Calcium-containing electron-dense structures in the axons of the squid giant synapse.

Following the Oschman and Wall technique, electron-dense structures (EDS) were found on unstained, unosmicated membranes of squid giant synapse axons. These densities contain high concentrations of calcium and phosphorus as identified by energy dispersive X-ray analysis. Based on the signal strength, the quantity is significantly greater than that of other regions of the membrane or tissue spaces. The calcium EDS occur as plaques or globules along the axonic membrane, and small globules are found between sheath cell processes. EDS also occur at the synaptic site. These densities were correlated with the opacity change seen in giant axons. It is proposed that these structures represent sites where the calcium-binding protein found by other investigators has become nearly saturated with calcium.

Morphological basis for a mechanical linkage in otolithic receptor transduction in the frog.

Observations made with a scanning electron microscope confirm the binding of the stereocilia to a matchhead-like bulbous terminal at the apex of the kinocilium in frog saccular receptor cells. Since the kinocilium is shown to rest on a portion of the receptor cell that lacks the rigid cuticular base of the stereocilia, movenment of the ciliary ensemble results in a "plunging-like" effect of the kinocilium which produces a distension of the membranc at its base. This membrane distension is envisaged as bringing about the ionic conductance changes necessary for the production of a generator potential and, thus, for the transduction of movement into vestibular nerve activity.

Dendritic spikes and their inhibition in alligator Purkinje cells.

Alligator Purkinje cells generate action potentials in the peripheral dendritic tree, after synaptic depolarization via superficial parallel fibers. These action potentials are inhibited at the dendrite level by preceding parallel-fiber volleys at close intervals. We conclude that this inhibition is produced by the activation of the inhibitory interneurons of the molecular layer, the stellate cells, which establish synaptic contacts with the dendrites of the Purkinje cells.

Inferior olive: its role in motor learing.

Specific chemical lesion of the rat inferior olive by intraperitoneal administration of 3-acetylpyridine prevents recuperation from motor abnormalities generated by unilateral labyrinthine lesion. Moreover, in animals that have recuperated from the balyrinthine lesion, 3-acetylpyridine produces a reversal of the symptoms within 2 hours of administration. These results indicate that the integrity of the olivo-cerebellar system is necessary for the acquisition and retention of this form of motor learning, but that the cerebellum itself is not the seat of such learning.

Distribution and functional significance of the P-type, voltage-dependent Ca2+ channels in the mammalian central nervous system.

In addition to the three types of voltage-dependent calcium channels presently recognized in the CNS, the L-, the T- and the N-types, a fourth distinct type known as the P-type channel has recently been described. This channel, initially recognized in Purkinje cells (and thus the name), is not blocked by dihydropyridines or by omega-conotoxin (GVIA), but is blocked by native funnel-web spider venom and by a polyamine (FTX) extracted from such venom. In addition, a synthetic polyamine (sFTX) has been produced that also specifically blocks P-channels in brain slices and at the neuromuscular junction, and blocks presynaptic Ca2+ currents in other vertebrate and invertebrate forms, as well as channels expressed in Xenopus oocytes following CNS mRNA injections. Using sFTX to form an affinity gel, a protein was isolated and reconstituted into lipid bilayers where it manifests single-channel properties that are electrophysiologically and pharmacologically similar to those of the native P-channels. Rabbits immunized with the isolated protein produced a polyclonal antibody that gave a positive western blot with the purified P-channel protein and generated a reaction product at specific sites in the CNS that agree with the physiological distribution of P-channel activity.

Dendritic morphology of central auditory neurons correlates with their tonotopic position.

We have investigated the morphology of dendritic arbors in a central auditory nucleus, the lateral superior olive, of the Mongolian gerbil. Morphometric observations were obtained directly from Golgi-impregnated material by using a microcomputer-based three-dimensional data acquisition system. In particular, measurements were made to determine the dendritic arborization across each of three axes: the tonotopic axis, the rostrocaudal axis, and the isofrequency axis (i.e., perpendicular to the tonotopic axis). The tonotopic position of each cell was computed on the basis of a topographic map that has been constructed for the gerbil LSO (Sanes et al.: J. Comp. Neurol. 279:436-444, 1989). It was found that the span of a dendritic arbor along the tonotopic axis was directly correlated with the neuron's tonotopic position: Low frequency neurons had much broader arborizations than high frequency neurons. Moreover, the distribution of frequency bandwidths to which single LSO neurons responded showed a striking similarity to dendritic arborizations across the tonotopic axis. Lower frequency neurons responded to a larger number of octaves than higher frequency neurons. There was no correlation between tonotopic position and dendritic arborization in the isofrequency or rostrocaudal axis. Nor was there any correlation between frequency and total dendritic length, number of primary dendrites, or soma area. However, there was a small but significant difference between the primary dendrite diameter of low and high frequency neurons. Low frequency neurons had significantly greater diameters. These results suggest that the frequency selectivity of central auditory neurons may employ, as one morphological substrate, the distance over which their dendrites arborize along the tonotopic axis.

Compartmentation of the cerebellar cortex by protein kinase C delta.

Six isozymes of protein kinase C were analysed in the rat cerebellum using immunohistochemistry. The results revealed a non-uniform distribution of protein kinase C delta among Purkinje, basket, and stellate cells. Serial-section mapping of the delta immunoreactivity revealed that (i) the number and intensity of labeled Purkinje cells increased from rostral to caudal while labeled basket-stellate cells decreased caudally; (ii) the majority of Purkinje cells were labeled in the nodulus, flocculus, and paraflocculus while the anterior lobules were mostly negative; and (iii) labeled Purkinje cells formed distinct parasagittal bands in lobules 6-9 of the paravermis and vermis. The banding of protein kinase C delta within subsets of Purkinje cells suggests units of cerebellar circuitry with specific signaling properties through protein phosphorylation. The visual-vestibular regions of the cerebellum contained the highest amount of the isozyme.

Distribution of glutamate receptors GluR 2/3 and NR1 in the developing rat cerebellum.

The distribution of glutamate receptors GluR2/3 and NR1 was analysed immunohistochemically during development of the rat cerebellum. GluR2/3 immunoreactivity appeared by postnatal day P0 in somata of Purkinje cells. Throughout P7, P15, P20 and adulthood, GluR2/3 immunoreactivity was found in the entire Purkinje cell dendritic arbor reaching to the external granular layer and, by P15, the surface of the cerebellum. By P7, the granular layer revealed scattered, mildly reactive, cells. NR-1 immunoreactivity first gained prominence about P7 in the region of the multi-layered Purkinje cell somata. By P15, NR1 was prominent in Purkinje cell somata and Golgi cells. The reaction product extended into the primary main dendrite of Purkinje cells. By P21, stellate and basket cells had intense reactivity throughout the molecular layer and reactive large-diameter dendrites of Golgi cells projected toward the molecular layer. Granule cells remained very weak among strongly reactive Golgi cell somata and dendrites. Ultrastructural immunohistochemistry revealed NR1 reaction product in Purkinje cell somata, in stellate cell somata and dendrites and on postsynaptic membranes of scattered spines throughout the molecular layer. The later appearance and restricted location of NR1 in somata and proximal dendrites of Purkinje cells contrasted markedly with GluR2/3 which appeared before birth and remained prominent throughout Purkinje cell dendritic arbors of adults. The time of NR1 expression correlated with the generation of granule cells, their synaptogenesis on Purkinje cells, the formation of stellate/baske cells and the shift of climbing fibre synapses from distal to proximal dendrites. The developmental appearance of stellate/basket cells and Golgi cells as well as their high reactivity remaining into adulthood suggest that these inhibitory molecular and granular layer interneurons are the principal targets of glutamate axons serving NR1 synaptic properties while Purkinje cells and brush type granule cells are targets for glutamate connections with GluR2/3 characteristics.

Perinatal methylazoxymethanol acetate uncouples coincidence of orientation of cerebellar folia and parallel fibers.

Perinatal administration of methylazoxymethanol acetate in the rat, as a one time injection on gestational day 21, postnatal days 0, 1 or 2, altered the parallel orientation of cerebellar folia. The effect persisted into adulthood. In animals injected on one of the postnatal days 3, 4 or 5, the folial pattern was not altered. Even when the injection was repeated for three days on postnatal days 3, 4 and 5, changes in the cerebellar surface were not found. However, in animals receiving a low protein diet during the last five days of gestation, the three injection regimen produced a distortion of the folial pattern. The surface of cerebella of animals injected on gestational day 21 through postnatal day 2 was covered with small blebs resembling the surface of a cauliflower head. In sagittal sections, islands of cortical laminae appeared to be isolated from the arbor vitae. However, serial reconstruction of the granular layer from sections revealed that these pieces were continuous with the arbor vitae. Surprisingly, cerebella having malaligned folia also had varying degrees of Purkinje cell somas distributed throughout the granule cell layer rather than in a single layer. This occurred even when the granule cell layer approached normal thickness. Analysis of cerebellar weight from the group injected on the day of birth revealed three levels of weight reduction: severe (greater than 40%), moderate (20-40%) and mild (less than 20%). The granule cell deficit was directly related to the weight reduction of the cerebella. In the severely-affected cerebella, areas of the cortex were virtually devoid of granule cells. The moderately-affected cerebella had a continuous granular layer which was thick and thin. In the mild type, the layer was relatively normal in thickness but, nevertheless, the cerebellar surface was highly distorted. In all animals treated with methylazoxymethanol acetate on days G21 through P5, parallel fibers were disoriented. This occurred even though the folia appeared normal in the G20, P3, P4, P5 and P3-5 injected groups. Bundles of parallel fibers crisscrossed in the plane of the cerebellar surface in all areas where a molecular layer was found.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural and molecular heterogeneity of astrocytes and oligodendrocytes in the gerbil lateral superior olive.

The goal of this study was to determine the distribution and diversity of astrocytes and oligodendrocytes within the lateral superior olive of the gerbil. We used morphometric analyses and several immunocytochemical markers to assess differences in glial cell composition between the lateral (low-frequency projection) and the medial (high-frequency projection) limb of the lateral superior olive. Cell counts from Toluidine-stained semithin sections revealed a similar density of total astrocytes in both the lateral and the medial limbs. However, based on cytologic features, there was a prevalence of fibrous-like astrocytes in the lateral limb and protoplasmic-like astrocytes in the medial limb. In a similar manner, glial fibrillary acidic protein staining of astrocytes was intense in the lateral limb, but was largely restricted to the nucleus borders in the medial limb of the lateral superior olive. While glial fibrillary acidic protein was largely restricted to astrocytic processes, glutamine synthetase and S100 protein staining occurred, for the most part, in glial cell bodies. The density of glutamine synthetase positive cell bodies was homogeneous between the two limbs, while the density of S100-positive somata was significantly greater in the lateral limb. Cell counts obtained from semithin sections demonstrated a greater density of oligodendrocytes in the lateral limb than in the medial limb of the lateral superior olive. In a similar manner, there was a 40% greater density of carbonic anhydrase-positive somata in the lateral limb compared to the medial limb. Transferrin immunostaining was restricted to oligodendrocytes, but the density of labeled somata was identical in the lateral and medial limbs. 2',3'-Cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein were also localized to the somata of oligodendrocytes, labeling both perisomatic and interfascicular cells. At the ultrastructural level, specialized contacts were found between pairs or clusters of oligodendrocytes. These results suggest that more than one type of astrocyte and oligodendrocyte is present within the gerbil lateral superior olive. Furthermore, glial cells were unevenly distributed, such that a greater density of oligodendrocytes and fibrous-like astrocytes were found in the low-frequency projection region. This heterogeneity is well correlated with known differences in the neuronal morphology within the lateral superior olive.

Immunohistochemical localization of Lyn (p56) protein in the adult rat brain.

Expression of a sarcoma proto-oncogene, c-lyn, was mapped in the adult rat brain using immunohistochemistry. Lyn protein was prevalent in restricted cell populations of the olfactory bulb and the basal forebrain which included nuclei of accumbens, fundal striatum, bed stria, ventral pallidum and central amygdala as well as deep entorhinal and pyriform cortices. Tightly packed Lyn-positive cells formed discrete multiple stripes crossing perpendicular to the rostral limb of the anterior commissure, and intense masses surrounding the caudal limb. In the thalamus, the habenula, anterodorsal nucleus and medial geniculate body, together with the paraventricular hypothalamic nuclei, had prominent reactive neuronal somata and dendrites in the neuropil. The lateral septal nucleus also had intense Lyn-positive neurons with overlapping dendritic fields. In addition, scattered neurons were evenly distributed throughout the striatum. The red, interpeduncular, auditory and trigeminal tract nuclei were intensely reactive. The cerebellar molecular layer was uniformly labeled except for a few isolated fiber bundles in the lowest part of this layer. The granule cells adjacent to the Purkinje cell layer appeared in reactive patches. In the spinal cord, the posteromarginal nucleus had intense labeling. The significance of this highly localized distribution pattern of Lyn protein may be related to connections forming functional compartments serving signal transduction within specific central nervous system circuitry.

Plasmalemmal ATPase calcium pump localizes to inner and outer hair bundles.

Recent studies demonstrate calcium ion influx at the tips of hair cell stereocilia during mechano-transduction. These ions must be either pumped from the cytosol into the extracellular space or endoplasmic envelope, or else sequestered by binding to specific proteins. A plasma membrane calcium pump (ATPase-type) was analysed in whole-mounts of rat organ of Corti using a monoclonal antibody to a large cytoplasmic loop of this protein. The reactivity was particularly high on the tips of longer stereocilia and was found along the shafts. Inner hair cell stereocilia had much less reactivity than outer hair cells. The reactivity lined the plasma membrane of inner hair cell bodies while a higher reactivity appeared in the cytoplasm of outer hair cells. Supporting cells were unreactive. Ultrastructural examination confirmed the plasma membrane calcium pump location on stereocilia and along the endolymph surface of receptor cells. Reaction product lined the plasma membrane of stereocilia as intense puncta. More reactive puncta occurred near the distal ends of stereocilia and the number decreased toward the ciliary base. The endolymph plasma membrane over the cuticular notch was especially reactive. The finding of more intense pump reactivity at the tips of stereocilia than the base is consistent with the hypothesis that during transduction, calcium ions enter stereocilia, distally, and the ATPase plasma membrane calcium pump rapidly extrudes these ions to the extracellular space.

Ultrastructural localization of the plasmalemmal calcium pump in cerebellar neurons.

In a previous study, fluorescence labeling of a plasmalemmal ATPase protein with the 5F10 monoclonal antibody revealed prominent antigen in the cerebellar molecular layer surrounding the somata and dendrites of Purkinje cells. In the present study, this antibody labeled with silver enhanced nano-sized gold particles on semithin plastic sections revealed a clearly demarcated plasma membrane outlining the somata and entire dendritic arbors of Purkinje cells including their spines. Ultrastructural analysis of horseradish peroxidase preparations showed reaction product along the plasmalemma and extending on to the sub-plasmalemmal endoplasmic reticulum. In the granular layer, somata of granule cells were reactive, as were their dendritic extensions into glomeruli where reactive claws surrounded voids formed by mossy fiber rosettes. Somata and dendrites of cerebellar nuclear cells also had reactive zones that were limited to the plasma membrane and a narrow zone of the sub-plasmalemmal endoplasmic reticulum. Comparative labeling of this protein and P channel protein revealed similar plasmalemmal locations. This study shows that a specific calcium ATPase pump protein is located on the plasmalemma of certain types of cerebellar neurons. The ultrastructural distribution of calcium pump and P channel antibodies occurred in punctate sites along the plasma membrane of dendrites and spines of Purkinje cells. The close association between P-type calcium channels and the plasma membrane calcium pump is consistent with rapid extrusion of intracellular calcium from neurons endowed with large numbers of voltage-gated calcium channels.

Computer-aided mapping of specific neuronal populations in the human brain.

Antibody-staining methods and computer-aided microscopic systems have been used to generate high-resolution panoramic maps of specific neuronal populations in the human brain (4,6,11). This report focuses on the problems inherent in attempting high-resolution mapping of large brain sections, and describes how they are solved by computer-aided mapping. Further applications of computers to the study of brain structure are considered.

Plasticity of cerebellar parallel fibers following developmental deficits in synaptic number.

As demonstrated previously, a deficit in the number of cerebellar granule cells that is induced by pre-and postnatal malnutrition, results in fewer but larger synapses on Purkinje cells. Here, we report that the axons of granule cells compensate this loss by generating additional dense projections enlarging the presynaptic grids. This presynaptic response is directly related to the availability of the postsynaptic contact area of the target neurons which reaches a relatively constant amount during development.

Plasticity of the parallel fiber-Purkinje cell synapse by spine takeover and new synapse formation in the adult rat.

Alteration in synaptic connectivity between Purkinje cell spines and parallel fibers of the cerebellum were studied following partial deafferentation of Purkinje cells in the the adult rat. Transection of parallel fibers by two lesions placed at a 1 mm interval on the folial crest were used to produce degeneration of these afferents. Ultrastructural analysis of synapses on Purkinje cell spines revealed degeneration with vacating of postsynaptic sites within 6 h. Reactive synaptogenesis as takeover of Purkinje cell spines by formation of new synapses from remaining parallel fibers occurred even before degenerating parallel fibers had vacated postsynaptic sites. This was accompanied by a marked increase in the number of dual innervations by reactive parallel fibers within one day. Some vacated postsynaptic sites were lost as indicated by a reduction in the number of synapses and others may have been taken over by newly formed synapses on spines. In addition, new synapses formed between the shafts of Purkinje cell branchlets and parallel fibers. Sprouting of parallel fibers occurred as small extensions without tubules while Purkinje cell spines reacted by forming elongated and multiple heads which contacted different parallel fibers. After 5 days degenerating boutons were rarely found. Enlarged spine heads were each capped by a proportionally enlarged parallel fiber bouton and joined by an elongated synaptic junction to parallel fibers. Some parallel fiber boutons were greatly enlarged and capped numerous profiles of spines. This study shows that formation of new pre- and postsynaptic sites takes precedence over reoccupation of original contacts and that multiple synapses on individual spines are being eliminated to give rise to single contacts with boutons. This elimination resulted in enlargement of synaptic contact areas between Purkinje cell spines and parallel fibers by taking over postsynaptic sites from some vacated and eliminated boutons.

Robust synaptic plasticity of striatal cells following partial deafferentation.

Partial ablation of the cerebral cortical input to the neostriatum generates a rapid lasting effect on the size of remaining synaptic sites. The neocortex was lesioned in adult rats and the neostriatum was analyzed for effects on remaining spines of principal cells during the period from 2 to 40 days. There was an increase in the size of spine heads, boutons and synaptic contact sites. The spine heads became very complex and a corresponding bouton enlargement was accompanied by an increase in the number of synaptic vesicles. By two days, the average profile length of postsynaptic membrane densities (PSDs) had increased by 25% representing an equivalent 50% increase in synaptic contact area. The number of synaptic sites was reduced on each principal neuron of the lesioned group. Comparison of the number of sites per unit volume to their average contact area revealed a reciprocal relationship indicating a conservation in the total synaptic contact area on each neuron. This effect was consistent for all postsurgical days. The lack of a significant return of synaptic number by 40 days indicates that axonal sprouting is not a major factor in neuronal plasticity in the adult striatum. The rapid increase in the size of spines, boutons and synaptic sites at remaining connections suggests that dendrites are the first to initiate the plasticity response in adult neurons through postsynaptic attachments and their corresponding receptor structure. The underlying mechanism of this plasticity may be through a conservation of macromolecules forming postsynaptic membrane specializations on target neurons. Remaining axons appear to follow the dendritic response with a plasticity generating presynaptic appositional specializations to match the contact area of the postsynaptic site.

Developmental factors related to abnormal cerebellar foliation induced by methylazoxymethanol acetate (MAM).

Quantitation of mid-sagittal sections of the molecular layer, and both the external and internal granular layers between control and methylazoxymethanol acetate (MAM)-treated rats, at various stages of cerebellar development revealed a much smaller area of these layers in sagittal profile, however, the fiber core was not significantly affected by the drug. The expansion of the pial surface length was parallel to the length of the Purkinje cell layer, although comparison of a fissure index revealed hypofissuration in the experimental group. In histological examination, there was perforation, patching, and agenesis of the external granular layer. Mushroom expansion of the external granular layer occurred at patches producing a gyrating folial pattern rather than parallel ones. The number of lobules and their basic pattern remained normal. We conclude that the deficits in the external granular layer interrupted the growth force that produces the normal rostrocaudal organization of parallel coronal foliation and this resulted in shallow periodic fissuration along the sagittal extent. Fissurations forming lobules arose largely independent of the external granular layer by directed expansion of the central fiber core while normal parallel foliation is an elaboration of the lobular surface controlled by growth forces defined by both distribution of the external granular layer and the underlying fiber core with associated Purkinje cells.

Transient c-fos expression and dendritic spine plasticity in hippocampal granule cells.

A transient expression of Fos protein occurs in nuclei of partially deafferented dentate granule cells within 1 h of transecting perforant path and fornix inputs. This was followed within 24 h by widening of spine necks, elaboration of the spine apparatus, and appearance of coated vesicles, multivesicular bodies, and ribosomes in remaining spines. This study supports the hypothesis that immediate early genes such as c-fos activate late response genes for generating building blocks of plasticity in partially deafferented neurons of adult rats.