Colocalization of (3H)-adenosine accumulation and GABA immunoreactivity in the chicken and rabbit retinas

Summary Using combined autoradiography and immunohistochemistry, we have compared (³H)-adenosine accumulation and GABA immunoreactivity in the chicken and rabbit retinas. Colocalization of the two markers was observed in a subset of amacrine cells and in certain cell bodies in the ganglion cell layer in both species and in a few horizontal cells in the chicken retina. Cells that contained only (³H)-adenosine or GABA were also seen. The degree of colocalization differed greatly between the two species. The results demonstrate a morphological relationship between the adenosine and GABA systems and provides information of the possible anatomical substrates underlying at teast some types of functional interactions.     

Glutamate Decarboxylase Activities in Single Vertebrate Neurons

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.     

多种神经活性物质在幼年猫和鸡视网膜神经...

An improved post-embedding immunocytochemical technique was used to examine the coexistence of multi-neuroactive substances in neurons of young cat and newly hatched chicken retinas. We found that two classical neurotransmitters-glycine (Gly) and r-aminobutyric acid (GABA) and one neuropeptide, neurotensin (NT) were located in the amacrine cells of cat and chicken retinas. Among them, some cells contain Gly and GABA (Gly/GABA AC), some cells contain Gly and NT (Gly/NT AC) or GABA and NT (GABA/NT AC), and the other cells, a few of them contain Gly, GABA and NT (Gly/GABA/NT AC). In addition, two classical neurotransmitters Gly and GABA were also located or coexisted in the horizontal cells of cat retina. These results indicated that the coexistence of two neurotransmitters or one neurotransmitter and one neuropeptide or two neurotransmitters and one neuropeptide was presented in neurons of developing can and chicken retinas.     

多种神经活性物质在幼年猫和鸡视网膜神经细胞中的共存(简报)

视网膜起源于前脑泡,结构简单层次分明,因此常被视为脑的简化模型。但近期工作证明,视网膜包含的神经活性物质(神经递质、调质和神经肽)种类之多、分布之复杂并不亚于脑。尤其是无长突细胞不仅包含多种递质和肽类,而且还有递质与肽类或肽类与肽类在一个细胞中的共存。对视网膜神经递质、调质和神经肽的研究将是探索脑奥秘的有效途径。     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum.

A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent Kmvalues for GABA neurons for high and low affinity uptake were 0.33 X 10(-6) M and 41.8 X 10(-4) M, respectively. For nonneuronal cells, the apparent Km for high affinity uptake was 0.29 X 10(-6) M. The apparent Vmax values for GABA neurons for high and low affinity uptake were 28.7 X 10(-6) mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent Vmax for high affinity uptake was 0.06 X 10(-6) mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 X 10(-9) M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by noneuronal cells was only slightly decreased. Most (75-85%) of the [3H]GABA (4.4 X 10(-6) M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

GABAergic Inhibition on Dopamine Cells of the Fish Retina: A [3H]Dopamine Release Study with Isolated Cell Fractions

Inner retinal cells including dopamine (DA) cells were isolated and fractionated from the carp (Cyprinus carpio) retina by an enzyme cell dissociation and metrizamide gradient centrifugation method. When gamma-aminobutyric acid (GABA) antagonists (bicuculline and picrotoxin) were added into the perfusate over such a cell fraction, they stimulated the release of [3H]DA which had been preloaded in the cell fraction. The action of GABA antagonists was dose and Ca2+ dependent. Their minimal effective concentration was very low (0.5 microM). A similar action was elicited by high K+. In the presence of excess GABA, this stimulatory action of GABA antagonists and high K+ on [3H]DA release was completely abolished. To interpret the action of GABA antagonists on DA cells, isolated cell fractions were preincubated with GABAse. After such a treatment, the stimulatory effects of GABA antagonists and high K+ on [3H]DA release were differentiated from each other; the former disappeared whereas the latter remained unchanged. The data strongly suggest that GABA inhibits the DA release from retinal DA cells and thus the GABA antagonists affect [3H]DA release from cell fractions not by a direct membrane action but by a disinhibition mechanism via GABA receptors on the DA cell bodies.     

Dopamine Inhibits Calcium-Independent γ-[3H]Aminobutyric Acid Release Induced by Kainate and High K+ in the Fish Retina

Kainic acid (KA) at micromolar concentrations stimulated the release of gamma-[3H]aminobutyric acid [( 3H]GABA) from a particulate fraction of the carp (Cyprinus carpio) retina. The KA action was dose-dependent but Ca2+-independent. A similar response was elicited by another glutamate receptor agonist, quisqualic acid, and high K+, but not by an aspartate agonist, N-methyl-D-aspartic acid. The stimulatory action of KA on the [3H]GABA release was selectively blocked by the KA blockers gamma-D-glutamylglycine and cis-2,3-piperidine dicarboxylic acid. Dopamine (DA), which is contained in DA interplexiform cells in the carp retina, inhibited the [3H]GABA release induced by KA and high K+ in a dose-dependent manner. 5-Hydroxytryptamine and two well-known GABA antagonists, bicuculline (Bic) and picrotoxin (Pic), also mimicked the DA effect on the GABA release at a comparable concentration. This inhibitory effect of DA as well as Bic and Pic on the [3H]GABA release evoked by KA was clearly antagonized by a DA blocker, haloperidol. The action of these agents (KA, DA, GABA antagonist) belonging to three different receptor categories on the GABAergic neurons (possibly external horizontal cells; H1 cells) is discussed in relation to other electrophysiological studies on the lateral spread of S-potentials between H1 cells.     

Inter- and intracellular relationship of substance P-containing neurons with serotonin and GABA in the dorsal raphe nucleus: combination of autoradiographic and immunocytochemical techniques

Double-labeling experiments were performed at the electron microscopic level in the dorsal raphe nucleus of rat, in order to study the inter- and intracellular relationship of substance P with gamma-aminobutyric acid (GABA) and serotonin. Autoradiography for either [3H]serotonin or [3H]GABA was coupled, on the same tissue section, with peroxidase-antiperoxidase immunocytochemistry for substance P in colchicine-treated animals. Intercellular relationships were represented by synaptic contacts made by [3H]serotonin-labeled terminals on substance P-containing somata and dendrites, and by substance P-containing terminals on [3H]GABA-labeled cells. Intracellular relationships were suggested by the occurrence of the peptide within [3H]serotonin-containing and [3H]GABA-containing cell bodies and fibers. Doubly labeled varicosities of the two kinds were also observed in the supraependymal plexus adjacent to the dorsal raphe nucleus. The results demonstrated that, in addition to reciprocal synaptic interactions made by substance P with serotonin and GABA, the dorsal raphe nucleus is the site of intracellular relationships between the peptide and either the amine or the amino acid.     

Characterization of the chicken erythrocyte anion exchange protein

The avian erythrocyte anion exchange protein (band 3), after labeling with [3H2]4,4'-diisothiocyanodihydrostilbene-2, 2'-disulfonic acid appears as a doublet of polypeptide chains with apparent Mr = 105,000 and 100,000 by sodium dodecyl sulfate gel electrophoresis. The structures of the two species are almost identical as determined by partial proteolysis. The copy number of band 3 molecules per chicken erythrocyte was determined to be 800,000 by quantitating the amount of [3H2]4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid covalently bound to the cell surface. A comparison of human and chicken band 3 has revealed differences in their structure. Chicken band 3 differs from the human polypeptide in isoelectric point and proteolytic patterns. Antisera raised against human and chicken band 3 do not cross-react, implying that the two sera do not recognize any common antigenic determinants. There is a 6.5-fold lower activity per cell in the rate of phosphate exchange in the chicken erythrocyte which can be entirely explained by the 1.5-fold decrease in copy number per cell and the increased size of the chicken erythrocyte. This would suggest that there is no difference in the enzyme turnover number between chicken and human band 3. A major functional difference resulting from the structural differences is the inability to bind glyceraldehyde-3-phosphate dehydrogenase, a function associated with the NH2 terminus of human band 3.     

Differential effects of zinc on native GABA(A) receptor function in rat hippocampus and cerebellum.

Hippocampal noradrenergic and cerebellar glutamatergic granule cell axon terminals possess GABA(A) receptors mediating enhancement of noradrenaline and glutamate release, respectively. The hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar one is not affected by benzodiazepine agonists, indicating the presence of an alpha6 subunit. We tested here the effects of Zn2+ on these two native GABA(A) receptor subtypes using superfused rat hippocampal and cerebellar synaptosomes. In the cerebellum, zinc ions strongly inhibited (IC50 approximately 1 microM) the potentiation of the K(+)-evoked [3H]D-aspartate release induced by GABA. In contrast, the GABA-evoked release of [3H]noradrenaline from hippocampal synaptosomes was much less sensitive to Zn2+ (IC50 > 30 microM). The effects of Zn2+ were then studied in two rat lines selected for high (ANT) and low (AT) alcohol sensitivity because granule cell GABA(A) receptors in ANT, but not AT, rats respond to benzodiazepine agonists due to a critical mutation in the alpha6 subunit. GABA increased the K(+)-evoked release of [3H]DCNS REGIONS-aspartate from cerebellar synaptosomes of AT and ANT rats, an effect prevented by the GABAA selective antagonist bicuculline. In AT rat cerebellum, the effect of GABA was strongly inhibited by Zn2+ (IC50 < or = 1 microM), whereas in ANT rats, the divalent cation was about 100-fold less potent. Thus, native benzodiazepine-sensitive GABAA receptors appear largely insensitive to functional inhibition by Zn2+ and vice versa. Changes in sensitivity to Zn2+ inhibition consequent to mutations in cerebellar granule cell GABA(A) receptor subunits may lead to changes in glutamate release from parallel fibers onto Purkinje cells and may play important roles in cerebellar dysfunctions.     

Interactions of glucagon and related peptides with chicken adipose tissue.

The effect of glucagon, Vasoactive Intestinal Polypeptide (VIP), secretin and gut glucagon on the cyclic adenosine 3'5' monophosphate (cAMP) level, and on the specific binding of these 125I-peptides to the adipocyte plasma membrane was measured in chicken adipocytes and compared to the results obtained in rat adipocytes. The displacement of 125I-glucagon from its specific sites was observed with about the same concentration of unlabeled hormone in fat cell plasma membranes of both species. However, the rise in cAMP induced by glucagon was much higher in chicken than in rat adipocytes. In chicken fat cells unlike rat fat cells, the cAMP accumulation elicited by glucagon was maintained during at least 60 min even in the absence of theophylline. Theophylline at 1-10 mM potentiated the glucagon-stimulated cAMP levels in rat fat cells, but had only a slight effect, if any, in chicken adiposyces. Porcine VIP, secretin or gut glucagon exerted no detectable action on the cAMP level of chicken adipocytes. The lack of cAMP accumulation was in good agreement with the absence of binding of 125I-VIP and 125I-secretin by chicken plasma membranes. These findings suggest that: 1) the difference of glucagon effect in rat and chicken fat cells results from variations in the rate of degradation of cAMP rather than from differences in the specific binding of glucagon between the two species; 2) the use of chicken fat cells is suitable to discriminate between glucagon and structurally related peptides from mammals.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Induced Release of γ-Aminobutyric Acid by a Carrier-Mediated, High-Affinity Uptake of L-Glutamate in Cultured Chick Retina Cells

[3H]gamma-aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse-stimulated (2 min) with 0.1 mM L-glutamate but not with D-glutamate. The EC50 for L-glutamate to evoke [3H]GABA release was approximately 15 microM. This value is close to the Km for high-affinity uptake of L-glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L-glutamate-induced release of GABA was abolished. Moreover, L-[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L-Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D-Aspartate, which is taken up by the same high-affinity uptake mechanism as L-glutamate, induced an increase in [3H]GABA efflux comparable to L-glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L-glutamate in eliciting [3H]GABA release. The addition of both GABA and L-glutamate to the incubation medium indicated that [3H]GABA efflux due to L-glutamate and GABA was additive. L-Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L-aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA-promoted GABA release-homoexchange, (b) a Ca2+-dependent L-aspartate-promoted release, and (c) a Ca2+-independent, Na+-dependent L-glutamate-evoked release. In addition, our data strongly suggest that the L-glutamate-promoted GABA release is due to a process of exchange of L-glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.     

High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.     

Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture

We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.     

Plant and animal histones are completely interchangeable in the nucleosome core

Nucleosome core particles were reconstituted using mixtures of plant (corn or tobacco) and animal (chicken erythrocytes) histones. We show by electron microscopy and sucrose gradient sedimentation that H3 and H4 from tobacco and chicken erythrocytes can be interchanged in the nucleosome kernel. Cross-linking experiments with the protein cross-linking reagent dimethylsuberimidate reveal that, despite structural differences between the histones of the two species, H2A and H2B can be interchanged provided the homologous H2A-H2B dimers are dissociated prior to the annealing.     

Characterization of separated cell types from developing rat cerebellum

The characteristics of [³H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [³H]GABA. In comparison with astrocytes, theV _max of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10⁶ cells) and the apparentK _t twofold greater (2 vs. 1 M). In contrast to these cell types, uptake was very low in granule cell-enriched preparations.cis-1,3-Aminocyclohexane carboxylic acid was a potent inhibitor of [³H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for -alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [³H]GABA transport was Na⁺ dependent in both cell classes. However, veratridine and ouabain selectively blocked [³H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [³H]GABA transport systems and the underlying metabolic processes.     

Differences in Receptor Specificity between the Influenza Viruses of Duck,Chicken, and Human

The affinity of the duck, chicken, and human influenza viruses to the host cell sialosides was determined, and considerable distinctions between duck and chicken viruses were found. Duck viruses bind to a wide range of sialosides, including the short-stem gangliosides. Most of the chicken viruses, like human ones, lose the ability to bind these gangliosides, which strictly correlates with the appearance of carbohydrate at position 158–160. The affinity of the chicken viruses to sialoglycoconjugates of chicken intestine as well as chicken, monkey, and human respiratory epithelial cells exceeds that of the duck viruses. The human influenza viruses have high affinity to the same cells but do not bind at all to the duck epithelial cell. This testifies to the absence of 6"-sialylgalactose residues from the duck cells, in contrast to chicken and monkey cells. The alteration of the receptor specificity of chicken viruses in comparison with duck ones results in the similarity of the patterns of accessible cells for chicken and human influenza viruses. This may be the cause of the appearance of the line of H9N2 viruses from Hong Kong live bird markets with receptor specificity similar to that of H3N2 human viruses, and of the ability of H5N1 and H9N2 chicken influenza viruses to infect humans.     

Mammalian Pol III Promoter H1 can Transcribe shRNA Inducing RNAi in Chicken Cells

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. RNAi based on DNA vector is not sufficiently established in chicken species. The present study was performed to evaluate RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 in the chicken cells by using a dual fluorescence reporter assay, a plasmid encoding GFP and a plasmid encoding RFP. The evaluation of RNAi efficiency was performed in two kinds of chicken cell type: primary CEF cells and chicken DT-40 cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescent microscopy, and their mRNAs content were analyzed by quantitative RT-PCR. The intensity of the green fluorescence generated by GFP was greatly suppressed by human H1 promoter transcribed GFP-shRNA. Quantitative RT-PCR analysis showed that normalized GFP mRNA expression was reduced to 37 and 32 in primary CEF and DT-40 cells, respectively. In contrast to GFP, the intensity of the red fluorescence generated by RFP protein and the RFP mRNA levels remained unchanged. Consequently, it was concluded that the RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 is applicable to suppress the gene expression specifically and efficiently in chicken cells. Jing Yuan and Xiaobo Wang - These authors contributed equally to this work.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.