Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice.

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. Here we generate mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2(-/-) mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2(-/-) mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases.

Structure of the allosteric regulatory enzyme of purine biosynthesis.

Multi-wavelength anomalous diffraction (MAD) has been used to determine the structure of the regulatory enzyme of de novo synthesis of purine nucleotides, glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase, from Bacillus subtilis. This allosteric enzyme, a 200-kilodalton tetramer, is subject to end product regulation by purine nucleotides. The metalloenzyme from B. subtilis is a paradigm for the higher eukaryotic enzymes, which have been refractory to isolation in stable form. The two folding domains of the polypeptide are correlated with functional domains for glutamine binding and for transfer of ammonia to the substrate PRPP. Eight molecules of the feedback inhibitor adenosine monophosphate (AMP) are bound to the tetrameric enzyme in two types of binding sites: the PRPP catalytic site of each subunit and an unusual regulatory site that is immediately adjacent to each active site but is between subunits. An oxygen-sensitive [4Fe-4S] cluster in each subunit is proposed to regulate protein turnover in vivo and is distant from the catalytic site. Oxygen sensitivity of the cluster is diminished by AMP, which blocks a channel through the protein to the cluster. The structure is representative of both glutamine amidotransferases and phosphoribosyltransferases.

Modification of AgNOR staining to reveal the nucleolus in thick sections specified for stereological and pathological assessments of brain tissue.

BACKGROUND: Various stains have been devised to reveal degenerative or reactive cell phenotypes, or the disintegrative and/or neuropathic lesions associated with Alzheimer's, Parkinson's, and Pick's diseases, Down's syndrome, or chemical toxicity. Utilization of silver staining has allowed researchers to elucidate neural pathways promoting a greater understanding of the functional connections between brain regions. All of these methods employing silver can be characterized as 'directed staining technologies'. NEW METHODS: The argyrophilic proteins (AgNOR) staining protocol was modified to stain nucleoli in thick sections prepared for stereological evaluation of brain tissue. Nucleoli appeared as black dots against a pale amber background. Tissue sections were counterstained with Toluidine Blue, or reduced-strength Tyrosine Hydroxylase immunohistochemistry to facilitate visualization of basic cellular morphology and regional nucleus identification. Here, we present a modified method for nucleolar staining in free-floating thick sections of brain embedded in a gelatin matrix. The modifications in our procedure include incubation in HCl to denature ('unravel') the DNA, a bleaching step to reduce non-specific background silver staining, and counterstaining with Toluidine Blue or reduced-strength tyrosine hydroxylase immunohistochemistry. COMPARISON WITH OLD METHODS: Prior to the development of immunohistochemistry, silver staining was used primarily to identify pathological profiles and trace axon pathways; however, in many cases, a combination of silver staining and immunohistochemistry are required to fully visualize pathomorphology. The mechanism of these stains requires the binding of silver ions to cellular components and the subsequent reduction of the ions to metallic silver. Dilutions of TH primary antibody were evaluated to maximize identification of neurons and the nucleolus amongst the soma and processes present in the thick section. The use of stereology as a tool to estimate cell number has become increasingly prevalent in neuroscience experiments. As requirements for the preparation of experimental tissue have been refined, researchers have begun to use thicker sections, between 40 to 80 microns, to increase the number of optical planes available for analysis. These thick sections require modified staining protocols to assure complete penetration of stains throughout the tissue section. CONCLUSIONS: This method is particularly useful in nucleolar identification for Stereology, and automated counting methods. Use of the nucleolus avoids some of the problems associated with use of the nucleus. The nucleolus is smaller than the nucleus and is less susceptible to transection during sectioning. It has a higher density than the nucleus and is easier to visualize. It is generally darker staining than the immunohistochemical reaction product that provides the identification marker for the cells to be counted. Examples of the method in several brain sections of the rat are shown, though the method has been also proven in other mammalian models.

The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity.

The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosome-linked PRPS1 gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.

Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR.

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.

Adaptation of an enzyme to regulatory function: structure of Bacillus subtilis PyrR, a pyr RNA-binding attenuation protein and uracil phosphoribosyltransferase.

BACKGROUND: The expression of pyrimidine nucleotide biosynthetic (pyr) genes in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR attenuation protein binds to specific sites in pyr mRNA, allowing the formation of downstream terminator structures. UMP and 5-phosphoribosyl-1-pyrophosphate (PRPP), a nucleotide metabolite, are co-regulators with PyrR. The smallest RNA shown to bind tightly to PyrR is a 28-30 nucleotide stem-loop that contains a purine-rich bulge and a putative-GNRA tetraloop. PyrR is also a uracil phosphoribosyltransferase (UPRTase), although the relationship between enzymatic activity and RNA recognition is unclear, and the UPRTase activity of PyrR is not physiologically significant in B. subtilis. Elucidating the role of PyrR structural motifs in UMP-dependent RNA binding is an important step towards understanding the mechanism of pyr transcriptional attenuation. RESULTS: The 1.6 A crystal structure of B. subtilis PyrR has been determined by multiwavelength anomalous diffraction, using a Sm co-crystal. As expected, the structure of PyrR is homologous to those proteins of the large type I PRTase structural family; it is most similar to hypoxanthine-guanine-xanthine PRTase (HGXPRTase). The PyrR dimer differs from other PRTase dimers, suggesting it may have evolved specifically for RNA binding. A large, basic, surface at the dimer interface is an obvious RNA-binding site and uracil specificity is probably provided by hydrogen bonds from mainchain and sidechain atoms in the hood subdomain. These models of RNA and UMP binding are consistent with biological data. CONCLUSIONS: The B. subtilis protein PyrR has adapted the substrate- and product-binding capacities of a PRTase, probably an HGXPRTase, producing a new regulatory function in which the substrate and product are co-regulators of transcription termination. The structure is consistent with the idea that PyrR regulatory function is independent of catalytic activity, which is likely to be extremely low under physiological conditions.

Regulation of the Bacillus subtilis pyrimidine biosynthetic operon by transcriptional attenuation: control of gene expression by an mRNA-binding protein.

The pyrimidine nucleotide biosynthetic (pyr) operon of Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which termination of transcription at points upstream of the genes being regulated is promoted by the binding of a regulatory protein, PyrR, to specific sequences in the pyr mRNA. Binding of PyrR to pyr mRNA is stimulated by uridine nucleotides and causes changes in the mRNA secondary structure. This model is supported by extensive molecular genetic analysis. PyrR, which is encoded by the first gene of the pyr operon, is also a uracil phosphoribosyltransferase, although it has little amino acid sequence resemblance to other bacterial uracil phosphoribosyltransferases. Purified B. subtilis pyrR promotes attenuation of pyr transcription in vitro and binds specifically to pyr RNA sequences. The crystallographic structure of PyrR demonstrates the similarity of its tertiary structure to other phosphoribosyltransferases and suggests the surface to which RNA binds. PyrR is widely distributed among eubacteria and appears to regulate pyr genes not only by the attenuation mechanism found in B. subtilis, but also by a coupled transcription-translation attenuation mechanism and by acting as a translational repressor. PyrR illustrates the concept that transcriptional attenuation is a much more widespread and mechanistically versatile mechanism for the regulation of gene expression in bacteria than is generally recognized.

Effects of glucose and nitrogen source on the levels of proteinases, peptidases, and proteinase inhibitors in yeast.

In Saccharomyces cerevisiae harvested from early exponential growth on glucose-containing media, the specifc activities of proteinases A and B, carboxypeptidase Y, and the inhibitors IA, IB, IC of these three proteinases, respectively, are found to be 10-30% of the specific activities observed in media without glucose, containing acetate as a carbon source; the activities of two aminopeptidases in glucose-grown cells were 30-50% of those in acetate-grown cells. In contrast to fructose-biphosphatase, phosoenolpyruvate carboxykinase, and cytoplasmic malate dehydrogenase, which are inactivated after the addition of glucose to derepressed cells, the proteinases and inhibitors are not inactivated after glucose addition, but appear to be repressed. Growth of the yeast on poor nitrogen sources or starvation for nitrogen results in 2-3 fold increases in the levels of most proteinases and peptidases, but this effect is not observed with glucose as the carbon source.

Mapping and reconstruction of domoic acid-induced neurodegeneration in the mouse brain.

Domoic acid, a potent neurotoxin and glutamate analog produced by certain species of the marine diatom Pseudonitzschia, is responsible for several human and wildlife intoxication events. The toxin characteristically damages the hippocampus in exposed humans, rodents, and marine mammals. Histochemical studies have identified this, and other regions of neurodegeneration, though none have sought to map all brain regions affected by domoic acid. In this study, mice exposed (i.p.) to 4 mg/kg domoic acid for 72 h exhibited behavioral and pathological signs of neurotoxicity. Brains were fixed by intracardial perfusion and processed for histochemical analysis. Serial coronal sections (50 microm) were stained using the degeneration-sensitive cupric silver staining method of DeOlmos. Degenerated axons, terminals, and cell bodies, which stained black, were identified and the areas of degeneration were mapped onto Paxinos mouse atlas brain plates using Adobe Illustrator CS. The plates were then combined to reconstruct a 3-dimensional image of domoic acid-induced neurodegeneration using Amira 3.1 software. Affected regions included the olfactory bulb, septal area, and limbic system. These findings are consistent with behavioral and pathological studies demonstrating the effects of domoic acid on cognitive function and neurodegeneration in rodents.

Strategies for assessing neurotoxicity.

The ultimate end point among the various measures of neurotoxicity is the death of neurons, since they do not regenerate. Experiments that accurately assess this degree of neurotoxicity must utilize a sensitive means of detection and control for the factors that are variables in the expression of neurotoxicity. Neurotoxic substances have the characteristic trait that each selectively destroys specific populations or subpopulations of neurons. The mechanisms underlying this selectivity are generally unknown. Without a priori knowledge of where in the brain to look, it is prudent to employ a histological procedure that imparts an easily detected marker to degenerating neuronal components. For example, the so-called "reduced silver" stains specifically impregnate degenerating neural elements. From studies of known neurotoxins, several factors have been shown to influence neurotoxicity: 1) species and strains; 2) age; 3) sex; 4) dose rate (acute vs. chronic); 5) survival time; and 6) sampling interval in the brain. Failure to include these factors in a screening paradigm could yield false-negative results and, thereby, invalidate extrapolations made to man.

Selective neurotoxic effects of nicotine on axons in fasciculus retroflexus further support evidence that this a weak link in brain across multiple drugs of abuse.

When administered continuously for several days at relatively low plasma levels, a variety of drugs of abuse with strong dopaminergic actions induce degeneration in axons traveling from the lateral habenula through the sheath of fasciculus retroflexus to midbrain monoaminergic nuclei. With some of these drugs, such as cocaine, this is virtually the only degeneration induced in brain. Nicotine given continuously also selectively induces degeneration in fasciculus retroflexus, but in the other half of the tract: the cholinergic axons running from medial habenula in the core of the tract to the interpeduncular nucleus. Fasciculus retroflexus appears to be a weak link in brain for diverse drugs of abuse when administered incessantly for several days. Alterations in this tract would be predicted to be especially important for the genesis of the symptomatology which develops during drug binges, residual effects of such binges, and the processes underlying relapse.

Synthesis and characterization of radioiodinated N-(3-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropanes: potential dopamine reuptake site imaging agents.

Methods have been developed for the preparation of radioiodinated N-substituted 2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropanes. The syntheses, physical properties, radiolabeling, and characterization of the pharmacological properties of N-(3(Z)-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropane (12) and N-(3(E)-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropane (13) are described. 2 beta-Carbomethoxy-3 beta-(p-substituted-phenyl)tropanes are potent ligands for the dopamine transporter. The radioiodinated derivatives are of interest because of the high uptake and prolonged striatal retention that may result from specific binding to low-capacity, high-affinity, dopamine reuptake sites. Radioiodine was introduced into the 3Z and 3E-position of N-(3-iodopropen-1-yl)-2 beta-carbomethoxy-3 beta-(4-chlorophenyl)tropane by iododemetalation of the corresponding 3-(tri-n-butylstannyl) derivatives. Competition binding data of various dopamine reuptake ligands with rat striatal tissue preparation for either [125I]-12 or [125I]-13 exhibited the following order of potency: E-13 > Z-12 > GBR 12909 >> mazindol >>> (-)-cocaine. Tissue distribution studies in rats showed that the E-13 was the best analogue. E-13 showed high striatal uptake (60 min, 1.23% dose/g; 120 min, 0.61% dose/g) and high striatal to cerebellum ratios (60 min, 15.9/1; 120 min, 16.5/1). These studies indicate that iodine-123-labeled E-13 is a potentially useful agent for imaging the dopamine reuptake sites by single-photon-emission computerized tomography.

The regional distribution and cellular localization of iron in the rat brain.

The regional distribution and cellular localization of iron throughout the rat brain was determined with iron histochemistry. Densitometry was used to measure the intensity of stain of 51 iron-concentrating sites. Among the areas of highest iron content are the circumventricular organs, islands of Calleja, globus pallidus, ventral pallidum, substantia nigra pars reticulata, interpeduncular nucleus, dentate nucleus, and interpositus nucleus. Iron occurs most commonly in oligodendrocytes and in the fibrous network of the neuropil, but is also found in the interstitial spaces of circumventricular organs and in the tanycytes of the organum vasculosum of the lamina terminalis, median eminence, and walls of the third ventricle. In diverse areas throughout the brain--among them, the islands of Calleja, dentate gyrus of the hippocampal formation, lateral septal nucleus, and central amygdala--iron is found in association with the perikarya and neuronal processes of nerve cells. The overlapping distribution patterns of iron and gamma-aminobutyric acid, enkephalin, and luteinizing hormone-releasing hormone suggest that the distribution of iron is related to its association with the metabolism of one or more neurotransmitters or neuroactive compounds.

Electroshock seizures protect against apoptotic hippocampal cell death induced by adrenalectomy.

Seizures evoked by electroshock induce rapid changes in the expression of several genes in the adult brain, including those encoding for neurotrophic factors. Some of the neurotrophic factors induced by brief seizures such as basic fibroblast growth factor and nerve growth factor have been shown to have neuroprotective action. We reasoned therefore that these seizures may protect against neural injury. To test this hypothesis, we examined the effect of electroshock-induced seizures on the vulnerability to cell death in the hippocampus. Cell death was induced by adrenalectomy, which results in a highly selective apoptotic neuronal death in the dentate granule cell layer of the hippocampus. Daily electroshock seizures were administered for seven days to sham-operated and adrenalectomized rats. Neuronal degeneration was evaluated by the highly sensitive and reliable cupric-silver impregnation method. Animals experiencing electroshock seizures were completely protected against adrenalectomy-induced cell death, whereas adrenalectomized animals not exposed to electroshock seizures exhibited substantial neuronal cell degeneration in the dentate granule cell layer. Daily restraint stress did not prevent the adrenalectomy-induced neuronal death, indicating that the neuroprotective effect of the seizure treatment is not accounted for by stress. We conclude that brief controlled seizure-evoked neural activation may allow the sparing of otherwise vulnerable neuronal populations in the injured adult brain. This prompts a need to explore the possibility that controlled administration of electroshock seizures may have therapeutic potential in treating neurodegenerative disorders.

The globus pallidus and its rostroventral extension into the olfactory tubercle of the rat: a cyto- and chemoarchitectural study.

The globus pallidus is characterized by a high iron content and the distribution of the ferric iron in the rat brain provides evidence that globus pallidus extends rostroventrally below the anterior commissure and into the olfactory tubercle. The extension of the globus pallidus into the olfactory tubercle is consistent with the notion of the ventral striatum,14 in the sense that it provides for an expected close proximity between the striatum and the globus pallidus throughout the dorsoventral extent of the corpus striatum. The distribution of enkephalin, and of acetylcholinesterase- and succinate dehydrogenase-positive neurons is also consistent with an extension of the ventral part of globus pallidus to the base of the forebrain in the rat. Since part of the ventral pallidum corresponds to a region that is usually referred to as the subcommissural part of the substantia innominata, it seems reasonable to restrict the term substantia innominata to the more caudally-located sublenticular part of the substantia innominata.

Dissimilar patterns of degeneration in brain following four different addictive stimulants.

Patterns of neural degeneration were compared following continuous administration of four drugs of addiction, each of which induces model psychoses in chronic addicts. D-amphetamine (D-Amph), cocaine (Coc), or phencyclidine (PCP) were administered continuously over a 5-day period. Both D-Amph and Coc induced pronounced degeneration in fasciculus retroflexus, but only D-Amph further induced substantial degeneration in striatum. Continuous PCP produced entirely different degeneration largely confined to the posterior entorhinal cortex, ventral dentate gyrus, and cingulate cortex. Methamphetamine (Meth) administered in the very high dose but less prolonged drug regimen often employed in studies of dopamine toxicity induced pronounced degeneration in striatum, but widespread degeneration in many other regions as well. These results indicate that drugs of abuse with psychotomimetic properties induce distinctively different patterns of neural degeneration, a finding with implications for theories of addiction and psychosis. They predict two different anatomical loci for alterations in psychosis: fasciculus retroflexus and ventral parahippocampus and hippocampus.

Neuroexcitatory and neurotoxic actions of the amnesic shellfish poison, domoic acid.

We have investigated the action of domoic acid in the mouse brain following systemic exposure. Domoic acid increased c-fos mRNA within 15 min and its translational product (c-Fos) within 1 h. c-Fos immunoreactivity was most prominent in the hippocampal formation, lateral septal nucleus, olfactory bulb, area postrema and the nucleus of the solitary tract. We next examined irreversible toxic effects of domoic acid. Domoic acid caused extensive degeneration in CA1-2 of the hippocampus, lateral septal nucleus and olfactory bulb. No degeneration was evident in the dentate gyrus or brain stem. These studies demonstrate that domoic acid has only neuroexcitatory effects on brain stem regions associated with visceral function whereas it has permanent neurotoxic effects on brain regions associated with memory formation.

Mutations in Bacillus subtilis PyrR, the pyr regulatory protein, with defects in regulation by pyrimidines.

The pyrimidine nucleotide biosynthetic (pyr) operon in Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which PyrR, a bifunctional pyr RNA-binding attenuation protein/uracil phosphoribosyltransferase, plays a crucial role. A convenient procedure for isolation of pyrR mutants with defects in the regulation of pyr operon expression is described. The selection is based on the selection of spontaneous mutations that convert the pyrimidine-sensitive growth of cpa strain (lacking arginine-repressible carbamyl phosphate synthetase) to pyrimidine resistance. Twelve such mutants were isolated and sequenced. All resulted from point mutations in the pyrR gene.

Short-term, D2 receptor blockade induces synaptic degeneration, reduces levels of tyrosine hydroxylase and brain-derived neurotrophic factor, and enhances D2-mediated firing in the ventral pallidum.

Repeated treatments with neuroleptics are associated with biochemical and morphological alterations in forebrain neurons as well as an upregulation of D2-mediated changes in neuronal function. The present study evaluated the histological and physiological effects of three once-daily treatments with two chemically divergent neuroleptics, haloperidol (1 mg/kg i.p./day) and eticlopride (3 mg/kg i.p./day), measured in rats 24 h after the last injection. It was determined that this short-term antagonism of D2-like receptors induced fiber and terminal degeneration and significantly decreased tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF) immunoreactivity in the ventral pallidum (VP), as determined by optical density measurements. While other forebrain regions demonstrated changes in TH and BDNF, the neurodegeneration profile was unique to the VP. This was accompanied by an enhancement in the efficacy of the D2 agonist quinpirole to increase spiking rate of VP neurons recorded in chloral hydrate-anesthetized rats. These data indicate that short-term treatments with D2 antagonists are sufficient to induce changes in the biochemical and morphological profiles uniquely within the VP. Moreover, the functional ramifications of these changes appear to include profound alterations in the way dopamine regulates neuronal activity in this region.

Auditory and vestibular pathology of seizure-prone chicks.

The mutant sex-linked lethal recessive px (paroxysmal) gene, expressed in White Leghorn chicks (Gallus domesticus) causes seizures beginning on approximately day 8 post-hatching. Seizures are spontaneous and inducible by auditory but not by photic stimulation. Prior to seizure onset px chicks are indistinguishable from non-px siblings. With seizure onset is a decrease in food intake which causes deterioration and death by 4-10 weeks of age. In a preliminary histological study conducted on 22-day-old seizure-prone chicks, brains were perfused and sections treated according to a modified cupric-silver staining technique. Nuclei and fiber tracts of px auditory and vestibular systems were extensively degenerated; control brains showed essentially no degeneration. A second experiment was conducted with preseizure chicks to determine whether and to what extent degeneration occurs prior to seizure onset. Deep nuclei of px cerebellum appeared to be the first seriously affected (5 days of age). Extent of degeneration progressed steadily over time through 20 days of age, by which time all components of the two sensory systems were maximally affected. Ambient noise did not affect onset or extent of degeneration, nor did it affect onset of seizures.