Phosphorylation of the presynaptic protein B-50 (GAP-43) is increased during electrically induced long-term potentiation.

The protein B-50 (F1, GAP-43) is a presynaptic-specific substrate of protein kinase C, functionally related to neurotransmitter release. An increase in phosphorylation of this protein has been proposed as a molecular mechanism underlying long-term potentiation (LTP). B-50 phosphorylation measured by quantitative immunoprecipitation in rat hippocampal slices incubated in the presence of radiolabeled inorganic phosphate was increased for at least 1 hr after the induction of LTP in the CA1 region. No significant changes in B-50 phosphorylation were observed in untetanized slices stimulated at low frequency. The direct demonstration of an increased phosphorylation of the protein B-50 during LTP is consistent with the hypothesis that presynaptic mechanisms contribute to maintenance of LTP.

Dendritic spine loss in hippocampus of aged rats. Effect of brain phosphatidylserine administration.

Dendritic spine density of pyramidal cells in region CA1 of the hippocampus has been evaluated in young (3 months), old (27 months) and old phosphatidylserine (BC-PS)-treated rats. BC-PS (50 mg/kg, suspended in tap water) was administered daily, starting at the age of 3 months until 27 months. Spine density was analyzed on Golgi-stained pyramidal neurons by a computerized analysis system. In 27-month-old rats, spine density showed with respect to 3-month-old animals, a significant decrease in both basal and apical dendrites (p less than 0.01; one-way ANOVA), with a mean loss of 12.11% in the basal dendrites and of 10.64% in the apical ones. In 27-month-old rats treated with BC-PS, values of spine density were not statistically different when compared to those of 3-month-old animals. The mechanisms underlying the beneficial effect of BC-PS treatment on neuronal connectivity might be explained on the basis of its pharmacological actions on neuronal membranes [9], neurotransmission [43] and/or interaction with NGF [7].

B-50/GAP-43 phosphorylation in hippocampal slices from aged rats: effects of phosphatidylserine administration.

Phosphorylation of the presynaptic protein B-50/GAP-43, a substrate of protein kinase C (PKC), has been implicated in neuronal mechanisms related to learning and memory. We evaluated both basal (5 mM KCl) and stimulated (30 mM KCl) B-50/GAP-43 phosphorylation in 32P-prelabeled hippocampal slices obtained from adult and senescent male Sprague-Dawley rats. The in situ B-50/GAP-43 phosphorylation was assayed by quantitative immunoprecipitation. There was no age-related difference in B-50/GAP-43 basal phosphorylation. However, B-50/GAP-43 phosphorylation in depolarized slices from aged rats was significantly decreased relative to that of adult animals. Aged rats were treated with either tris buffer or sonicated suspension of phosphatidylserine (PS) in tris buffer (15 mg/kg IP for 7 and 17 days). PS did not affect basal and high K(+)-induced B-50/GAP-43 phosphorylation in the 7-day treatment. However, after 17 days, PS restored the K(+)-induced B-50/GAP-43 phosphorylation. It is proposed that repeated PS administrations might be beneficial to the age-induced deterioration of endogenous B-50/GAP-43 phosphorylation by acting on Ca++ homeostatic mechanisms and/or PKC.

Unipolar brush cell axons form a large system of intrinsic mossy fibers in the postnatal vestibulocerebellum.

The unipolar brush cells (UBCs), a class of neurons recently identified in the granular layer of the vestibulocerebellum, receive excitatory synaptic input from mossy fibers (MFs) in the form of a giant glutamatergic synapse. UBCs are provided with axons that bear synaptic endings situated at the center of glomeruli, similar to cerebellar MF afferents. A single MF stimulus evokes a prolonged train of action potentials in the UBC (Rossi et al., 1995), which is presumably distributed to postsynaptic targets. Knowledge of the synaptic connections of UBC axons is essential to define the role of these cells in the integration of vestibular signals in the cerebellar circuitry. To evaluate these connections, the nodulus (folium X) was isolated from vermal slices of postnatal day 8 mice, cultured for 2-4 or 15-30 days in vitro, and studied by electron and fluorescence microscopy. The peak of degeneration of extrinsic MF terminals, which have been severed from the parent cell bodies, was observed at 2 days in vitro (DIV). Quantification of degenerating and nondegenerating (e.g., intrinsic) MF terminals indicated that about half of the MF terminals were provided by local UBC axons synapsing on dendrites of granule cells and other UBCs. The proportion of nondegenerating vs. degenerating MF terminals terminating on UBCs also indicated that approximately two-thirds of the intrinsic MFs are involved in UBC-UBC connections. In long-term cultures, the granular layer appeared well preserved and the UBC axons formed an extensive system of MF collaterals. It is suggested that UBCs may act by spatially amplifying vestibular inputs carried by extrinsic MFs.

Compartmental organization of Purkinje cells in the mature and developing mouse cerebellum as revealed by an olfactory marker protein-lacZ transgene.

In a line of transgenic mice (HpY-1), the pattern of expression of an olfactory marker protein (OMP)-lacZ fusion gene was analyzed in the cerebellum, where, in adult mice, OMP-lacZ was expressed primarily in Purkinje cells (PCs) of the posterior lobe. The transgene-expressing PCs were organized in parasagittal bands, with a boundary of expression roughly corresponding to the primary fissure that separates the cerebellum into anterior and posterior compartments. The regional expression of the lacZ gene was also analyzed during embryonic and postnatal development of the cerebellum. Within the cerebellum-isthmus region, transgene expression first was detected at embryonic day 13.5 (E13.5) in a cluster of postmitotic cells. By E14.5, lacZ was also expressed by a subpopulation of migrating PCs in the postisthmal and lateral cerebellar primordium, and, by E16.5, transgene-positive PCs formed caudally four sagittal bands symmetric to the medial embryonic fissure. The caudal pattern was retained in postnatal cerebella, where, by postnatal day 0 (P0), transgene-positive PCs in vermal lobules VIII and IX appeared to be organized in two prominent parasagittal compartments on either side of a negative midline band. In early postnatal animals, the transgene was expressed transiently in the anterior lobe vermis. Hence, from P5 onward, transgene expression appeared mostly restricted to the posterior lobe, where it followed a caudal-to-rostral gradient. In the paraflocculus, transgene-expressing PCs were confined to the rostrodorsal portion. The results indicate that the anterior and posterior cerebellar lobes are regulated by distinct ontogenetic programs, and PCs of functionally distinct cerebellar regions express the transgene differentially. Furthermore, the data suggest that ectopic expression of OMP-lacZ in the cerebellum is under the control of regulatory elements that provide positional information for the regional specification of PC subsets.

Cholecystokinin-immunoreactive cells form symmetrical synaptic contacts with pyramidal and nonpyramidal neurons in the hippocampus.

The ultrastructural features and synaptic relationships of cholecystokinin (CCK)-immunoreactive cells of rat and cat hippocampus were studied using the unlabeled antibody immunoperoxidase technique and correlated light and electron microscopy. CCK-positive perikarya of variable shape and size were distributed in all layers and were particularly concentrated in stratum pyramidale and radiatum: the CCK-immunoreactive neurons were nonpyramidal in shape and the three most common types had the morphological features of tufted, bipolar, and multipolar cells. Electron microscopic examination revealed that all the CCK-positive boutons established symmetrical (Gray's type II) synaptic contacts with perikarya and dendrites of pyramidal and nonpyramidal neurons. The origin of some of the boutons was established by tracing fine collaterals that arose from the main axon of two CCK-immunostained cells and terminated in the stratum pyramidale; these collaterals were then examined in the electron microscope. The axon of one such neuron exhibited a course parallel to the pyramidal layer and formed pericellular nets of synaptic boutons upon the perikarya of pyramidal neurons. This pattern of axonal arborization is very similar to that of some of the basket cells, previously suggested to be the anatomical correlate for pyramidal cell inhibition. Typical dendrites of pyramidal cells also received symmetrical synaptic contacts from CCK-immunoreactive boutons, and some of these boutons could be shown to originate from a local neuron in stratum radiatum. Many CCK-immunoreactive cells received CCK-labeled boutons upon their soma and dendritic shafts. Synaptic relationship, established by multiple "en passant" boutons, was observed between CCK-positive interneurons of the stratum lacunosum-moleculare and radiatum. The soma and dendrites of the CCK-immunostained neurons also received symmetrical and asymmetrical synapses from nonimmunoreactive boutons. These results indicate that the CCK-immunoreactive neurons participate in complex local synaptic interactions in the hippocampus.

Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex.

Unipolar brush cells (UBCs) of the mammalian vestibulocerebellum receive mossy fiber projections primarily from the vestibular ganglion and vestibular nuclei. Recently, the axons of UBCs have been shown to generate an extensive system of cortex-intrinsic mossy fibers, which resemble traditional cerebellar mossy fiber afferents and synapse with granule cell dendrites and other UBCs. However, the neurotransmitter used by the UBC axon is still unknown. In this study, we used long-term organotypic slice cultures of the isolated nodulus (lobule X) from postnatal day 8 mouse cerebella to identify the neurotransmitter and receptors at synapses of the UBC axon terminals, relying on the notion that, in these cultures, all of the cortex-extrinsic fibers had degenerated during the first few days in vitro. Quantification of glutamate immunogold labeling showed that the UBC axon terminals have the same high gold-particle density as the glutamatergic parallel fiber varicosities. Furthermore, UBCs identified by calretinin immunoreactivity expressed the glutamate receptor subunits GluR2/3, NMDAR1, and mGluR2/3, like they do in the mature mouse cerebellum in situ. Evoked excitatory postsynaptic currents (EPSCs), spontaneous EPSCs, and burst discharges were demonstrated in UBCs and granule cells by patch-clamp recording. Both the evoked and spontaneous EPSCs were blocked by ionotropic glutamate receptor antagonists CNQX and D-AP5. We conclude that neurotransmission at the UBC axon terminals is glutamatergic. Thus, UBCs provide a powerful network of feedforward excitation within the granular layer, which may amplify vestibular signals and synchronize activity in clusters of functionally related granule cells which project vertically to patches of Purkinje cells.

Effects of phosphatidylserine in age-associated memory impairment.

We treated 149 patients meeting criteria for age-associated memory impairment (AAMI) for 12 weeks with a formulation of phosphatidylserine (100 mg BC-PS tid) or placebo. Patients treated with the drug improved relative to those treated with placebo on performance tests related to learning and memory tasks of daily life. Analysis of clinical subgroups suggested that persons within the sample who performed at a relatively low level prior to treatment were most likely to respond to BC-PS. Within this subgroup, there was improvement on both computerized and standard neuropsychological performance tests, and also on clinical global ratings of improvement. The results suggest that the compound may be a promising candidate for treating memory loss in later life.

Vesicular glutamate transporters VGLUT1 and VGLUT2 define two subsets of unipolar brush cells in organotypic cultures of mouse vestibulocerebellum.

Different isoforms of a vesicular glutamate transporter (VGLUT) mediate glutamate uptake into synaptic vesicles of excitatory neurons. There is agreement that the VGLUTs are differentially expressed in brain, and that two isoforms, VGLUT1 and VGLUT2, are localized to excitatory axon terminals in the cerebellar cortex. While granule cells express solely VGLUT1, there is no report about the VGLUT(s) of the unipolar brush cell (UBC), the second type of glutamatergic interneuron residing in the cerebellar granular layer. In the mouse, UBCs are particularly numerous in the uvula (lobule IX) and nodulus (lobule X). These folia contain two distinct subsets of UBCs: one kind expresses the calcium-binding protein calretinin (CR), and the other kind expresses the metabotropic glutamate receptor (mGluR) 1alpha. UBCs give rise to an extensive system of intrinsic mossy fibers (MF), whose terminals innervate granule cells and other UBCs, altogether similar to those formed by the extrinsic MFs. The presence of both extrinsic and intrinsic MFs in the vestibulocerebellum makes it difficult to determine which type of VGLUT is contained in MFs formed by the UBC axons. Hence, the nodulus was isolated from sagittal cerebellar slices from postnatal day 10 mice, and cultured for 15-20 days in vitro. Double immunofluorescence and confocal microscopy showed that mossy terminals of CR-positive (CR(+)) UBCs were immunoreactive for VGLUT1 and VGLUT2, while mossy terminals of mGluR1alpha-positive (mGluR1alpha(+)) UBCs were provided with VGLUT1 only. Moreover, CR(+) dendritic brushes were contacted by mossy terminals provided with both transporters, while mGluR1alpha(+) dendritic brushes were contacted by mossy terminals immunopositive for VGLUT1 and immunonegative for VGLUT2. These data indicate that the two UBC subsets use different modalities of vesicular glutamate storage and form separate networks. We consider it possible that expressions of CR with VGLUT1/VGLUT2 and mGluR1alpha(+) with VGLUT1 in the two subsets of vestibulocerebellar UBCs are determined by specific vestibular inputs, carried by groups of primary and/or secondary vestibular afferents.

Muscle reinnervation--II. Sprouting, synapse formation and repression.

Extensor digitorum longus muscle is reinnervated by the regenerating neurites at the end-plate region; as soon as the contact is made, the rate of neurite elongation inside the cleft decreases about 1000-fold while interfibre growth and sprout formation proceed unchanged. Polyinnervation reaches the maximum level 7-10 days after reinnervation, then synaptic repression begins. The elimination of redundant innervation takes place when the biophysical properties of the muscle are again normal. There is no sign of either phagocytosis or degeneration, therefore the process of synaptic repression is probably due to retraction, as neurites do when in culture. The role of Schwann cells and nerve sheath in the process of maintenance is suggested.

Cerebellar input to magnocellular neurons in the red nucleus of the mouse: synaptic analysis in horizontal brain slices incorporating cerebello-rubral pathways.

We studied the synaptic input from the nucleus interpositus of the cerebellum to the magnocellular division of the red nucleus (RNm) in the mouse using combined electrophysiological and neuroanatomical methods. Whole-cell patch-clamp recordings were made from brain slices (125-150 microm) cut in a horizontal plane oriented to pass through both red nucleus and nucleus interpositus. Large cells that were visually selected and patched were injected with Lucifer Yellow and identified as RNm neurons. Using anterograde tracing from nucleus interpositus in vitro, we examined the course of interposito-rubral axons which are dispersed in the superior cerebellar peduncle. In vitro monosynaptic responses in RNm were elicited by an electrode array placed contralaterally in this pathway but near the midline. Mixed excitatory post-synaptic potentials (EPSPs)/inhibitory post-synaptic potentials (IPSPs) were observed in 48 RNm neurons. Excitatory components of the evoked potentials were studied after blocking inhibitory components with picrotoxin (100 microM) and strychnine (5 microM). All RNm neurons examined continued to show monosynaptic EPSPs after non-N-methyl-D-aspartate (NMDA) glutamate receptor components were blocked with 10 microM 6,7-dinitroquinoxaline-2,3-dione or 5 microM 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX; n=12). The residual potentials were identified as NMDA receptor components since they (i) were blocked by the addition of the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (APV), (ii) were voltage-dependent, and (iii) were enhanced by Mg(2+) removal. Inhibitory components of the evoked potentials were studied after blocking excitatory components with NBQX and APV. Under these conditions, all RNm neurons studied continued to show IPSPs. Blockade of GABA(A) receptors reduced but did not eliminate the IPSPs. These were eliminated when GABA(A) receptor blockade was combined with strychnine to eliminate glycine components of the IPSPs. Thus, IPSPs evoked by midline stimulation of the superior cerebellar peduncle, while blocking alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA receptors, raise the possibility of direct inhibitory inputs to RNm from the cerebellum. In summary we propose that the special properties of the NMDA receptor components are considered important for the generation of RNm motor commands: their slow time course will contribute a steady driving force for sustained discharge and their voltage dependency will facilitate abrupt transitions from a resting state of quiescence to an active state of intense motor command generation.

A new type of specific interneuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells.

By means of Golgi staining and gold-toning, we have found an interneuron in the pyramidal cell layer of the hippocampus which forms synapses exclusively on the axon initial segments of pyramidal neurons. An individual initial segment receives up to 30 symmetrical synapses from one axo-axonic cell. Each axo-axonic cell is in synaptic contact with the axon initial segments of several hundred pyramidal neurons. The interneuron is thus ideally situated to synchronize the output of a large population of pyramidal cells and so might be involved in the generation of rhythmic activity and in epileptogenesis.

T system in ascidian muscle: organization of the sarcotubular system in the caudal muscle cells of Botryllus schlosseri tadpole larvae.

The organization of the sarcotubular system has been examined in the caudal muscle cells of the ascidian. Botryllus schlosseri. At variance with striated muscle of other protochordates. Botryllus muscle cells are endowed with a well-developed T system, which has a peculiar laminar structure. The thin T laminae are in continuity with the plasma membrane and extend longitudinally in the intermyofibrillar spaces. At the level of the I-band the T laminae are focally associated with SR cisternae in dyad junctions similar to those observed in invertebrate muscles. These findings are discussed in relation to the origin of the sarcotubular system in vertebrate muscle.

Spontaneous isolated lipoma of the choroid plexus in the baboon.

A case of symptomless, solitary lipoma of the choroid plexus is described. The tumour was found in the left lateral ventricle of an adult female baboon (Papio papio) in the course of post-mortem examination. Routine histological investigation showed that the tumour was composed exclusively of characteristic adipose cells with scarce collagen septa and without other hamartoma-like constituents, such as glial cells, neurons, cartilage or muscle fibres. The tumour mass was lined by a typical single layer of cuboidal cells; no calcifications were observed either inside the tumour or in the adjacent periventricular regions. This case is reported in view of the rarity of such tumours of the choroid plexus in man and animals, and to throw light on their possible origin.