Chronic intrastriatal injection of the excitatory amino acid receptor antagonist L-kynurenic acid in rat produces selective neuron sparing lesions.

Heritable neurodegenerative diseases may be associated with one or more endogenous neurotoxins whose actions on neurons lead to the degenerative changes. One metabolite of tryptophan, the amino acid L-kynurenic acid (L-KYN), was chronically injected into the striatum of the male rat to test its potential as an endogenous neurotoxin. L-KYN, at concentrations of approximately five times its normal brain levels, produced a large lesion with relative selective neuron sparing. The L-KYN-induced lesion presented three concentric regions: a central necrotic zone, a thin pyknotic zone, and an outermost spongiose zone. The number of GABA-ergic neurons were markedly reduced (approximately 76%), while cholinesterase-positive neurons were also lost. The NADPH diaphorase-positive neurons were the most resistant to L-KYN neurotoxicity and were spread throughout the spongiose zone. The brain levels of L-KYN are abnormal in patients with the neurodegenerative disorder Huntington's disease and as a neurotoxin L-KYN may play a role in the etiology of this disease. Of further significance, the fact that L-KYN is neurotoxic contraindicates the use of this excitatory amino acid receptor antagonist as a therapeutic agent in the treatment of neurodegenerative disorders.

Morphological diversity of ependymal cells in tissue culture.

Ependymal cells were visualized in primary cultures of cerebral cortex from rat using an immunohistochemical staining technique. Five different morphological subtypes of cuboidal ependyma were recognized: 1) round, 2) triangular, 3) columnar, 4) cone- and spindle-shaped, and 5) large pleomorphic cells. These cells varied in size and almost all possessed cilia. Two distinct forms of tanycyte ependyma were detected based on the presence of cilia. These features reflect a significant level of development of the ependymal cells in culture and may correspond to functional diversity within this group.

Chronic intrastriatal L-pyroglutamate: neuropathology and neuron sparing like Huntington's disease.

Continuous injection of L-pyroglutamate (L-PGA) into the rat striatum induces a lesion with three regions: a necrotic core, a rim of pyknotic cells, and a peripheral spongiose region. The L-PGA was administered through an implanted intrastriatal cannula coupled to an Alzet osmotic pump loaded with one of three doses of L-PGA (3, 5, or 13 times the normal amount of L-PGA/g wet wt rat forebrain (23 nmol/g). The magnitude of the lesion was dependent upon the concentration of buffered L-PGA and the duration of continuous pumping. The necrotic region contained macrophages and neutrophils, while condensed neurons and oligodendroglial cells were present in the pyknotic region. The spongiose region contained vacuolated neuropil and degenerating nerve cells and oligodendroglia. The spongiose region blends with the normal neuropil. A population of aspiny neurons were identified throughout the spongiose region. These neurons stained positive for NADPH diaphorase and demonstrated a somatostatin-like immunoreactivity similar to that of the aspiny neurons spared in Huntington's disease and in the neurotoxin-induced striatal-lesioned rat models of Huntington's disease.

The toxin kainic acid: a study of avian nerve and glial cell response utilizing tritiated kainic acid and electron microscopic autoradiography.

Three questions are asked regarding the toxin kainic acid (KA). Does it destroy specific glial cells as well as neurons? Does KA gain access to the cytoplasm in intact cells and to which organelles does it bind? Intracerebral injections of tritiated KA into the pigeon (Columba livia) paleostriatal complex (basal ganglia) coupled with electron microscopic autoradiography revealed the following major points. Kainic acid destroyes oligodendrocytes, with pathophysiology apparent by 30 min after challenge with KA leading to cell destruction by 4 h. The response of astrocytes at the longest observation period (4 h) involves swelling of perivascular endfeet and processes in the neuropil. Reactive microglial-like cells show an accumulation of label in their cytoplasm, but no apparent morphological changes. The label appears in the cytoplasm of intact cells, both glia and neurons early after challenge with the toxin. Label is associated (bound) with mitochondria at an incidence significantly above chance at 30 min, 2 and 4 h after challenge with KA. Two hours after exposure to KA is the critical period where metabolic, physiological and morphological changes occur that lead to cell death. Cell destruction may be a consequence of KA-induced energy depletion. Kainate may interfere with adequate energy production by uncoupling glycolysis and the Krebs cycle in the mitochondria.

Presence and development of ependymal cells in primary tissue cultures derived from embryonic rat cerebral cortex.

Using indirect immunohistochemistry, a secondary antibody was detected in a commercial preparation of antiserum against vasoactive intestinal polypeptide. The secondary antibody selectively stained ependymal cells during the first 3 weeks in vitro in cultures of dissociated cerebral cortical tissue from rat. This staining provided a convenient mechanism for investigating the development and properties of these cells in cultures. The overall level of immunofluorescent staining during the initial 3-week time period appeared to directly reflect the proliferation and development of ependymal cells. Fluorescent staining was initially detected in cells which appeared to correspond to matrix cells or progenitor cells from the ependyma. These cells underwent rapid cell division, as evidenced by distinct morphological stages, to yield daughter cells which were the precursors of mature ependymal cells. Three different morphological classes of mature ependymal cells were observed in the cortical cultures. These classes corresponded to the cuboidal, tanycyte and secretory ependymal cell types described in vivo. Direct counting of stained cells showed that these morphological classes were represented in the cultures in roughly the same proportions seen in vivo (cuboidal 75%, tanycyte 19% and secretory 6%). The temporal aspects of ependyma development permitted the staining of developmental stages corresponding to the various morphological classes or types. The morphological sequence of development of the cuboidal cell and tanycyte from the precursor cell or matrix cell--daughter cell was determined. These two cell types displayed marked differences in their developmental sequence. The developmental sequence of the secretory cell could not be resolved; however, what appeared to be multiple morphological subtypes of this cell class were encountered.(ABSTRACT TRUNCATED AT 250 WORDS)

Thalamic arterial pattern: an endocast and scanning electron microscopic study in normotensive male rats.

Rat cerebral vasculature serves as a model for study of the pathophysiology of stroke in humans. Human thalamic arteries show a high incidence of stroke. The objective is to describe the thalamic arterial vascular pattern in normotensive male rats as the initial step for quantitative histochemical studies of enzyme activities in the walls of these vessels. Intracardiac injections of methyl methacrylate monomer provide detailed vascular endocasts. The thalamic vascular bed defined by in situ dissection, serial reconstruction, and light and scanning electron microscopy of endocasts contained four groups of vessel: ventral medial thalamic arteries, thalamic branches from the posterior cerebral artery, and ventral lateral and ventral anterior thalamic arteries. Thalamic vessels are muscular arterioles that, after three to four bipinnate branches, feed into a continuous capillary bed (no loops). The parent vessels and their subsequent branches have been evaluated in terms of their mean internal diameters, mean interbranch intervals, and branch angles. The arterial patterns to rat and human thalami are very similar, with the exception of the anterior choroidal artery which is missing in the rat. The branches supplying the thalamus in both the rat and human are closely associated with the circle of Willis; however, the constituent parts of the circle in rat vary from the pattern in human brain. The rat thalamic arteries show morphological features similar to those seen in the stroke-prone ganglionic arteries in the human basal ganglia.

Ultrastructure of ependymal cells in primary cultures of cerebral cortex.

Ultrastructural features of ependymal cells growing in primary cultures of dissociated cerebral cortical tissue were investigated using electron microscopy. The ependyma exhibited a specific orientation in tissue culture such that the cell surface corresponding to the apical surface in situ was directed toward the culture medium. The membrane on the apical surface of cultured ependyma was characterized by the presence of cilia and microvilli. The lateral margins were marked by a variety of junctions: zonula occludens, zonula adherens, and membrane interdigitations. The cell cytoplasm contained pleomorphic mitochondria, Golgi profiles, clusters of ribosomes, rough endoplasmic reticulum, and particularly profuse scattered microfilaments and microtubules. These features support the identity of the cells as ependymal in the cultures and establish them as a relatively accurate reflection of ependyma in situ.

A histochemical study of cerebral cortical vessels and ganglionic vessels of the caudatoputamen in aging normotensive rats.

The goal was to describe the metabolic profile of ganglionic and cortical arteries and arterioles in aging normotensive male rats. Five enzymes indicative of key metabolic pathways in the vessel walls were semiquantitatively evaluated using bright-field histochemical microscopy. Lactate dehydrogenase showed significant reactivity which increased with vessel diameter in cortical and ganglionic vessels in all age groups tested. Succinate dehydrogenase and cytochrome oxidase showed little reactivity in both cortical and ganglionic vessels, suggesting a reduced role for aerobic metabolic pathways. Myosin ATPase reactivity was high in cortical and ganglionic vessels. Only this enzyme showed an increased reactivity that was correlated with the age and diameter of the vessel. Glucose-6-phosphate dehydrogenase reactivity was more pronounced in cortical than ganglionic vessels, suggesting that the hexose-monophosphate-shunt may be more active in the cortical vessels. There were no regional differences in enzyme reactivity throughout the caudatoputamen. In conclusion, both the cortical and ganglionic vessels are metabolically active, with significant anaerobic glycolysis, and reduced, but observable capacity for aerobic metabolism. The decreased myosin ATPase reactivity and the low level of glucose-6-phosphate dehydrogenase reactivity in the ganglionic arterioles of senescent rats may contribute to the susceptibility of these vessels to cerebrovascular accidents.

L-pyroglutamate: an alternate neurotoxin for a rodent model of Huntington's disease.

Intrastriatal injections of L-Pyroglutamate (L-PGA) in mice produced behavioral and neuropathological effects that resemble in part the kainate-injected rat striatal model of Huntington's Disease (HD). The behavioral responses induced after unilateral injections of L-PGA included circling, postural asymmetry of head and trunk and possible dyskinesias. The neuropil in the injected striatum contained dilated profiles, degenerating neurons and oligodendroglia, and numerous phagocytic microglial-like cells. A dose response relation existed. The size of the lesion (expressed as a percent volume of the striatum destroyed) ranged from 1 +/- 0.18% at 0.02 mumoles to 20.2 +/- 3.97% at 200 mumoles L-PGA (pH = 7.3). L-PGA is a weak neurotoxin when compared to kainic acid. Several factors raise interest in the possible role of L-PGA in HD, including the recently reported elevated plasma levels of L-PGA in some HD patients, and these considered in the discussion.

The TPc, the avian substantia nigra: pharmacology and behavior.

The nucleus tegmenti pedunculo-pontinus, pars compacta (TPc) may be the avian analogue of the mammalian substantia nigra (SN). The analogy is suggested by both comparative neuroanatomical and neurohistochemical observations. To test the proposed analogy certain drugs (agonists or antagonists of putative transmitters that modulate the dopaminergic and GABAergic systems in rat) were injected into the TPc of the pigeon and the behavior effects observed. Muscimol (a GABA agonist) injected into the caudal TPc induced contralateral rotation and postural asymmetries. Pretreatment with subcutaneous injections of apomorphine hydrochloride enhanced and haloperidol suppressed the rotatory response, while the postural asymmetries were not altered by either drug. Muscimol injected into the rostral TPc induced contralateral rotation, marked ataxia, and postural asymmetries, particularly the head and neck, legs and wings. Whereas apomorphine (subcutaneous injection) was without effect on the rotatory response to muscimol, haloperidol suppressed the rotatory response. Neither drug effected the postural asymmetry. Following drug injections into either the pigeon TPc or the rat SN the behaviors induced in both bird and rat suggest that the TPc and SN are analogous.

Vascular supply pattern to rat caudatoputamen and globus pallidus: scanning electronmicroscopic study of vascular endocasts of stroke-prone vessels.

The caudatoputamen (CP) and globus pallidus (GP) are supplied by vessels often involved with stroke in both rat and human. The pattern of vascular supply to the CP and GP in rat has, in contrast to humans, been only partially described. The vascular pattern to the rat CP and GP is described utilizing vascular endocasts and scanning electronmicroscopy in aging, normotensive rats. Endocasts were produced by intra-cardiac infusion of Batson's Corrosion Compound. The vascular pattern is complex, involving 1) recurrent vessels from the anterior cerebral artery, 2) branches from the arterial circle rostral or caudal to the origin of the middle cerebral artery (MCA), 3) up to 6 branches from the MCA, and 4) 2 major branches from the caudal part of the arterial circle. The vessels in groups 1--3 were serpentine, their luminal diameters abruptly reduced at branch points, and the angle of departure from the parent vessels approximated 90 degrees. These vessels supplied much of the CP and GP, while group 4 supplied the caudal CP with vessels arranged in a lattice-like fashion from the 2 penetrating parental arteries.

Necrotizing effects of kainic acid on neurons in the pigeon brain: histological observations.

The neurotoxin kainic acid (KA) has been shown to destroy neurons in the pigeon paleostriatal complex (PC), the avian analogue of the caudato-putamen and globus pallidus. In this earlier study the movement disorders in pigeons were strikingly similar to those reported by others in rats following intracerebral injection of KA into the corpus striatum. The toxic influences of KA on other parts of the pigeon brain have not been described. Therefore, KA was injected into areas of the telencephalon, diencephalon, mesencephalon and cerebellum. Areas sensitive to KA showed a marked cell loss and the neuropil exhibited spaces that contained fragments of necrotic neurons. The injection sites were invaded by glia and granulocytes. Kainic acid had a local necrotizing effect; for example, it destroys neurons in the PC, nucleus rotundus, nucleus spiriformis lateralis, nucleus ruber and neurons of the cerebellar cortex. An apparent long-distance effect of KA was also observed, since intracerebral injections of KA into the PC was followed by cell loss in the ipsilateral nucleus of the ansa lenticularis. Kainic acid has proved to be a potent neurotoxin with a pronounced necrotizing effect upon neurons in the pigeon brain.

A technique for improved fixation of the pigeon central nervous system for electron microscopy.

The central nervous system (CNS) of the pigeon has been difficult to fix with consistency, and consequently this problem has impeded ultrastructural studies of various parts of the pigeon brain. Here we describe a method for effective fixation of the pigeon CNS and discuss the three principal problems associated with good fixation of this animal's brain. The animal was deeply anesthetized and the thoracic cavity was opened without collapsing the pectoral girdle upon the brachiocephalic trunks and the common carotids. The perfusion pressure was raised to 140-150 mm Hg to overcome the high resistance of the small diameter, long common carotids. Heparin was added to the wash buffer to retard coagulation of blood in the vascular bed of the brain. The method is not foolproof, but with care excellent fixation can be achieved.

Forebrain projections of the pigeon olfactory bulb.

The olfactory system of the pigeon (Columba livia) was examined. Our electrophysiological and experimental neuroanatomical (Fink-Heimer technique) data showed that axons from the olfactory bulb terminated in both sides of the forebrain. The cortex prepiriformis (olfactory cortex), the hyperstriatum ventrale and the lobus parolfactorius comprised the uncrossed terminal field. The crossed field included the paleostriatum primitivum and the caudal portion of the lobus parolfactorius, areas which were reached through the anterior commissure. In this report the relationships between areas that receive olfactory information and the possible roles that olfaction plays in the birds' behavior are discussed.