Three-Dimensional Fermi Surface of Overdoped La-Based Cuprates.

We present a soft x-ray angle-resolved photoemission spectroscopy study of overdoped high-temperature superconductors. In-plane and out-of-plane components of the Fermi surface are mapped by varying the photoemission angle and the incident photon energy. No k_{z} dispersion is observed along the nodal direction, whereas a significant antinodal k_{z} dispersion is identified for La-based cuprates. Based on a tight-binding parametrization, we discuss the implications for the density of states near the van Hove singularity. Our results suggest that the large electronic specific heat found in overdoped La_{2-x}Sr_{x}CuO_{4} cannot be assigned to the van Hove singularity alone. We therefore propose quantum criticality induced by a collapsing pseudogap phase as a plausible explanation for observed enhancement of electronic specific heat.

Surface antigens of brain synapses: identification of minor proteins using polyclonal antisera.

Antigenic proteins of brain synaptic plasma membranes (SPM) and postsynaptic densities (PSD) were characterized using antisera raised against SPM. Immunostaining of brain sections showed that the antigens were restricted to synapses, and electron microscopy revealed staining at both presynaptic terminals and PSDs. In primary brain cell cultures the antisera were also neuron-specific but the antigens were distributed throughout the entire neuronal plasma membrane, suggesting that some restrictive influence present in whole tissue is absent when neurons are grown dispersed. The antigenic proteins with which these antisera react were identified using SDS gel immunoblots. SPM and PSD differed from one another in their characteristic antigenic proteins. Comparison with amido-black stained gel blots showed that in both cases most of these did not correspond to known abundant proteins of SPM or PSDs revealed by conventional biochemical techniques. None of the antigens revealed by the polyclonal antisera were detected by any of a large series of monoclonal antibodies against SPM.

Two-dimensional type-II Dirac fermions in layered oxides.

Relativistic massless Dirac fermions can be probed with high-energy physics experiments, but appear also as low-energy quasi-particle excitations in electronic band structures. In condensed matter systems, their massless nature can be protected by crystal symmetries. Classification of such symmetry-protected relativistic band degeneracies has been fruitful, although many of the predicted quasi-particles still await their experimental discovery. Here we reveal, using angle-resolved photoemission spectroscopy, the existence of two-dimensional type-II Dirac fermions in the high-temperature superconductor La1.77Sr0.23CuO4. The Dirac point, constituting the crossing of [Formula: see text] and [Formula: see text] bands, is found approximately one electronvolt below the Fermi level (EF) and is protected by mirror symmetry. If spin-orbit coupling is considered, the Dirac point degeneracy is lifted and the bands acquire a topologically non-trivial character. In certain nickelate systems, band structure calculations suggest that the same type-II Dirac fermions can be realised near EF.

Direct observation of orbital hybridisation in a cuprate superconductor.

The minimal ingredients to explain the essential physics of layered copper-oxide (cuprates) materials remains heavily debated. Effective low-energy single-band models of the copper-oxygen orbitals are widely used because there exists no strong experimental evidence supporting multi-band structures. Here, we report angle-resolved photoelectron spectroscopy experiments on La-based cuprates that provide direct observation of a two-band structure. This electronic structure, qualitatively consistent with density functional theory, is parametrised by a two-orbital ([Formula: see text] and [Formula: see text]) tight-binding model. We quantify the orbital hybridisation which provides an explanation for the Fermi surface topology and the proximity of the van-Hove singularity to the Fermi level. Our analysis leads to a unification of electronic hopping parameters for single-layer cuprates and we conclude that hybridisation, restraining d-wave pairing, is an important optimisation element for superconductivity.

Glial-restricted precursors: patterns of expression of opioid receptors and relationship to human immunodeficiency virus-1 Tat and morphine susceptibility in vitro.

Recent evidence suggests that human immunodeficiency virus (HIV)-induced pathogenesis is exacerbated by opioid abuse and that the synergistic toxicity may result from direct actions of opioids in immature glia or glial precursors. To assess whether opioids and HIV proteins are directly toxic to glial-restricted precursors (GRPs), we isolated neural stem cells from the incipient spinal cord of embryonic day 10.5 ICR mice. GRPs were characterized immunocytochemically and by reverse transcriptase-polymerase chain reaction (RT-PCR). At 1 day in vitro (DIV), GRPs failed to express mu opioid receptors (MOR or MOP) or kappa-opioid receptors (KOR or KOP); however, at 5 DIV, most GRPs expressed MOR and KOR. The effects of morphine (500 nM) and/or Tat (100 nM) on GRP viability were assessed in GRPs at 5 DIV by examining the apoptotic effector caspase-3 and cell viability (ethidium monoazide exclusion) at 96 h following continuous exposure. Tat or morphine alone or in combination caused significant increases in GRP cell death at 96 h, but not at 24 h, following exposure. Although morphine or Tat caused increases in caspase-3 activity at 4 h, this was not accompanied with increased cleaved caspase-3 immunoreactive or ethidium monoazide-positive dying cells at 24 h. The results indicate that prolonged morphine or Tat exposure is intrinsically toxic to isolated GRPs and/or their progeny in vitro. Moreover, MOR and KOR are widely expressed by Sox2 and/or Nkx2.2-positive GRPs in vitro and the pattern of receptor expression appears to be developmentally regulated. The temporal requirement for prolonged morphine and HIV-1 Tat exposure to evoke toxicity in glia may coincide with the attainment of a particular stage of maturation and/or the development of particular apoptotic effector pathways and may be unique to spinal cord GRPs. Should similar patterns occur in vivo then we predict that immature astroglia and oligodendroglia may be preferentially vulnerable to HIV-1 infection or chronic opiate exposure.

Caspase-3 activity is reduced after spinal cord injury in mice lacking dynorphin: differential effects on glia and neurons.

Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin(-/-) mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin(-/-) mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3-4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6-7 mm, there were fewer caspase-3(+) oligodendrocytes and astrocytes than at 3-4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin(-/-) oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg(6)-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.

Differential involvement of p38 and JNK MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite degeneration in striatal neurons.

The role of p38 and c-jun-N-terminal kinases 1/2, members of the mitogen-activated protein kinase family, in mediating the toxic effects of human immunodeficiency virus-1 transactivator of transcription (Tat) and gp120 were explored in primary mouse striatal neurons in vitro. Both Tat and gp120 caused significant increases in p38 and c-jun-N-terminal kinase mitogen-activated protein kinase phosphorylation, caspase-3 activity, neurite losses and cell death in striatal neurons. Tat-induced increases in caspase-3 activity were significantly attenuated by an inhibitor of c-jun-N-terminal kinase (anthra[1,9-cd]pyrazol-6(2H)-one), but not by an inhibitor of p38 ([4-(4-fluorophenyl)-2-(4-methylsul-finylphenyl)-5-(4-pyridyl)1 H-imidazole]), mitogen-activated protein kinase. However, despite preventing increases in caspase-3 activity, c-jun-N-terminal kinase inhibition failed to avert Tat-induced neuronal losses suggesting that the reductions in caspase-3 activity were insufficient to prevent cell death caused by Tat. Alternatively, gp120-induced increases in caspase-3 activity, neurite losses and neuronal death were prevented by p38, but not c-jun-N-terminal kinase, mitogen-activated protein kinase inhibition. Our findings suggest that gp120 induces neuronal dysfunction and death through actions at p38 mitogen-activated protein kinase, while Tat kills neurons through actions that are independent of p38 or c-jun-N-terminal kinase mitogen-activated protein kinase, or through the concurrent activation of multiple proapoptotic pathways.

Genetic background regulates semaphorin gene expression and epileptogenesis in mouse brain after kainic acid status epilepticus.

The host response to neural injury, which can include axonal sprouting and synaptic reorganization is likely to be under tight genetic regulatory control at the level of the genome and may be implicated in epileptogenesis. Despite its importance, however, the molecular basis of synaptic reorganization is unclear. We have studied the development of synaptic reorganization, semaphorin gene expression, and epileptogenesis in hippocampus of epileptogenic sensitive (FVB/NJ) and epileptogenic resistant (C57BL/6J) mice (i.e. distinct genetic backgrounds) after kainic acid-induced status epilepticus. Our results support the hypothesis that disruption of transcriptional regulation of axon guidance genes leads to a differential loss of tonic neuropilin-2 dependent activation of semaphorin 3F receptors on hippocampal neurons on distinct genetic backgrounds. This results in rearranged synaptic circuitry and thus promotes epileptogenesis. These findings may define biologic principles underlying the role of semaphorin signaling which may broadly apply to other systems undergoing neural regeneration.

Molecular targets of opiate drug abuse in neuroAIDS.

Opiate drug abuse, through selective actions at mu-opioid receptors (MOR), exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the CNS by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Neurons are affected directly and indirectly by opiate-HIV interactions. Although most opiates drugs have some affinity for kappa (KOR) and/or delta (DOR) opioid receptors, their neurotoxic effects are largely mediated through MOR. Besides direct actions on the neurons themselves, opiates directly affect MOR-expressing astrocytes and microglia. Because of their broad-reaching actions in glia, opiate abuse causes widespread metabolic derangement, inflammation, and the disruption of neuron-glial relationships, which likely contribute to neuronal dysfunction, death, and HIV encephalitis. In addition to direct actions on neural cells, opioids modulate inflammation and disrupt normal intercellular interactions among immunocytes (macrophages and lymphocytes), which on balance further promote neuronal dysfunction and death. The neural pathways involved in opiate enhancement of HIV-induced inflammation and cell death, appear to involve MOR activation with downstream effects through PI3-kinase/Akt and/or MAPK signaling, which suggests possible targets for therapeutic intervention in neuroAIDS.

Sensitization of enteric neurons to morphine by HIV-1 Tat protein.

BACKGROUND: Gastrointestinal (GI) dysfunction is a major cause of morbidity in acquired immunodeficiency syndrome (AIDS). HIV-1-induced neuropathogenesis is significantly enhanced by opiate abuse, which increases proinflammatory chemokine/cytokine release, the production of reactive species, glial reactivity, and neuronal injury in the central nervous system. Despite marked interactions in the gut, little is known about the effects of HIV-1 in combination with opiate use on the enteric nervous system. METHODS: To explore HIV-opiate interactions in myenteric neurons, the effects of Tat ± morphine (0.03, 0.3, and 3 µM) were examined in isolated neurons from doxycycline- (DOX-) inducible HIV-1 Tat(1-86) transgenic mice or following in vitro Tat 100 nM exposure (>6 h). KEY RESULTS: Current clamp recordings demonstrated increased neuronal excitability in neurons of inducible Tat(+) mice (Tat+/DOX) compared to control Tat-/DOX mice. In neurons from Tat+/DOX, but not from Tat-/DOX mice, 0.03 µM morphine significantly reduced neuronal excitability, fast transient and late long-lasting sodium currents. There was a significant leftward shift in V(0.5) of inactivation following exposure to 0.03 µM morphine, with a 50% decrease in availability of sodium channels at -100 mV. Similar effects were noted with in vitro Tat exposure in the presence of 0.3 µM morphine. Additionally, GI motility was significantly more sensitive to morphine in Tat(+) mice than Tat(-) mice. CONCLUSIONS & INFERENCES: Overall, these data suggest that the sensitivity of enteric neurons to morphine is enhanced in the presence of Tat. Opiates and HIV-1 may uniquely interact to exacerbate the deleterious effects of HIV-1-infection and opiate exposure on GI function.

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

Subplasmalemmal Ca-stores in Paramecium tetraurelia. Identification and characterisation of a sarco(endo)plasmic reticulum-like Ca(2+)-ATPase by phosphoenzyme intermediate formation and its inhibition by caffeine.

Considering increasing interest in calcium stores in protozoa, including parasitic forms, and specifically in subplasmalemmal stores in higher eukaryotes, we have isolated subplasmalemmal calcium stores (alveolar sacs) from the ciliated protozoan, Paramecium tetraurelia. Using antibodies against established sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCAs) we detected in Western blots of subcellular fractions a band of approximately 106 kDa size selectively in alveolar sacs--but not, for example, in plasma membranes--and concomitant restriction of immunofluorecence labelling to the cell cortex of permeabilised cells. These results are the same as with ABs against a peptide derived from a cloned SERCA-like gene from Paramecium [Hauser K., Pavlovic N., Kissmehl R., Plattner H. Molecular characterization of a sarco(endo)plasmic reticulum Ca(2+)-ATPase gene from Paramecium tetraurelia and localisation of its gene product to subplasmalemmal calcium stores. Biochem J 1998; 334: 31-38]. When such isolated alveolar sacs were now tested for phosphoenzyme intermediate (EP) formation, a phosphoprotein of the same apparent molecular mass (approximately 106 kDa) as in blots could be identified in gel autoradiograms. This EP corresponds to that formed in the reaction cycle of different SERCA-types, with dependency on Ca2+ and Mg2+, sensitivity to La3+ or insensitivity towards calmodulin, calmodulin antagonists and vanadate. However, EP formation in alveolar sacs is not inhibited by established SERCA inhibitors (e.g. thapsigargi[ci]n tested up to 100 microM). Surprisingly, caffeine, which is frequently used to mobilise Ca2+ from intracellular stores, strongly inhibits EP formation. In parallel experiments, we did not find any similar effect with sarcoplasmic reticulum isolated from skeletal muscle. We conclude that the approximately 106 kDa protein of alveolar sacs in Paramecium may represent a SERCA-like Ca(2+)-ATPase with some unorthodox features, which might be relevant also for some other protozoan systems. In this case, the established Ca(2+)-mobilizing effect of caffeine may be amplified by inhibiting store refilling.

Expression of the green fluorescent protein in Paramecium tetraurelia.

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.

Purification of the inducible alpha-agglutinin of S. cerevisiae and molecular cloning of the gene.

The alpha-agglutinin responsible for mating type-specific agglutination of S. cerevisiae alpha-cells has been purified to homogeneity. The glycoprotein released from the cell surface under mild conditions has a relative molecular mass of 200 to 300 kDa as determined by SDS-gel electrophoresis. The protein moiety corresponds to 68.2 kDa. With an oligonucleotide corresponding to the N-terminal amino acid sequence, the alpha-agglutinin gene has been cloned and sequenced. From the DNA sequence, a protein of 631 amino acids with 12 potential N-glycosylation sites is predicted. The carboxy terminal one-third of the protein is not required for agglutination activity.

A novel, calcium-inhibitable casein kinase in Paramecium cells.

This is the first identification of a Ca2+-inhibitable casein kinase (CPK) which we have isolated from the 100000 x g supernatant of Paramecium cell homogenates. The 1000-fold enriched CPK activity depends on millimolar Mg2+ and is inhibited by low concentrations of heparin or by > or = 100 microM Ca2+. Enzyme activity is stimulated by polylysine or polyarginine with either casein or with specific casein kinase-2 (CK-2) peptide substrates (RRRDDDSDDD and RREEETEEE). The enzymic properties are similar with GTP instead of ATP. CPK does not undergo autophosphorylation. In gel kinase assays, enzyme activity is associated with a 36 kDa band. Calmodulin as another characteristic substrate for mammalian CK-2 has not been phosphorylated by this protein kinase. Besides casein, CPK phosphorylates in vitro the catalytic subunit of bovine brain calcineurin (CaN), a typical substrate of type 1 mammalian casein kinase (CK-1) in vitro. Again this phosphorylation is significantly reduced by Ca2+. Thus, CPK combines aspects of different casein kinases, but it is clearly different from any type known by its Ca2+ inhibition. Since CPK also phosphorylates the exocytosis-sensitive phosphoprotein, PP63, in Paramecium, which is known to be dephosphorylated by CaN, an antagonistic Ca2+-effect during phosphorylation/dephosphorylation cycles may be relevant for exocytosis regulation.

Autoradiographic studies of cerebellar histogenesis in the premetamorphic bullfrog tadpole: II. Formation of the interauricular granular band.

This study examines the origin of cells in the interauricular granular band (iagb) in the cerebellum of the frog tadpole during early stages of development by means of histological and autoradiographic methods. Premetamorphic bullfrog tadpoles were exposed to multiple doses of 3H-thymidine (10 microCi/g body weight per exposure) at developmental stages ranging from 1 week to 1 year and were killed at either 6 or 12 months of age. The autoradiographic data were examined to determine the time when cells of the iagb were generated. Our findings show that initial generation of iagb cells begins at week 3 and that a peak in the formation of postmitotic neurons is reached at the age of 10 weeks. This is followed by other peaks of cell generation at the ages of 16 weeks, 10 months, and 11.5 months. The generation cycles of iagb cells are interrupted by periods of quiescence when label cannot be detected in any of the cells. These quiescent periods occur at the ages of 20-26 weeks, 7 months, and 12 months. These findings indicate that cells of the iagb are generated in a cyclical manner over the entire 1-year period which was studied. Comparison of our present data on iagb cell formation with the generation of cells in the EGL shows that the production of these two groups of cells is overlapping, but cells of the iagb begin and cease production before those of the EGL. On the basis of our findings we propose that the cells of the iagb and the EGL belong in separate cell groups which are generated by distinct subpopulations of germinal cells in the neuroepithelial cap.

Autoradiographic studies of cerebellar histogenesis in the premetamorphic bullfrog tadpole: I. Generation of the external granular layer.

This study examines the time of origin of cells in the external granular layer (EGL) in the frog cerebellum during early stages of development. Premetamorphic bullfrog tadpoles were given multiple intraperitoneal injections of 3H-thymidine (10 microCi/g body weight per injection) at developmental stages ranging from 4 weeks to 1 year and were killed at either 6 or 12 months of age. Autoradiograms were analyzed to determine the time when cells of the EGL were generated by an examination of the labeling pattern in the neuroepithelial cap where EGL cells were presumably formed and in the EGL into which they migrated. The developmental stage of the cerebellum in the 6-month-old tadpole was essentially the same as that of the 12-month-old animal except for an increased size in the older tadpole. The cerebellum in both age groups contained a distinct neuroepithelial cap and an EGL, which was somewhat better formed in the 12-month-old tadpole. Some heavily labeled cells were found in the neuroepithelial caps of 6-month-old tadpoles from injection times of 6 weeks to 6 months. In the cerebella of 12-month-old tadpoles, however, heavily labeled cells were found in the neuroepithelial cap only with the injection time of 12 months; with injection times from 7 to 11 months, the cells were labeled lightly. Labeled EGL cells were found in the cerebella of 6-month-old tadpoles from an injection time of 6 weeks on; with injection times from 10 weeks to 6 months some EGL cells contained heavy amounts of label. In the cerebella of 12-month-old tadpoles, labeling of EGL cells was not detectable with injection times of 7-9 months; they contained light to medium labeling with injection times of 10 and 11 months and heavy labeling when injected at 12 months. These results indicate that EGL cells are generated continuously in premetamorphic tadpoles from the age of 6 weeks to 12 months. Furthermore, these results suggest that the rate of EGL cell formation is faster during the second half-year of development than during the first.

Stellate cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Stellate cell development was studied in the bullfrog cerebellum during spontaneous and thyroxine-induced metamorphosis using the Golgi-Kopsch method and electron microscopy. Cells that possessed axosomatic synapses and resembled stellate cells were present even in the incipient molecular layer of the cerebellum in the premetamorphic tadpole. These cells may have originated from the early, transient wave of external granule cells that have been reported in the cerebellum of premetamorphic tadpoles in the first 6 months of development, and may constitute the variant population of stellate cells that are present later during development or the degenerating cells that have been observed during metamorphosis as scattered dying cells in the molecular layer. Typical stellate cells, whose development resembled the genesis and differentiation of stellate cells in birds and mammals, were initially observed at the outer border of the molecular layer that is adjacent to the external granular layer during the onset of metamorphosis. These stellate cells were bipolar with processes extending in a plane perpendicular to elongating parallel fibers, and with progressive development, became multipolar with dendrites oriented in various directions with respect to the pia. Stellate cell axons innervate the dendrites and somata of Purkinje cells and other stellate cells, and can be categorized into two types: (1) axons with extensive branching near the soma of origin, and (2) long axons with few branches that occasionally terminate in the Purkinje cell layer. Atypical neurons that did not resemble typical stellate cells were also present in the molecular layer; these might be classified as a stellate cell variant. The generation and differentiation of stellate cells can be induced 1 to 2 years prematurely by administering thyroid hormone to premetamorphic tadpoles. Like most events of cerebellar histogenesis in the frog, stellate cell development also appears to be largely a thyroid-dependent phenomenon.

Granule cell development in the frog cerebellum during spontaneous and thyroxine-induced metamorphosis.

Granule cell maturation in the cerebellum of bullfrog tadpoles was studied during both spontaneous and thyroxine-induced metamorphosis by using electron microscopy and Golgi-impregnated preparations. The production of cerebellar microneurons, a majority of which are granule cell precursors, was quantitatively compared during spontaneous and thyroxine-induced metamorphosis by using stereological methods and biochemical measurements of DNA. Granule cell migration and differentiation appeared morphologically similar during spontaneous and thyroxine-induced metamorphosis. In both instances, granule cells migrated tangentially along the pial surface, migrated into the internal granular layer, developed dendritic arbors, and formed synaptic contacts with the processes of Golgi cells and with mossy fibers. These events are similar to developmental processes that have been described in detail in other animals. Quantitative stereological measurements demonstrated similar overall patterns of change during spontaneous and thyroxine-induced metamorphosis. Most notably, increases in the volume of the external granule layer correlated with increases in the relative and total amounts of DNA. However, measurements of total DNA were consistently reduced during the period of accelerated change that occurs in thyroxine-induced metamorphosis, although external granular layer volume was greater in these tadpoles after 2 and 3 weeks of thyroxine treatment than in spontaneously metamorphosing tadpoles. While granule cell development in the frog is largely dependent on thyroid hormone, differences between thyroid-hormone-induced and spontaneously metamorphosing tadpoles suggest that normal patterns of cerebellar development are also dependent on events that occur in premetamorphic tadpoles in the absence of thyroid hormone.