Effects of age on nerve fibers in the rhesus monkey optic nerve.

During normal aging there is a reduction in white matter volume in the cerebral hemispheres and structural abnormalities in myelin in some parts of the central nervous system, but whether nerve fibers are lost with age and whether the myelin changes are ubiquitous is not known. Studying the optic nerve, which is a circumscribed bundle of nerve fibers, offers an opportunity to gain further insight into the effects of normal aging on white matter. The present study examined the optic nerves from young (4-10 years) and old (27-33 years) rhesus monkeys using light and electron microscopy. These nerves had been perfused transcardially to obtain optimal preservation of the tissue. Varying degrees of degeneration were encountered in all the optic nerves from the old monkeys. The changes included myelin abnormalities, similar to those reported in other parts of the central nervous system; the presence of degenerating axons and their sheaths; changes in neuroglial cells; and thickening of the trabeculae of connective tissue in the nerve. The total number of nerve fibers was reduced from an average of 1.6 x 10(6) in the young optic nerves to as few as 4 x 10(5) in one old monkey, and with one exception in all of the old optic nerves the packing density of nerve fibers was less than in any of the young optic nerves. The degenerative changes were most marked in those optic nerves that contained the fewest nerve fibers.

Inherited retinal degeneration and apoptosis in mutant zebrafish.

The mechanism of retinal cell death was studied in mutant zebrafish (Danio rerio) which undergo inherited degeneration of the retina and the brain. The shrunken head (shr(m33)) mutation was isolated as part of a large scale mutagenesis screen. The yellow head (yhd) mutation arose spontaneously among inbred wild type zebrafish. Although the mutants share many morphological features, including small eyes, a small brain and an enlarged pericardial sac, crossing shr(m33) and yhd heterozygotes results in phenotypically normal fish. The retinae of both mutant lines of fish begin to develop normally and then undergo massive degenerative changes. Pyknotic cells first appear in the retina of the shr(m33) fish by 3 days post-fertilization and in the yhd fish by 1.5 days post-fertilization. By 5 days post-fertilization the outer nuclear layer containing the photoreceptor cells has largely disappeared in both mutants. The inner nuclear layer and ganglion cell layer are also severely affected. By 6-7 days post-fertilization, the retina has been largely cleared of pyknotic cells by retinal pigment epithelial cells and by rare macrophage-like cells. Both mutations are lethal by 7-8 days post-fertilization. Two independent measures, TdT-mediated dUTP-biotin nick end label (TUNEL) and transmission electron microscopy, indicate that the pyknotic cells in the mutant retinae are apoptotic. Apoptosis is very rarely observed during normal development of the teleost retina and was not observed in age-matched wild type zebrafish retinae examined for comparison. Our results indicate that a genetic defect can induce massive apoptosis in cell populations that do not normally undergo apoptosis during development.

GABA as a developmental signal in the inner retina and optic nerve.

Gamma-aminobutyric acid (GABA) acts as an inhibitory neurotransmitter in the mature vertebrate retina, where it is localized predominantly in amacrine cells, and to a lesser extent in other cell types. During development, GABA is expressed transiently in additional cells, including retinal ganglion cells and horizontal cells. Elements of the GABA system, including GABA uptake and release mechanisms and GABA receptors, are also expressed early in retinal development, well in advance of the onset of visual function. The GABA transporter is a major component of the GABA system in the mature retina, and is most likely responsible for GABA release early in development, prior to the establishment of vesicular synaptic transmission. GABA, produced by amacrine cells and retinal ganglion cells, may serve a developmental role in the establishment of circuitry in the retinal inner plexiform layer and may also be involved in the formation of appropriate central connections by retinal ganglion cell axons.

The distribution of neurotrophin receptor TrkC-like immunoreactive fibers and varicosities in the rhesus monkey brain.

The distribution of immunoreactivity for the neurotrophin receptor tyrosine kinase TrkC was examined in the brain of the adult rhesus monkey. TrkC-like immunoreactivity was widespread and consisted primarily of varicose fibers. The most dense populations of fibers were in the basal forebrain (in the cholinergic cell groups Ch1, Ch2 and Ch4), in the raphé complex throughout its rostrocaudal extent, and in the locus coeruleus. Other fibers were present in the thalamus, hypothalamus, central gray matter of the midbrain, dorsal midline of the brainstem and the cerebral cortex. The only neuronal cell bodies with consistent labeling were located in the lateral hypothalamus. Purkinje cells in the cerebellum showed variable labeling. Specific labeling of varicosities and cell bodies was abolished by omission of the primary antiserum or by preabsorption with the TrkC peptide antigen. We conclude that TrkC-like immunoreactivity can be detected in a wide variety of subcortical locations in the adult rhesus monkey. Labeling was particularly prominent in the vicinity of the major cholinergic, serotonergic and adrenergic nuclei, known from other studies to be vulnerable in the ageing brain. This suggests that the ligand for TrkC, neurotrophin-3, may persist as a survival factor for critical neurons into adulthood.

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish.

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo.

Zebrafish TrkC1 and TrkC2 receptors define two different cell populations in the nervous system during the period of axonogenesis.

We identified previously five distinct trk genes in the zebrafish. The structures of two of these, TrkC1 and TrkC2, are most similar to mammalian TrkC. Detailed sequence comparisons reported here indicate that although the similarities to TrkC are greatest in those regions of the extracellular domain implicated in ligand binding, the two sequences also differ significantly in these regions. Whole-mount in situ hybridization experiments in the early embryo revealed full-length trkC1 but no trkC2 transcripts in the cranial ganglia and in a subset of Rohon-Beard neurons. At the same time, full-length trkC2 but no trkC1 transcripts were detected laterally in the spinal cord, in the caudal hindbrain, in reticulospinal neurons of rhombomeres 4, 5, and 6, and in the midbrain. Both types of transcripts were expressed in clusters of cells in the dorsal telencephalon and the nucleus of the tract of the postoptic commissure. These results suggest distinct functions of trkC1 and trkC2 in nervous system development. The expression patterns define two different neuronal populations in the zebrafish.

Apolipoprotein E is synthesized in the retina by Müller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells.

We have investigated the synthesis and transport of apoE, the major apolipoprotein of the central nervous system, in the retina of the living rabbit. Four hours after the injection of [35S]methionine/cysteine into the vitreous, 44% of [35S]Met/Cys-labeled apoE is in soluble and membrane-enclosed retinal fractions, while 50% is in the vitreous. A significant amount of intact [35S]Met/Cys-labeled apoE is rapidly transported into the optic nerve and its terminals in the lateral geniculate and superior colliculus within 3-6 h in two distinguishable vesicular compartments. Müller glia in cell culture also synthesize and secrete apoE. Taken together, these results suggest that apoE is synthesized by Müller glia and secreted into the vitreous. ApoE is also internalized by retinal ganglion cells and/or synthesized by these cells and rapidly transported into the optic nerve and brain as an intact molecule. We discuss the possible roles of retinal apoE in neuronal dynamics.

Five Trk receptors in the zebrafish.

Using a homology-based cloning strategy we have identified five members of the Trk family in the zebrafish Danio rerio. They are homologous to the three mammalian Trk receptors in their conserved intracellular kinase regions and the organization of their extracellular regions. The five trk genes are differentially expressed in the developing brain, spinal cord, cranial ganglia, and retina. Full-length forms of three of the trk genes are expressed when neurons pioneer the major axon tracts, whereas the two other trk genes have a later onset of expression. Truncated transcripts and forms containing an extracellular juxtamembrane region insert were found. The degree of sequence variation and expression differences within the family suggest that each of the five zebrafish Trk receptors have a functionally distinct role. These findings demonstrate that the vertebrate Trk family is larger than previously appreciated.

The development of neurotrophin receptor Trk immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The purpose of this study was to examine the cellular distribution of the Trk family of neurotrophin receptors in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Semithin sections from zebrafish retinae were examined immunohistochemically for the presence of Trk polypeptides using commercially available antisera that cross-react with the fish. Cross-reactivity was confirmed by Western blot. Trk polypeptides were detected at about 1 day of age on the surfaces of retinal neuroblasts and faint Trk immunoreactivity was observed in the primordial optic nerve at 1.5 days. By 2 days the optic nerve was clearly positive for Trk and at 2.5 days Trk immunoreactivity was found in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers, as well as in the optic nerve. At 3 days and 4 days the location of Trk immunoreactivity was unchanged but by 4 days it had diminished in intensity. In the adult zebrafish retina Trk immunoreactivity was found in the same locations as in the embryonic fish, as well as in a population of cells in the middle of the inner nuclear layer and in photoreceptors. We conclude that Trk neurotrophin receptors are present in the zebrafish eye during development and that their persistence in the adult may support the continuous neural reorganization that accompanies the growth of the eye in the fish.

The development of GABA immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The goal of this study was to determine the pattern of gamma-aminobutyric acid (GABA) expression in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Zebrafish embryos were fixed at intervals between 1 and 4 days postfertilization, and semithin plastic sections were prepared for postembedding immunocytochemistry with antisera against GABA. Sections were also prepared from several adult zebrafish eyes for comparison. GABA immunoreactivity first appeared in the optic nerve at 2 days postfertilization, and by 2.5 days the inner nuclear layer (INL), inner plexiform layer (IPL), retinal ganglion cell layer, and optic nerve were all positive for GABA. The GABA expression in the retinal ganglion cell layer and optic nerve was transient, however, and these structures were largely unlabeled by 4 days postfertilization. The pattern of GABA immunoreactivity at 4 days resembled that seen in the adult zebrafish: A large population of presumptive amacrine cells was labeled at the base of the INL, and the IPL was positive for GABA, as were occasional cells in the ganglion cell layer. Horizontal cells, particularly at the retinal margins, were also GABA positive beginning at about 3 days postfertilization. The transient expression of GABA in retinal ganglion cells and their axons during the period when synaptic contacts are being established both within the retina and between the retina and central targets suggests that GABA may have a role in the development of this system, in addition to serving as a classical neurotransmitter.

Developmental variation in the structure of the retina.

Events traditionally called "developmental errors" are known to occur in both vertebrate and invertebrate nervous systems. This study was concerned with the frequency and mode of generation of such events in the mammalian retina. We studied three anomalous structures observed in the rabbit's retina after staining of the cell populations that accumulate indoleamines: type 3 cells, stray processes in the optic fiber layer, and displaced cells. They were counted in rabbit retinas prepared as whole-mounts, and in most cases topological maps were made. For comparison, the conventional indoleamine-accumulating amacrine cells and the tyrosine hydroxylase (TH)-positive cells, which are members of the mammalian retina's recognized complement of amacrine cells, were also counted. A further comparison was made with the number and distribution of TH-positive amacrine cells in highly inbred mice. The ordinary amacrine cells did not vary much in number from animal to animal. Especially in inbred mice, the reproducibility was striking: the extreme variation in number of TH amacrine cells between any two of the 14 retinas studied was 22%, and the mean difference between two eyes of individual mice was 2.5 +/- 1.7%. The three anomalous structures were rare and variable. Their numbers varied more than fourfold from animal to animal. However, their numbers in two eyes from the same animal varied by an overall average of only 14 +/- 10%. The anomalous structures were present in all rabbits, and their morphology was the same in all cases: they are under precise control by the developmental program. The anomalous cells share many phenotypic features with the regular amacrine cells of the indoleamine-accumulating class.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized.

We have investigated the synthesis, axonal transport, and processing of the beta-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a t1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]-methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is approximately 110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.

Co-release of acetylcholine and GABA by the starburst amacrine cells.

Rabbit retinas were isolated from the eye and maintained in vitro. When they were incubated for 60 min in the presence of 3H-GABA, subsequent autoradiography showed radioactivity to be present primarily in amacrine cells. Under these conditions, most of the radioactivity contained in the retinas remained in the chemical form of GABA. Autoradiography and immunohistochemistry of alternate sections showed the amacrine cells that accumulate 3H-GABA to be the same cells that contain endogenous GABA immunoreactivity. These include the starburst cells, the indoleamine-accumulating cells, and other, as yet unidentified amacrine cells. The localization confirms previous immunohistochemical findings. When retinas containing 3H-GABA were expressed to elevated concentrations of K+, their content of 3H-GABA decreased. Autoradiography showed a reduced 3H-GABA content in all of the cells that contained 3H-GABA. Since those include the starburst cells, previously shown to be cholinergic, the finding demonstrates that the starburst cells release both ACh and GABA. Retinas simultaneously labeled with 14C-GABA and 3H-ACh were superfused, and the release of radioactive compounds from the retina was studied. Depolarization by elevated K+ caused an increased recovery of both ACh and GABA in the superfusate, but the predominant mechanisms of their release appeared to be different. The stimulated release of ACh was entirely Ca2+ dependent, while the release of radioactivity originating from GABA was much less so. A concentration-dependent counterflux (homoexchange) of intracellular GABA was demonstrated by raising the extracellular concentration of GABA (or nipecotic acid). These results suggest that a large outward flux of GABA occurs via the GABA transporter, probably by the potential-sensitive mechanism studied by Schwartz (1982, 1987). Stimulation of double-labeled retinas by flashing light or moving bars always increased the release of ACh, and the release was entirely dependent on the presence of extracellular Ca2+. Stimulation with light never caused a detectable release of GABA. This was unexpected, since the two neurotransmitters are present in the same amacrine cells: stimulation adequate to release one neurotransmitter should release both.(ABSTRACT TRUNCATED AT 400 WORDS)

Connections of indoleamine-accumulating cells in the rabbit retina.

To study the connections of the neurons of the rabbit retina that accumulate indoleamines, we injected 5,7-dihydroxytryptamine into the vitreous body. It accumulated within a subset of amacrine cells and could be visualized there by aldehyde-induced fluorescence. The fluorescent labeling was photo-converted to an insoluble, osmiophilic product by irradiation in the presence of diaminobenzidine, and the tissue was examined by electron microscopy. Preservation of the structure of the tissue after photoconversion was satisfactory and the dendrites of the indoleamine-accumulating cells could easily be identified. They form a dense plexus near the junction of the inner plexiform and ganglion cell layers, where they exhibit large synaptic endings that occupy a substantial fraction of the surface of rod bipolar terminals. The dendrites of the indoleamine-accumulating cells receive input from rod bipolars at dyad synapses, where the other postsynaptic partner is a dendrite of a narrow-field, bistratified amacrine cell; in addition, they receive amacrine cell input throughout the inner plexiform layer. The only outputs we observed are reciprocal synapses onto the rod bipolar endings. Thus, these amacrine cells appear to exert an important effect on the transmission of scotopic information through the retina.

Shape and distribution of an unusual retinal neuron.

Rabbit retinas were exposed to exogenous indoleamines and fixed with mixed aldehydes. The indoleamines were accumulated by two types of amacrine cell and by an unusual cell (type 3) that branches widely in both plexiform layers. The type 3 cells were studied after immunohistochemistry, photooxidation of the fluorescent label, or injection with Lucifer Yellow. Their cell bodies are located at the scleral margin of the inner nuclear layer. The cells' arbors in the outer plexiform layer range from 800 to 1,500 microns in diameter. A descending process crosses the inner nuclear layer and branches in layer 5 of the inner plexiform layer. The arbor in the inner retina can exceed 500 microns in diameter. The distribution of type 3 cells was mapped in a series of retinal whole mounts. The number of type 3 cells ranged from 58 to 270 in different retinas. In two retinas from a single animal, however, it was virtually identical. Type 3 cells are concentrated in the region ventral to the visual streak, so that large areas of the retina are not covered by any type 3 cell. Because of their incomplete retinal coverage and variable number from animal to animal, the type 3 cells appear to be developmental anomalies. Paradoxically, their generation must be precisely controlled because of the numerical symmetry between an individual animal's two eyes.

Indoleamine accumulation by retinal neurons exposed to blood.

Exposing rabbit retinas for one minute to an incubation medium containing 10 microliters of blood diluted in 20 ml of medium was sufficient to produce serotonin-like immunoreactivity in some of the retinal indoleamine-accumulating neurons. Retinas from rabbits that had been perfused before the eyes were removed had no detectable immunoreactivity. Our results support the conjecture that the serotonin sometimes detected in the retina originates in the blood. Why the cells have a carrier for a molecule that they do not normally contain remains unclear.

Photoconversion of some fluorescent markers to a diaminobenzidine product.

Retinal whole mounts, brain sections, and astrocyte cultures were labeled with various fluorescent markers. Tissues or cells were then irradiated by light in the presence of diaminobenzidine. Irradiation initiated a reaction in which specific fluorescent labeling was replaced by an insoluble diaminobenzidine product. The diaminobenzidine product is more stable than the original fluorescent labeling and can be processed for electron microscopy. In some cases, the reaction product reveals cellular detail that cannot be resolved in the fluorescent labeling. The 10 fluorescent markers tested have widely differing structures, span a broad range of wavelengths, and label several different cellular elements. The photoconversion reaction was successful with all markers and tissues tested.

The effect of frontal eye field and superior colliculus lesions on saccadic latencies in the rhesus monkey.

Rhesus monkeys were trained to make saccadic eye movements to visual targets using detection and discrimination paradigms in which they were required to make a saccade either to a solitary stimulus (detection) or to that same stimulus when it appeared simultaneously with several other stimuli (discrimination). The detection paradigm yielded a bimodal distribution of saccadic latencies with the faster mode peaking around 100 ms (express saccades); the introduction of a pause between the termination of the fixation spot and the onset of the target (gap) increased the frequency of express saccades. The discrimination paradigm, on the other hand, yielded only a unimodal distribution of latencies even when a gap was introduced, and there was no evidence for short-latency "express" saccades. In three monkeys either the frontal eye field or the superior colliculus was ablated unilaterally. Frontal eye field ablation had no discernible long-term effects on the distribution of saccadic latencies in either the detection or discrimination tasks. After unilateral collicular ablation, on the other hand, express saccades obtained in the detection paradigm were eliminated for eye movements contralateral to the lesion, leaving only a unimodal distribution of latencies. This deficit persisted throughout testing, which in one monkey continued for 9 mo. Express saccades were not observed again for saccades contralateral to the lesion, and the mean latency of the contralateral saccades was longer than the mean latency of the second peak for the ipsiversive saccades. The latency distribution of saccades ipsiversive to the collicular lesion was unaffected except for a few days after surgery, during which time an increase in the proportion of express saccades was evident. Saccades obtained with the discrimination paradigm yielded a small but reliable increase in saccadic latencies following collicular lesions, without altering the shape of the distribution. Unilateral muscimol injections into the superior colliculus produced results similar to those obtained immediately after collicular lesions: saccades contralateral to the injection site were strongly inhibited and showed increased saccadic latencies. This was accompanied by a decrease of ipsilateral saccadic latencies and an increase in the number of saccades falling into the express range. The results suggest that the superior colliculus is essential for the generation of short-latency (express) saccades and that the frontal eye fields do not play a significant role in shaping the distribution of saccadic latencies in the paradigms used in this study.(ABSTRACT TRUNCATED AT 400 WORDS)

A system of indoleamine-accumulating neurons in the rabbit retina.

The indoleamine-accumulating neurons of the rabbit retina were labeled by intraocular injection of 5,7-dihydroxytryptamine (5,7-DHT). The retinas were fixed with 2.5% paraformaldehyde and 0.2% glutaraldehyde and inspected by fluorescence microscopy. Five kinds of cell accumulated the indoleamine. They were labeled to essentially the same brightness and remained so despite variations in the concentration at which 5,7-DHT had been applied or the duration of its application. Experiments in which 5,7-DHT was applied to retinas incubated in vitro gave identical results. To see the whole shape of the cells, we visually guided micropipettes to the fluorescent cell bodies and injected the cells with Lucifer yellow CH. To study the cells as a population, we used a new method in which the fluorescence of 5,7-DHT is photochemically converted to an insoluble diaminobenzidine product. The dendrites of all of the indoleamine-accumulating cells were then simultaneously visible. Used together, these techniques revealed an interrelated system of indoleamine-accumulating neurons. All of the cells contribute processes to a dendritic plexus that lies at the inner margin of the inner plexiform layer. The plexus is roughly 4 micron thick. It is pierced by the stalks of the Müller cells and is occasionally interrupted by ganglion cell bodies, where they extend above the average margin of the ganglion cell layer. Otherwise it fills much of the space at the junction of the plexiform and ganglion cell layers. The type 1 and type 2 cells are amacrine cells with cell bodies at the inner margin of the inner nuclear layer. They have 5-8 radially branching primary dendrites which extend horizontally across the inner plexiform layer before descending to join the dendritic plexus. They differ from each other in cell body shape, dendritic morphology, and the course of their dendrites within the inner plexiform layer. Each has a "displaced" counterpart, with a morphology similar to the type 1 or type 2 cell but with a cell body located in the ganglion cell layer. The displaced cells are separate functional elements because, in contrast to the type 1 and type 2 cells, they have no dendrites (and hence can have no synaptic connections) in the outer part of the inner plexiform layer. The fifth kind of cell (type 3) appears not to have been described before. Its cell body is located at the outer margin of the inner nuclear layer.(ABSTRACT TRUNCATED AT 400 WORDS)

NADPH diaphorase histochemistry in the macaque striate cortex.

The distribution of the enzyme dihydronicotinamide adenine dinucleotide phosphate (NADPH) diaphorase was examined in the striate cortex of the rhesus monkey. The pattern of activity in the neuropil matched that of cytochrome oxidase in adjacent sections and the enzymes were similarly modulated by monocular deprivation. Scattered individual cells were also intensely positive for NADPH diaphorase. Labelled cells were most common in the white matter and layers 2 and 3; they were least common in layers 4 and 5. Diaphorase cells were morphologically diverse, but no pyramidal or spiny cells were labelled. Labelled cells often had multiple varicose processes, which extended laterally for over 1 mm. Although the function of this enzyme is unknown, the morphology and distribution of the diaphorase-positive cells resembles published reports of cortical cells containing somatostatin, avian pancreatic polypeptide, and neuropeptide Y-like immunoreactivity, and NADPH diaphorase is colocalized with these substances in the rodent striatum (S.R. Vincent, O. Johansson, T. Hökfelt, L. Skirboll, R.P. Elde, L. Terenius, J. Kimmel, and M. Goldstein, J. Comp. Neurol. 217:252-263, '83).