Evidence for degenerative and regenerative changes in neostriatal spiny neurons in Huntington's disease.

Golgi impregnations of neostriatum from deceased Huntington's disease patients and controls were examined. In all cases of Huntington's disease the morphology of dendrites of medium-sized spiny neurons was markedly altered by the appearance of recurved endings and appendages, a decrease or increase in the density of spines, and abnormalities in the size and shape of spines. Pathological changes were rarely observed in medium-sized and large aspiny neostriatal neurons. The findings provide evidence for simultaneous degeneration and growth of spiny neurons in Huntington's disease and support the view that a specific population of neostriatal neurons is selectively involved in its pathogenesis.

Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.

The cause of neurodegeneration in Huntington's disease (HD) is unknown. Patients with HD have an expanded NH2-terminal polyglutamine region in huntingtin. An NH2-terminal fragment of mutant huntingtin was localized to neuronal intranuclear inclusions (NIIs) and dystrophic neurites (DNs) in the HD cortex and striatum, which are affected in HD, and polyglutamine length influenced the extent of huntingtin accumulation in these structures. Ubiquitin was also found in NIIs and DNs, which suggests that abnormal huntingtin is targeted for proteolysis but is resistant to removal. The aggregation of mutant huntingtin may be part of the pathogenic mechanism in HD.

Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons.

The gene defective in Huntington's disease encodes a protein, huntingtin, with unknown function. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking.

Excitotoxic injury of the neostriatum: a model for Huntington's disease.

Intrastriatal lesions with excitatory amino acids mimic some of the neurochemical and neuropathological characteristics of Huntington's disease (HD); this has led to the hypothesis that an endogenous excitotoxin may be involved in the disease. Recent advances in understanding the metabolic pathways of endogenous excitotoxins and the distribution and function of excitatory amino acid receptors have helped to refine the excitotoxin hypothesis, which is still inadequate to explain some aspects of the disease. However, as an experimental model for producing neuronal depletion in the neostriatum, excitotoxic injury has allowed the study of other neuronal characteristics of HD such as progressive atrophy and regeneration; it has also permitted extensive exploration of the anatomical and functional recovery induced by intrastriatal grafts. Moreover, adaptation of the rodent model to the non-human primate has enabled investigators to examine lesion-induced motor dysfunctions that are more comparable to those in HD. Thus, beyond its potential importance in the pathogenesis of HD, excitotoxic injury as an experimental tool promises to help further elucidate the pathological and functional alterations characteristic of the disease.

Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy.

An expansion of polyglutamines in the N terminus of huntingtin causes Huntington's disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons. How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur. We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink. Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin. Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes, mitochondria, and nuclear membranes. Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes. Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles. We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.

Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington's disease.

Neurons in Huntington's disease exhibit selective morphological and subcellular alterations in the striatum and cortex. The link between these neuronal changes and behavioral abnormalities is unclear. We investigated relationships between essential neuronal changes that predict motor impairment and possible involvement of the corticostriatal pathway in developing behavioral phenotypes. We therefore generated heterozygote mice expressing the N-terminal one-third of huntingtin with normal (CT18) or expanded (HD46, HD100) glutamine repeats. The HD mice exhibited motor deficits between 3 and 10 months. The age of onset depended on an expanded polyglutamine length; phenotype severity correlated with increasing age. Neuronal changes in the striatum (nuclear inclusions) preceded the onset of phenotype, whereas cortical changes, especially the accumulation of huntingtin in the nucleus and cytoplasm and the appearance of dysmorphic dendrites, predicted the onset and severity of behavioral deficits. Striatal neurons in the HD mice displayed altered responses to cortical stimulation and to activation by the excitotoxic agent NMDA. Application of NMDA increased intracellular Ca(2+) levels in HD100 neurons compared with wild-type neurons. Results suggest that motor deficits in Huntington's disease arise from cumulative morphological and physiological changes in neurons that impair corticostriatal circuitry.

Axonal transport of N-terminal huntingtin suggests early pathology of corticostriatal projections in Huntington disease.

Aggregation of N-terminal mutant huntingtin within nuclear inclusions and dystrophic neurites occurs in the cortex and striatum of Huntington disease (HD) patients and may be involved in neurodegeneration. We examined the prevalence of inclusions and dystrophic neurites in the cortex and striatum of 15 adult onset HD patients who had mild to severe striatal cell loss (grades 1, 2 or 3) using an antibody that detects the N-terminal region of huntingtin. Nuclear inclusions were more frequent in the cortex than the striatum and were sparse or absent in the striatum of patients with low-grade striatal pathology. Dystrophic neurites occurred in both regions. Patients with low-grade striatal pathology had numerous fibers with immunoreactive puncta and large swellings within the striatal neuropil, the subcortical white matter, and the internal and external capsules. In the globus pallidus of 3 grade 1 cases, N-terminal huntingtin markedly accumulated in the perinuclear cytoplasm and in some axons but not in the nucleus. Findings suggest that in the earlier stages of HD, accumulation of N-terminal mutant huntingtin occurs in the cytoplasm and is associated with degeneration of the corticostriatal pathway.

An improved approach to prepare human brains for research.

We describe two protocols for preparing human brains collected for research and diagnosis. In both protocols, one half brain is processed for research and the other for neuropathological evaluation. Clinical, neuropathological and tissue mRNA retention data are used for sample categorization. In protocol 1, coronal, whole hemisphere slices cut at standardized landmarks are frozen with a cooling device at -90 degrees C, which yields discrete anatomical structures. In selected instances, small blocks of brain are frozen at -160 degrees C in liquid nitrogen vapor. Cooling device or liquid nitrogen vapor frozen samples are suitable for in situ hybridization, protein blotting or immunohistochemistry. Morphological freezing artifacts are minimal. In protocol 2, one half brain is frozen en bloc on dry ice; this tissue is suitable for regional evaluation of gene expression or neurochemistry. Morphological freezing artifacts are severe. In both protocols, the other half brain is fixed in formalin prior to sectioning and diagnostic evaluation. The standardized selection of paraffin blocks from each brain allows precise diagnoses to be established, including identification of dangerous infectious processes; moreover, it makes it possible to produce a set of uniformly selected blocks and slides for comparative studies. These protocols lead to standardized tissue preparation for research and reduce variables impairing interpretation and comparison of data.

Early and progressive accumulation of reactive microglia in the Huntington disease brain.

Microglia may contribute to cell death in neurodegenerative diseases. We studied the activation of microglia in affected regions of Huntington disease (HD) brain by localizing thymosin beta-4 (Tbeta4), which is increased in reactive microglia. Activated microglia appeared in the neostriatum, cortex, and globus pallidus and the adjoining white matter of the HD brain, but not in control brain. In the striatum and cortex, reactive microglia occurred in all grades of pathology, accumulated with increasing grade, and grew in density in relation to degree of neuronal loss. The predominant morphology of activated microglia differed in the striatum and cortex. Processes of reactive microglia were conspicuous in low-grade HD, suggesting an early microglia response to changes in neuropil and axons and in the grade 2 and grade 3 cortex, were aligned with the apical dendrites of pyramidal neurons. Some reactive microglia contacted pyramidal neurons with huntingtin-positive nuclear inclusions. The early and proximate association of activated microglia with degenerating neurons in the HD brain implicates a role for activated microglia in HD pathogenesis.

Synaptic organization of cholinergic neurons in the monkey neostriatum.

Cholinergic neurons in the monkey neostriatum were examined at the light and electron microscopic level by immunohistochemical methods in order to localize choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. At the light microscopic level a sparse distribution of cholinergic neurons was identified throughout the caudate nucleus. Neurons had large (25-30 microns) somata, eccentric invaginated nuclei, primary dendrites of unequal diameters, and varicosities on distal dendritic branches. Ultrastructural study showed that the cholinergic cells had a cytoplasm abundant in organelles. Within dendritic branches, mitochondria and cisternae were localized primarily to varicosities. Synaptic inputs were distributed mostly to the dendrites and at least four types that formed symmetric or asymmetric synapses were observed. Immunoreactive fibers were relatively numerous within the neuropil and exhibited small diameters (0.1-0.15) micron) and swellings at frequent intervals. Cholinergic boutons that formed synapses were compared to unlabeled terminals making asymmetric synapses with dendritic spines. Results showed that ChAT-positive axons had significantly smaller cross-sectional areas, shorter synaptic junctions, and a higher density and surface area of mitochondria than the unlabeled boutons. Cholinergic axons formed symmetric synapses mostly with dendritic spines (53%) and the shafts of unlabeled primary and distal dendrites (37%). A relatively small proportion of the boutons contacted axon initial segments (1%) and cell bodies (9%) that included medium-sized neurons with unindented (spiny) and indented (aspiny) nuclei. The majority of dendritic spines contacted by cholinergic axons were also postsynaptic to unlabeled boutons forming asymmetric synapses. The results suggest that cholinergic neurons in the primary neostriatum belong to a single morphological class corresponding to the large aspiny (type II) interneuron identified in previous Golgi studies. Present results along with earlier Golgi-electron microscopic observations from this laboratory suggest that neostriatal cholinergic cells integrate many sources of intrinsic and extrinsic inputs. The observed convergence of ChAT-immunoreactive boutons and unlabeled axons onto the same dendritic spines suggests that intrinsic cholinergic axons modulate extrinsic inputs onto neostriatal spiny neurons at postsynaptic sites close to the site of afferent input.

Large neurons in the primate neostriatum examined with the combined Golgi-electron microscopic method.

Large neurons in the monkey neostriatum were examined in the electron microscope in tissue treated with the rapid-Golgi impregnation method followed by the gold-toning procedure. Two types of large neurons were investigated: an aspiny neuron (aspiny type II; N = 5) with numerous varicose dendrites and a spiny cell (spiny type II; N = 1) with few sparsely spined dendrites. The large aspiny neurons had variably shaped somata, an eccentric highly invaginated nucleus, and a cytoplasm rich in organelles. Mitochondria were distributed unevenly in dendrites and were localized primarily in varicosities. Some mitochondria exhibited dense bodies 80-300 nm in size. Most synapses (84%) onto large aspiny neurons occurred 20 micron or more from the cell body and contacted dendritic varicosities (63%). A smaller proportion of boutons (21%) contacted constricted portions of varicose segments. A low incidence of synaptic boutons was observed on smooth primary and secondary dendrites (11%), cell bodies (3%), and branch points (2%). Seven percent of the axons that synapsed with large aspiny neurons also contacted nearby dendrites or spines of medium-sized spiny neurons. At least eight morphologically distinct types of axons making synapses with large aspiny neurons were identified and included both symmetric and asymmetric types. The large spiny neuron was different from the large aspiny neuron in its subcellular characteristics. Synapses were found on all portions of the cell, including the axon initial segment, but fewer types of axonal inputs were identified. These findings confirm that the two types of large neurons identified in Golgi impregnations of the primate neostriatum are also different at the ultrastructural level, both in their cytological features and in their synaptic organization. The large aspiny neuron integrates synaptic inputs that innervate a relatively large area of caudate neuropil and appear to arise from a variety of extrinsic and intrinsic sources. The high density of synaptic inputs to dendritic varicosities suggests that they have an important functional role.

Immunoreactive leu-enkephalin in the monkey hypothalamus including observations on its ultrastructural localization in the paraventricular nucleus.

The distribution of immunoreactive leu-enkephalin neurons and fibers in the monkey hypothalamus, including ultrastructural localization in the paraventricular nucleus (PVN), was examined with the peroxidase-antiperoxidase immunocytochemical method. Immunoreactive leu-enkephalin cell bodies and fibers were present in the PVN, the region of the dorsal nucleus and nucleus of the anterior commissure, the dorsomedial nucleus, ventromedial nucleus, and lateral hypothalamus. Within the PVN labeled cells were found mostly in the medial parvocellular region, and a smaller proportion including some large cells was present in the lateral, and dorsolateral zones. Immunoreactive neurons contained numerous large granular vesicles (LGV) which ranged from 63 to 235 nm in size, suggesting that at least some enkephalin-containing neurons belong to the population of neurosecretory cells. Positive neurons were postsynaptic to four types of unlabeled axon terminals. Leu-enkephalin-containing fibers (some of which were myelinated) and boutons contained small clear vesicles and numerous LGV. Axon terminals made synaptic contacts with the cell bodies, primary and distal dendrites of unlabeled neurons. The findings show that enkephalin-containing neurons in the PVN integrate a variety of neuronal inputs and provide morphological evidence for the inhibiting influence of enkephalins on the firing rate of PVN neurons. It may be speculated that the effects of opioids on the release of vasopressin and other substances possibly originating from PVN neurons may be regulated in part within the nucleus by locally synapsing axons belonging to enkephalin-containing neurons.

Early postnatal development of the monkey neostriatum: a Golgi and ultrastructural study.

Paired specimens of the neostriatum were taken from monkeys at zero (newborn), one, two, four, eight, and 16 weeks of age, and prepared for Golgi impregnations and electron microscopy. Light microscopy shows that in the first postnatal week, the structure contains the five neuronal types and four categories of afferent axons described in the adult, as well as some cells too undifferentiated to classify. Most neurons exhibit immature dendritic features, including local enlargements, terminal growth cones with filopodia, and filiform processes. In spiny type I cells, various levels of maturity may coexist in regions of a single dendrite, in different dendrites of the same neuron, and among individual cells. Spine density increases progressively with age, but the relative distribution of spine types remains about the same. Spiny type II neurons show some decline in spine density, and generally mature sooner than spiny type I cells. The long axons of spiny neurons have varicosities which disappear at about eight weeks. In younger animals (newborn and one week), the dendrites of aspiny neurons (types I, II, and III) may have a "spiny" appearance, exhibiting many spine-like and filiform processes. Concurrently, the short axons vary in degree of arborization from very immature to well developed. Electron microscopy corroborates the developmental features recognized in the Golgi material: dendritic and axonal growth cones, filopodia and varicosities, as well as various stages of maturation in somata and dendrites. Degenerating elements, mostly of an axonal nature, are seen up to eight weeks. The synapses which reach maturity at birth are of the asymmetric axospinous type, in which the axonal profile contains small round vesicles, and of the symmetric axodendritic class, with the presynaptic elements having pleomorphic vesicles. Some synapses are slower to mature and appear at one to eight postnatal weeks. These include those made by profiles with pleomorphic vesicles, forming either symmetric contacts with somata and axon initial segments, or asymmetric contacts with spines. The same applies to the asymmetric axodendritic synapses made by elements containing small round vesicles. Finally, profiles containing large round or flat vesicles are the latest to participate in mature synapses formation. Findings indicate that a considerable degree of qualitative and quantitative change takes place in the monkey neostriatal neuropil during early postnatal development, especially in the first eight-week period.

A Golgi and ultrastructural study of the monkey globus pallidus.

Golgi preparations reveal that the most frequent type of pallidal neuron (principal cell), which has been recognized in all previous reports, is large (20-50 microns), fusiform, with dendrites up to 700 microns long. Large neurons of globular shape are less frequently impregnated. The morphology of dendrites varies considerably within the same neuron. Some exhibit numerous spines and protrusions and are seen to terminate in elaborate arborizations. A small interneuron (12 microns), with relatively short dendrites, up to 150 microns, and a short sparsely branching axon is observed less frequently. At least two types of afferent axons are present. A small-diameter fiber from the neostriatum enters the pallidum in bundles and gives rise to numerous thin branching processes with varicosities about 1 micron in size. The axon collaterals are oriented orthogonal to the main axon and parallel to the dendrites of principal cells. A large-caliber fiber with clusters of 2-3 microns swellings can also be seen in close proximity to large pallidal dendrites. Ultrastructurally, principal cell dendrites (trunks, spines, and protrusions) are totally covered by synapsing axon terminals. In contrast, some small dentrites, presumed to belong to interneurons, form very few synapses. At least six categories of profiles containing vesicles are observed. One group has cytologic features of dendrites and participates in serial and triadic synapses with other profiles in the pallidal neuropil. Results suggest that the synaptic organization of the globus pallidus may be viewed as a repetitive, geometric arrangement of striatal and other afferent axons ensheathing and synapsing with the dendrites of principal cells. This pattern is interrupted by the presence of presynaptic dendrites, probably belonging to interneurons, which participate in complex synaptic arrangements.

Ultrastructural localization of immunoreactive calbindin-D28k in the rat and monkey basal ganglia, including subcellular distribution with colloidal gold labeling.

Normal cellular function depends on the controlled flux of Ca++ within intracellular compartments and across the plasma membrane. Proteins that bind Ca++ are thought to contribute to the regulation of intracellular Ca++ and, perhaps more importantly, signal functional changes in cell activity. In the brain, calbindin-D28k is among a class of calcium-binding proteins that are widely and heterogeneously distributed in select populations of neurons, among them neostriatal cells, but whose function is largely unknown. In this study of the monkey and rat neostriatum and globus pallidus, calbindin-D28k was localized with immunoperoxidase and immunogold methods in order to identify striatal cell populations that contain this protein and the subcellular compartments in which it is likely to function. Light and electron microscopy showed intense and extensive labeling of immunoreactive calbindin-D28k in the cell bodies, dendrites, and spines of medium-sized neostriatal spiny neurons and in their axon terminals which end in the globus pallidus. More discrete labeling with a gold-conjugated second antibody showed that the predominant site of calbindin-D28k was the matrix of the cytoplasm. Gold label was also associated with the karyoplasm of spiny cells and with the neurofilaments and axoplasmic matrix of striatopallidal axons and terminals, respectively. Membranes were either sparsely labeled (endoplasmic reticulum, mitochondria) or devoid of gold particles (nuclear envelope and plasmalemma). Radioimmunoassays of striatal subcellular fractions supported the anatomical findings by indicating that the soluble fractions of neostriatal tissue homogenates contained most of the calbindin-D28k immunoreactivity and that washes from forebrain synaptosomes treated with Triton X-100 yielded high levels of immunoreactive calbindin-D28k. These findings show that immunoreactive calbindin-D28k is localized to spiny neurons of the striatopallidal pathway and are consistent with previous observations on subcellular localization in nonneuronal tissues. If, as recently speculated, calbindin-D28k regulates calcium concentrations in neostriatal spiny neurons, this feature may be particularly involved with the high density of glutamatergic inputs to these cells. More work is needed to determine whether calbindin-D28k, when complexed to Ca++ in neostriatal spiny cells, signals the activation of protein kinases, phosphorylation, and/or neurotransmitter release, as has been shown for other Ca++-binding proteins in mammalian tissues.

Localization of immunoreactive GABA and enkephalin and NADPH-diaphorase-positive neurons in fetal striatal grafts in the quinolinic-acid-lesioned rat neostriatum.

Fetal striatal tissue grafts have been shown to partially reverse the biochemical and behavioral deficits induced by excitotoxic lesions. To determine if grafted striatal neurons contain neurochemical markers similar to those in neurons in the caudate nucleus and to establish the morphological characteristics and relative frequency of labeled neurons in the grafts, the localization of immunoreactive GABA and leucine-enkephalin (ENK) and of NADPH-diaphorase (NADPH-d) activity was examined in fetal striatal grafts at the light and electron microscopic levels. Striatal tissue from 17-day fetuses was grafted into the caudate nucleus of adult rats 1 week after intracaudate injections of either a low or high dose of quinolinic acid. At the light microscopic level, immunoreactive GABA and ENK and NADPH-d-positive neurons, processes, and punctate structures were present within adjacent sections of the same grafts. The frequency and morphological features of these labeled cell populations were similar in grafts placed into either minimally or extensively lesioned striata. Immunoreactive GABA and ENK neurons in the grafts constituted 28% and 13.5%, respectively, of the neuronal population of the graft and their mean diameters were 22 and 14% larger, respectively, than neostriatal neurons that contained the same chemical markers. NADPH-d-positive neurons in the grafts formed 3.5% of total grafted neurons and exhibited characteristics of neostriatal NADPH-d-containing aspiny cells, including medium-sized somata, indented nuclei, and varicose dendrites. At the electron microscopic level most GABA-positive neurons in the grafts contained indented nuclei and most immunoreactive ENK somata had unindented nuclei. Dendrites and dendritic spines with GABA or ENK immunoreactivity were present in the grafts where they were postsynaptic to unlabeled axons. Immunoreactive GABA and ENK axon terminals formed synapses with unlabeled neuronal profiles in the grafts. These findings demonstrate that fetal striatal grafts contain chemically defined neuronal populations that form synaptic connections within the graft and share some features with corresponding cell groups in the neostriatum. These results provide an anatomical basis for the graft-induced recovery from behavioral and biochemical deficits caused by instrastriatal lesions reported in other studies.

A Golgi study of the human neostriatum: neurons and afferent fibers.

The neostriatum of 20 adult humans was examined in Golgi-Kopsch and rapid Golgi preparations. At least five types of neurons and four types of afferent fibers are described. Neurons of medium size with spine-rich dendrites (spiny type I) are the most frequent type. These cells exhibit a greater morphological diversity than those previously studied in the monkey. Also, quantitative data show that, compared to the monkey, spiny type I neurons in man have a greater somal size and dendritic field radius. Although the types of spines are similar, the mean density and radial distribution of spines along dendrites differ in the two species. Morphologic features of the axon, which is usually long with extensive collateral branches, suggest that more than one process from the axon may be efferent. Medium to large neurons with sparsely spined dendrites (spiny type II) differ from type I neurons in having a poorer branching and greater radial spread of their dendrites and a lower density of spines. They also differ from spiny type I neurons in their distribution and relative proportion of various spine types. The axon of the spiny type II neuron is long and has collaterals which are poorly arborized in comparison to those of spiny type I cells. Aspiny neurons are of medium (aspiny type I) and large (aspiny type II) size. They have varicose, curved dendrites and a short axon which arborizes mostly within the dendritic field. A group of smaller neurons with more variable dendritic morphology is also seen. Observations suggest that in the human brain the proportion of medium-sized aspiny neurons and small neurons may be greater than in other species.

Cytochrome oxidase activity in the rat caudate nucleus: light and electron microscopic observations.

Cytochrome oxidase (CO) activity was examined in the neostriatum of normal adult rats at the light and electron microscopic level. At the light microscopic level a heterogeneous distribution of CO activity was observed and was characterized by patches of high activity ranging in size from 200 to 800 microns surrounded or adjacent to regions of lower activity. The most dorsomedial and ventromedial regions of the caudate nucleus appeared to be consistently high in activity in all animals. At the ultrastructural level CO reaction product was localized to the membranes and intracristal spaces of mitochondria. The most reactive mitochondria (those containing the denest precipitates of reaction product) were found within the dendrites of spiny neurons in all caudate regions. In areas of high CO activity the mitochondria within bundles of myelinated fibers and in many axon terminals were also highly reactive whereas those in neuronal somata, primary dendrites, and glial cells and processes exhibited relatively little activity. Quantitative study showed that mitochondria within dendrites accounted for most of the CO activity in caudate neuropil. The mitochondria within dendrites and axon terminals were more reactive in regions of high CO activity than in regions of low CO activity. No differences in the density of synapses or in the proportions of axospinous and axodendritic synapses were observed between CO-rich and CO-poor areas. Heterogeneity in the distribution of CO activity in the caudate nucleus may be related to the "patchy" pattern of localization previously observed for some neostriatal afferents, enzymes, transmitters, peptides, and receptor ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive GAP-43 in the neuropil of adult rat neostriatum: localization in unmyelinated fibers, axon terminals, and dendritic spines.

GAP-43 is a neuron-specific phosphoprotein that has been implicated in neuronal development, axonal regeneration, and synaptic plasticity. Although in mammals the caudate-putamen is among those brain areas that retain a high content of GAP-43 throughout life, the role of the phosphoprotein in the neostriatum is unknown. In order to understand better the possible function(s) of GAP-43 in the adult striatum, its cellular localization was examined with immunohistochemistry at the light and electron microscopic levels by using a sheep polyclonal antibody. At the light microscopic level immunoreactive GAP-43 was abundant throughout the neostriatal neuropil but was absent from neuronal somata. At the ultrastructural level, labeling was most prevalent in small unmyelinated axons (0.12-0.15 microns diameter). Reaction product was distributed along fibers in discrete patches about 1 micron apart and in preterminal sites from which vesicle-filled boutons arose. Staining was also present in small (0.35 microns) axon terminals that contained round vesicles and formed asymmetric synapses, mostly with thin spines. Following unilateral cortical lesions, some degenerating cortical axons in the neostriatum exhibited GAP-43 labeling. Unexpectedly, in normal striatum, GAP-43 was also occasionally found in the heads of dendritic protrusions and in thin spines that received asymmetric contacts. We speculate that in the adult neostriatum, the protein may be important in the remodeling of synapses onto medium spiny neurons that involve, in part, the corticostriatal pathway.

A Golgi study of the monkey paraventricular nucleus: neuronal types, afferent and efferent fibers.

The neuronal organization of the paraventricular nucleus (PVN) was examined in Golgi impregnations of adult monkey. Results showed that at least six types of neurons could be identified in the nucleus on the basis of morphological features of the somata, dendrites, and axons. Four types of neurons with sparse to densely spined cell bodies and dendrites exhibited long axons and included large neurons (types I and II), medium-sized to large neurons (type III), and small to medium-sized cells (type IV). Axons of type I, III, and IV neurons had different diameters and were followed out of the PVN. Axon collaterals that arborized within the PVN were seen on the axons of types III and IV cells. Two types of interneurons with small somata were also found. One (type V) exhibited varicose dendrites and a profusely arborizing local axon. The other cell (type VI) had recurved dendrites with long appendages and no impregnated axon. Afferent fibers were also identified. Type 1 was a fine-caliber axon that coursed long distances in the PVN and exhibited numerous short branches. Additional observations suggested that type 1 afferents originated from the stria terminalis. The other afferent axon (type 2) was thicker and gave rise to terminal arborizations containing clusters of small swellings. The efferent fibers of the PVN were also examined in impregnations of the paraventriculosupraopticohypophysial tract. Fibers formed an extensive plexus as they coursed ventrally and passed through the lateral hypothalamus. Axons coursing more laterally in the tract were much larger than those more medially located. Our findings show a diverse organization of neuronal types within the monkey PVN with evidence for intrinsic connections through axon collaterals of efferent neurons and the locally arborizing axons of interneurons. Correlations are proposed between morphological subtypes of neurons seen in this Golgi study and the known functional output pathways of the PVN.