobed nuclei observed in the absence of prominent fibrillar bundles and in only two cases were nuclei of a more regular outline seen in association with many conspicuous bundles of cytoplasmic fibrils. No correlation was found between the apparent degree of maturity of the nuclei, as assessed by the degree of chromatin condensation, and the absence or abundance of fibrillar bundles.

Crystal structure of Auer rods in acute myeloblastic leukaemia (AMyL).

Ultrathin sections containing Auer rods from cases of acute myeloblastic leukaemia (AMyL) were tilted in the goniometer stage of the electron microscope and the resulting series of electronmicrographs analysed in an optical diffractometer illuminated by laser. The results showed that Auer rods of AMyL show a truly three dimensional crystal structure. Measurements from the optical diffraction patterns were consistent with a monoclinic unit cell, the unit cell edge lengths a, b, and c being 6.6 [SD) 0.5) nm, 8.6 (0.2) nm, and 9.6 (1.0) nm, respectively; the angle between a and c being 120 (7) degrees. This structure was quite distinct from the "tubular" substructure reported by others in the Auer rods of acute promyelocytic leukaemia (APL), although it was consistent with periodicities measured by others in Auer rods of AMyL. A complete understanding of the three dimensional structures of Auer rods in the different types of acute myeloid leukaemia (AML) could well prove to be of considerable diagnostic importance.

Differential effects of two NMDA receptor antagonists on cognitive-behavioral development in nonhuman primates I.

The present experiment examined effects of chronic exposure to remacemide (an N-methyl-D-aspartate [NMDA] antagonist which also blocks fast sodium channels) or MK-801 (which blocks NMDA receptors, exclusively) on learning and motivation in young rhesus monkeys. Remacemide (20 or 50 mg/kg/day) or MK-801 (0.1 or 1.0 mg/kg/day) was administered every day to separate groups of animals via orogastric gavage for up to 2 years. Immediately prior to dosing, 5 days per week (M--F), throughout the 2-year dosing period, an incremental repeated acquisition (IRA) task was used to assess learning and a progressive ratio (PR) task was used to assess motivation. The results indicate an effect of 50 mg/kg/day remacemide to impair learning (IRA) which persisted even after drug treatment was discontinued. MK-801 had no effect on learning but transiently increased motivation. Because the effects of remacemide occurred independently of changes in motivation or response rates, they are likely due to specific cognitive impairments and are not due to an inability of subjects to fulfill the motoric requirements of the task. The fact that MK-801 did not alter learning suggests that NMDA antagonism alone may be insufficient to produce learning deficits in young monkeys and that such deficits may rely on the ancillary blockade of fast sodium channels.

Differential effects of two NMDA receptor antagonists on cognitive--behavioral performance in young nonhuman primates II.

The present experiment examined the effects of chronic exposure to remacemide (an NMDA antagonist that also blocks fast sodium channels) or MK-801 (which blocks NMDA receptors more selectively) on the acquisition of color and position discrimination and short-term memory behavior in juvenile rhesus monkeys. Throughout the 2-year dosing period, a conditioned position responding (CPR) task was used to assess color and position discrimination and a delayed matching-to-sample (DMTS) task was used to assess memory. Chronic exposure to high doses of either drug delayed the acquisition of accurate color and position discrimination without altering response rates. In the case of MK-801, these effects abated within 6 months of the start of treatment. In the case of remacemide, the effects persisted for 17 months of dosing. Neither compound significantly altered performance of the short-term memory task at any time point or at any dose tested. The fact that the effects of remacemide on behavioral performance were more persistent than those seen for MK-801 suggests that tolerance may develop to the behavioral effects of MK-801, which does not develop to the effects of remacemide. Alternatively, these results may suggest that the concurrent antagonism of NMDA receptors and fast sodium channels may have more profound consequences for behavior than does the antagonism of NMDA receptors alone.

Assessing the potential toxicity of MK-801 and remacemide: chronic exposure in juvenile rhesus monkeys.

The present experiment examined the effects of chronic exposure to either 0.1 or 1.0 mg/kg MK-801 [a selective N-methyl-D-aspartate (NMDA) receptor antagonist] or 20.0 or 50.0 mg/kg remacemide (an NMDA receptor antagonist which also blocks fast sodium channels) in juvenile rhesus monkeys. Endpoints were monitored to provide a general index of subjects' health and included measures of clinical chemistry, hematology, ophthalmology, spontaneous home-cage behavior, and peak drug plasma levels. In general, both drugs were well tolerated and produced no treatment-related effects during 2 years of dosing and assessment. Periodic plasma drug level determinations provided limited evidence that both compounds may induce their own metabolism. The present results contrast sharply with previously reported effects of long-lasting impairments in the acquisition of incremental learning and in the development of color and position discrimination in these same subjects. These observations highlight the importance of collecting a broad range of toxicology data, including tests of cognitive function, to make comprehensive assessments of new drug safety. In the present case, the less obvious effects of these drugs on cognition defined the toxicologic response.

Auer rods and myeloperoxidase: an approach for investigating changes at the molecular level in acute myeloid leukaemia (AML).

Auer rods are cytoplasmic inclusions found only in the leukaemic cells of some cases of acute myeloid leukaemia (AML). The ultrastructure of Auer rods is different in different sub-types of the disease and it is suggested here that this may be a consequence of the Auer rods consisting of different crystalline forms of the enzyme myeloperoxidase. There is statistical evidence to indicate that patients with Auer rods have a more favourable prognosis than those without. It is proposed that an investigation of the factors that determine the form, if any, of the crystallisation of myeloperoxidase will lead to a better understanding of the molecular events underlying the different types of AML. In the long term this may in turn lead to a therapeutic advance.

Ultrastructural features in 100 cases of acute myeloid leukaemia.

Ultrastructural features thought to be significant in acute myeloid leukaemia (AML) have been examined in 100 cases entered into the Medical Research Council 9th AML Trial. It is concluded that, of the features examined, only rarely is there a striking correlation with diagnosis which could be usefully employed with confidence. This is a consequence of there being both a large overlap of similar ultrastructural features between the different sub-types of the disease and also wide variations in ultrastructure within the conventionally diagnosed sub-types of AML. Thus the similarities between cells from different cases which allow them to be classified by light microscopy do not necessarily extend to the ultrastructural level. However, it is suggested that a classification on ultrastructural grounds might have a prognostic significance which would only be revealed by a long-term study.

Crystalline inclusions in the cytoplasm and nuclei of cells of acute myeloid leukaemia.

In a survey by electron microscopy of peripheral blood and/or bone marrow from 230 adult patients with acute myeloid leukaemia, five were observed to contain crystalline inclusions in the cytoplasm of the leukaemic cells and a sixth contained crystals in the nuclei. In four cases, two of FAB type M2 and two of M4, the cytoplasmic crystals were hexagonal in section and 1-2 micron long. Two examples showed internal periodicities in the range 3.3-4.0 nm when the electronmicrographs were analysed by optical diffractometry. A single case of M1 contained smaller trapezoidal crystals with a 4.9nm periodicity. The sixth patient, with unusual cytological abnormalities and a rare t(3; 6) chromosomal translocation, contained six-sided crystals in the nuclei of some relatively undifferentiated cells. To the best of our knowledge such intranuclear crystals have not previously been reported in leukaemia. The relevance of the crystals to the leukaemic process is discussed.

Electron microscopic observations on the development and cytochemistry of the large granule complexes in chicken erythrocyte nuclei.

Large granule complexes are structures found in a small percentage of chicken erythrocyte nuclei when observed in ultra-thin sections in the electron microscope. They consist of an amorphous region associated with a number of large (approximately 30 min) granules. We have shown, by a novel use of phenylhydrazine to synchronize populations of chicken erythrocytes in vivo, that large granule complexes do not occur in the nuclei until the cells have reached one-third to one-half of their normal intravascular lifespan. The mature large granule complexes are formed by aggregation of pre-existing fibrillar, granular and amorphous material, and their presence is correlated with the presence of another ultrastructural feature of the nucleus, the so-called "filled cavities' in the chromatin. Digestion of ultra-thin sections of erythrocytes embedded in the hydrophilic resin glycol methacrylate (GMA) has shown that the major component of the amorphous region is a rather acidic protein that is not haemoglobin, the most abundant protein in the erythrocyte. The large granules also contain protein and, almost certainly, RNA. The problems encountered in reaching this conclusion have emphasized the lack of unambiguous cytochemical tests for use on ultra-thin sections. We have shown that the large granule complex differs in many respects from the nucleolus in the erythrocyte series, even though the two organelles have certain superficial similarities such as their overall dimensions and the presence of granular and fibrillar regions. The most likely function of the large granule complex is as a repository for material, including RNA, the processing of which has ceased in the inactivated erythrocyte nucleus.

A critical evaluation of Bernhard's EDTA regressive staining technique for RNA.

The EDTA regressive staining procedure to detect RNA (Bernhard's technique) is based on the proposition that after staining ultrathin sections with uranyl the stain is preferentially removed from DNA rather than RNA by the action of the chelating agent EDTA. Whilst attempting to use the EDTA regressive staining procedure to detect the presence of RNA in the large granule complexes of chicken erythrocyte nuclei, certain anomalous staining patterns were observed in the chromatin of these nuclei. Essentially, these were that the edges of condensed chromatin bodies stained positively for RNA even though this molecule is known not to be present there in significant quantities. The staining patterns suggested that chromatin was retaining its stain in a manner expected of RNA but not DNA as a consequence of EDTA-containing species failing to pass freely through the section. This hypothesis was tested by carrying out the EDTA procedure on embedded specimens of a DNA-containing virus, simian virus 40 (SV40), small enough not to be exposed at the surface of the section. In this way it has been shown that virus particles completely surrounded by resin destain so much more slowly than chromatin, which is accessible at the surface of the section, that without any other information it would be concluded that the viruses contained RNA, not DNA. This apparently anomalous result arises because the difficulties encountered by stain or EDTA molecules in passing through a plastic section were not appreciated at the time of the initial publication of the technique. The observations are discussed in the light of recent knowledge that has been gained on the kinetics of staining by measuring the electron-scattering densities of stained sections, similar measurements having been made on sections stained by Bernhard's technique. A model for the mechanism of the EDTA regressive staining technique consistent with the experimental observations is proposed and the conditions under which Bernhard's staining procedure retains its specificity are defined. Briefly, these conditions are that: (a) the sections be stained for only a short period with uranyl before treating them with EDTA even though such brief staining is undesirable for quantitative measurements of stain uptake into biological material; and (b) that sections stained in lead only be compared as controls with sections stained by Bernhard's technique so that any specificity of lead for sub-cellular components is not confused with a positive indication of the presence of RNA. Unless these conditions are fulfilled, results obtained by the use of the regressive staining technique may be highly misleading.

Use of light microscopic immunotechniques in selecting preparation conditions and immunoprobes for ultrastructural immunolabelling of lactoferrin.

Successful postembedding immunolabelling for electron microscopy is sometimes difficult to achieve. We propose that light microscopy can be used (1) to detect quickly processing steps which have an adverse effect on the tissue antigenicity and (2) to check the specific reactivity of the immunogold detecting system normally employed at the ultrastructural level. The individual steps of fixation, dehydration and embedding were tested for their ability to preserve antigenicity by light microscopic peroxidase--anti-peroxidase cytochemistry. Steps that severely reduced antigenicity were replaced by less destructive alternatives compatible with reasonable ultrastructural preservation. The specific reactivity of the immunogold detecting system was assessed by using the light microscopic immunogold-silver staining method. We studied the antigen lactoferrin in human neutrophilic granulocytes from patients with chronic myeloid leukaemia. We obtained strong immunolabelling of specific granules and good ultrastructural preservation using routine methods at room temperature. For lactoferrin the method of choice was to fix in 3% paraformaldehyde/0.1% glutaraldehyde followed by 1% OsO4, dehydrate in 70% ethanol, embed in LR White resin and polymerize at 40 degrees C for 40 h. These conditions may not be suitable for all antigens and we emphasize that for each new antigen a similar study should be carried out.

Ultrastructure and cytogenetics in seven cases of acute promyelocytic leukaemia (APL).

In the seven cases of acute promyelocytic leukaemia (APL) presented here we have studied the ultrastructural and cytogenetic features which are thought to be of particular significance in this disease. On the basis of our findings from the seven cases of APL described in detail, and our unreported results obtained for a large number of myeloid leukaemias other than APL, we conclude the following. Stellate rough endoplasmic reticulum, certain inclusion bodies and Auer rods having a tubular substructure are, if present, probably diagnostic of APL. However, these structures are not always observed in APL. Inflated rough endoplasmic reticulum is highly indicative of APL while slender cytoplasmic projections, convoluted or lobed nuclei and conspicuous bundles of cytoplasmic fibrils are very common in the abnormal promyelocytes of this disease. There is a strong correlation between the presence of conspicuous bundles of cytoplasmic fibrils and convoluted or lobed nuclei. Most of the APL cases showed the characteristic translocation t(15;17) and we could find no ultrastructural difference between the cases with the translocation and the single example of a normal karyotype.

Histone H5 promotes the association of condensed chromatin fragments to give pseudo-higher-order structures.

We describe two distinct situations in which chicken erythrocyte chromatin fragments associate in solution. The erythrocyte-specific histone H5 is implicated since chromatins that do not contain H5 do not show this behaviour. Well-defined oligomers of between approximately 6 and approximately 18 nucleosomes prepared at low ionic strength condense and associate when the ionic strength is raised to 75 mM, forming pseudo-higher-order structures. The associated forms, probably predominantly dimers, are stabilized by migration of about 10% of the H5, and of the minor lysine-rich histone H1, from the non-associated forms, probably reflecting the preference of H5 for higher-order structures observed previously [Thomas, J. O. and Rees, C. (1983) Eur. J. Biochem. 134, 109-115]. Since the final (H1 + H5) content of the aggregate at 75 mM is never higher than that of the fragment prepared at low ionic strength, migration is probably to a small proportion of sites that have inevitably become vacant due to handling losses at the higher (but not at low) ionic strength. H5 thus maximizes its interactions in the condensed state of chromatin and even maintains the association of two or more fragments without continuity of the DNA. Aggregates of oligomers larger than about 18 nucleosomes may be too long to withstand hydrodynamic shear forces in the absence of such continuity. During nuclease digestion of nuclear chromatin, H5 and, to a lesser extent, H1, are released from the ends of very short fragments and bind to larger oligomers of various sizes leading to heterogeneous aggregates that survive exposure to low ionic strength. These aggregates, in contrast to those described above, have up to 60% more H5 and 20% more H1 than chromatin prepared at low ionic strength. Whether the excess H5 and H1 bind non-specifically or to a second low-affinity binding site on each nucleosome is not known. The associated forms described above (1) are well defined and potentially useful for structural studies, whereas the other aggregates (2) seem less likely to be directly relevant to the native structure of chromatin.

Retinoic acid induces granulocytic differentiation of myeloid progenitors in congenital agranulocytosis bone marrow cells.

Congenital agranulocytosis is a rare fatal infantile disease characterised by recurrent bacterial infections, persistent absence of neutrophils and maturation arrest at the promyelocyte/myelocyte stage. The effectiveness of retinoic acid in inducing differentiation of congenital agranulocytosis marrow myeloid progenitor cells was studied. Non-adherent mononuclear marrow cells were treated in an in vitro culture with retinoic acid at various concentrations from 1nM to 1 microM for seven days. Morphological and functional differentiation into mature granulocytes was induced by retinoic acid in a dose-response stimulation with a maximum response at a concentration of 1 microM. These results suggest a potential therapeutic role for retinoic acid in the treatment of congenital agranulocytosis.

Higher-order structure of nucleosome oligomers from short-repeat chromatin.

Sedimentation measurements and electron microscopy at a series of ionic strengths suggest that chromatin from neurons of the cerebral cortex is able to form condensed structures in vitro that are probably several turns of a solenoid with about six nucleosomes per turn. Since neuronal chromatin has a short nucleosomal repeat (approximately 165 bp) allowing virtually no linker DNA between nucleosomes, and yet forms apparently 'normal' elements of solenoid, the packing of nucleosomes in the solenoid must be highly constrained. This permits only a limited number of possible models, and enables tentative suggestions to be made about the location of the linker DNA in the typical solenoid.