Josef Špaček

Dendritic spine pathology: Cause or consequence of neurological disorders?

Altered dendritic spines are characteristic of traumatized or diseased brain. Two general categories of spine pathology can be distinguished: pathologies of distribution and pathologies of ultrastructure. Pathologies of spine distribution affect many spines along the dendrites of a neuron and include altered spine numbers, distorted spine shapes, and abnormal loci of spine origin on the neuron. Pathologies of spine ultrastructure involve distortion of subcellular organelles within dendritic spines. Spine distributions are altered on mature neurons following traumatic lesions, and in progressive neurodegeneration involving substantial neuronal loss such as in Alzheimers disease and in Creutzfeldt-Jakob disease. Similarly, spine distributions are altered in the developing brain following malnutrition, alcohol or toxin exposure, infection, and in a large number of genetic disorders that result in mental retardation, such as Downs and fragile-X syndromes. An important question is whether altered dendritic spines are the intrinsic cause of the accompanying neurological disturbances. The data suggest that many categories of spine pathology may result not from intrinsic pathologies of the spiny neurons, but from a compensatory response of these neurons to the loss of excitatory input to dendritic spines. More detailed studies are needed to determine the cause of spine pathology in most disorders and relationship between spine pathology and cognitive deficits.

Three-Dimensional analysis of dendritic spines

SummaryA total of 212 dendritic spines (108 from the visual and 104 from cerebellar cortices of the mouse) were analyzed in serial sections. Dendritic spines (DS) and synaptic active zones (SAZ) were classified according to their shape, and the following quantitative data were measured: DS stalk and bulb diameters, DS length and volume, number of cisterns of the spine apparatus, DS and SAZ surface areas and their mutual proportions. Quantitative relationships between the spine apparatus and the size of DS and SAZ, between the volume and surface area of DS and between the size of DS and the size of SAZ were studied. Thin, mushroom-shaped and stubby DS with simple (circular or oval), complex (perforated, annulate or horseshoe-shaped) and multifocal SAZ were found on terminal branches of pyramidal cell apical dendrites and club-shaped DS with simple (circular or oval) SAZ on spiny branchlets of Purkinje cells.Statistically significant differences were found between all values measured on various DS types in the visual cortex. Linear dependencies of the DS surface area on DS volume and of the SAZ surface area on the DS surface area were established. Only a limited area of DS plasma membrane (7–10%) was occupied by SAZ. This finding indicates a possible functional importance of the SAZ/DS (and possibly also of the total SAZ/total postsynaptic membrane) surface ratio.

THREE-DIMENSIONAL ORGANIZATION OF CELL ADHESION JUNCTIONS AT SYNAPSES AND DENDRITIC SPINES IN AREA CA1 OF THE RAT HIPPOCAMPUS

Recent work has emphasized the role of adhesion molecules in synaptic plasticity, including long‐term potentiation in the hippocampus. Such adhesion molecules are concentrated in junctions that are characterized by dense thickenings on both sides of the junction and are called puncta adhaerentia (PA). Reconstruction from serial electron microscopy was used to determine the location and size of PA in the stratum radiatum of hippocampal area CA1, where many of the previous functional studies have been performed. PAs were found at the edges of synapses on 33% of dendritic spines. The areas occupied by PA were variable across different types of synapses, occupying 0.010 ± 0.005 μm2 at macular synapses and 0.034 ± 0.031 μm2 at perforated synapses. Another zone, called a vesicle‐free transition zone (VFTZ), was identified. Like the PA, this zone also had no presynaptic vesicles and was located at the edges of synapses; however, unlike the PA, the presynaptic thickening was less than the postsynaptic thickening. Together, 45% of spine synapses had PA and/or VFTZ occupying 23 ± 11% of the total junctional area between axons and spines. PA also occurred at nonsynaptic sites involving neuronal as well as glial elements. Most (64%) of these PAs occurred between nonsynaptic portions of dendritic spines and neighboring astrocytic processes. Smooth endoplasmic reticulum was often apposed to one or both sides of the synaptic and the nonsynaptic PA. These findings provide further data as a structural basis for understanding the roles of cell adhesion junctions in hippocampal synaptic function and plasticity. J. Comp. Neurol. 393:58–68, 1998.

Trans-Endocytosis via Spinules in Adult Rat Hippocampus

Locations of a distinctive mode of trans-endocytosis involving dendrites, axons, and glia were quantified through serial section electron microscopy. Short vesicular or long vermiform evaginations emerged from dendrites and axons and were engulfed by presynaptic or neighboring axons, astrocytes, and, surprisingly, a growth cone to form double-membrane structures called spinules. In total, 254 spinules were evaluated in 326 μm3 of stratum radiatum in area CA1 of mature rat hippocampus. Spinules emerged from spine heads (62%), necks (24%), axons (13%), dendritic shafts (1%), or nonsynaptic protrusions (<1%) and invaginated into axons (∼90%), astrocytic processes (∼8%), or a growth cone (∼1%). Coated pits occurred on the engulfing membrane at the tips of most spinules (69%), and double-membrane structures occurred freely in axonal and astrocytic cytoplasm, suggesting trans-endocytosis. Spinule locations differed among mushroom and thin spines. For mushroom spines, most (84%) of the spinules were engulfed by presynaptic axons, 16% by neighboring axons, and none by astrocytic processes. At thin spines, only 17% of the spinules were engulfed by presynaptic axons, whereas 67% were engulfed by neighboring axons and 14% by astrocytic processes. Spinules engulfed by astrocytic processes support the growing evidence that perisynaptic glia interact directly with synapses at least on thin spines. Spinules with neighboring axons may provide a mechanism for synaptic competition in the mature brain. Trans-endocytosis of spinules by presynaptic axons suggest retrograde signaling or coordinated remodeling of presynaptic and postsynaptic membranes to remove transient perforations and assemble the postsynaptic density of large synapses on mushroom spines.

Detailed evaluation of 2959 allogeneic and xenogeneic dense connective tissue grafts (fascia lata, pericardium, and dura mater) used in the course of 20 years for duraplasty in neurosurgery

SummarySurgical experience with 2959 allogeneic and xenogeneic dense connective tissue grafts (1767 of fascia lata, 909 of pericardium, and 283 of dura mater), used in 2665 neurosurgical operations performed in the course of 20 years (1976 to 1995) is reported.Duraplasty using either allogeneic or xenogeneic grafts has had a similar, and favourable clinical outcome. Nevertheless, the pliable deep frozen fascia lata grafts, which could be used in any location, have been reserved for sella turcica plugging, anterior cranial base plasty, aneurysmal wrapping, and surgery of lipomyelomeningocele. Pericardium and dura mater grafts were in the majority of cases used over the brain convexity and posterior cranial fossa. Ovine pericardium proved to be superior to bovine and allogeneic pericardia because of its workability, flexibility, reduced thickness, and better transparency.Postsurgical complications occurred in 7.3%, and they were: 1) cerebrospinal fluid fistulas in 2.8%; 2) meningites in 2.3% (aseptic 1.4%, bacterial 0.8%, and tumoural 0.1% meningites); 3) pseudomeningoceles in 2.2%; 4) wound infections in 0.6%; 5) malresorptive hydrocephalus in 0.5%; and 6) adhesions to nerve tissue in 0.5%. The majority of complications healed without surgery. Forty-eight grafts (1.6%) failed to fulfil the requirements of the surgeon, and 46 of them were re-operated upon. Though another thirty-nine grafts healed successfully, 39 shunts (1.5%) had to be performed for malresorptive hydrocephalus (0.9%), and/or for a big pseudomeningocele (0.6%). So, the pure complication rate in 2665 duraplasties was 3.1%.The complex evaluation of the allogeneic and xenogeneic grafts (fascia, pericardium, and dura mater), used for duraplasty in neuro-surgery during the last 20 years proved them, as remarkably good, with a success rates of 96.9%.

Extracellular sheets and tunnels modulate glutamate diffusion in hippocampal neuropil.

Although the extracellular space in the neuropil of the brain is an important channel for volume communication between cells and has other important functions, its morphology on the micron scale has not been analyzed quantitatively owing to experimental limitations. We used manual and computational techniques to reconstruct the 3D geometry of 180 μm3 of rat CA1 hippocampal neuropil from serial electron microscopy and corrected for tissue shrinkage to reflect the in vivo state. The reconstruction revealed an interconnected network of 40–80 nm diameter tunnels, formed at the junction of three or more cellular processes, spanned by sheets between pairs of cell surfaces with 10–40 nm width. The tunnels tended to occur around synapses and axons, and the sheets were enriched around astrocytes. Monte Carlo simulations of diffusion within the reconstructed neuropil demonstrate that the rate of diffusion of neurotransmitter and other small molecules was slower in sheets than in tunnels. Thus, the non‐uniformity found in the extracellular space may have specialized functions for signaling (sheets) and volume transmission (tunnels). J. Comp. Neurol. 521:448–464, 2013.

Relationships between synaptic junctions, puncta adhaerentia and the spine apparatus at neocortical axo-spinous synapses. A serial section study.

SummaryA total of 80 cotical axo-spinous synaptic junctions were reconstructed from serial sections and about 100,000 were analyzed in single sections. Special attention was paid to the occurrence of puncta adhaerentia associated with perforated, annulate or horseshoe-shaped (=complex) synaptic junctions and to the presence and proximity of the spine apparatus. Further evidence is presented that the spine apparatus has no relationship to simple (round or oval) synaptic specializations, but is present in association with at least 91% of complex junctions. The spine apparatus points towards the punctum adhaerens which in at least 71% of cases seems to be an integral part of the complex synapse. Direct continuity was found between the dense material of the spine apparatus and the punctum adhaerens. It is suggested, in accordance with other recent studies, that expansion of the synaptic active zone occurs by the addition and transformation of puncta adhaerentia. The spine apparatus may participate in this dynamic process as a possible donor of specific postsynaptic proteins.

Three-dimensional reconstructions of astroglia and oligodendroglia cells

SummaryThree-dimensional reconstructions of portions of astrocyte and oligodendrocyte bodies and processes are presented. The reconstructions have been made from series of ultrathin sections, and have been compared with glial cells in Golgi-impregnation preparations.The surface of the cell bodies and processes of protoplasmic astrocytes is uneven, wrinkled, and appears to be very well adapted to the surrounding structures. The plasma membrane of protoplasmic astrocytes intervenes between nerve processes, shows a tendency to surround individual axons or their bundles, and forms sheaths for some axodendritic synapses. The surface of the bodies and processes of fibrous astrocytes is much smoother.Oligodendrocytes too, have a smooth surface, and their fine processes do not give rise to expansions similar to those of astrocytes. Reconstruction of the endoplasmic reticulum of a nerve cell associated with a subsurface cistern is presented.It is concluded that three-dimensional reconstruction of glial cells at the electron microscopic level supports the result obtained by the Golgi impregnation.

Dynamics of the Golgi method: a time-lapse study of the early stages of impregnation in single sections

SummaryThis paper describes the early stages of impregnation by the Golgi method. Sections of aldehyde-fixed and potassium dichromate-treated cerebral cortex were mounted on glass slides and cover slipped. The dichromate solution was replaced by silver nitrate solution and events in the section were followed and recorded by time lapse microphotography and video recording until stopped by replacement of silver nitrate solution by glycerol. The sections were subsequently prepared for electron microscopy (EM) study.In sections about 2×2 mm and 100 μm thick a fine, dark granular precipitate formed at the edges within the first minutes of exposure to silver nitrate and a wave of brownish colouration spread to a depth of about 0.3 mm. After approximately 7 min, shrub-like focal precipitates (nucleation centres) appeared in the sections. From these nucleation centres (but also from the section edges) thread-like ‘outgrowths’, usually identified as dendrites, spread into somata. Sometimes impregnation began within the soma and spread into dendrites. The rate of impregnation (i.e., of silver chromate deposition within dendrites) was typically 1–7 μm mins-1, faster in the earlier stages (up to 3 μm ss-1) and very slow after 30 min, by which time many neurons were more or less fully impregnated.The dimensions of the section, the width of an agar frame around the sections, and the frequency with which the silver nitrate in the sections was replenished all affected the extent and time course of the impregnation.By EM the earliest intracellular deposits consisted of tubulolamellar formations which did not cross plasma or endocellular membrane boundaries and which contained irregularly shaped and scattered granules, initially about 10 nm in diameter. The latter progressively enlarged and coalesced as the tubulolamellar formations extended, eventually to fill the cross-sectional area of neuronal processes and cell bodies.These observations shed light on why so few neurons become impregnated with the Golgi method. Impregnation occurred only in those cells a part of which was within a nucleation centre.

Posterior cranial fossa surgery in 454 children Comparison of results obtained in pre-CT and CT era and after various types of management of dura mater

Abstract At the Department of Neurosurgery, Hradec Králové, 454 children (aged under 18 years) were operated on for posterior cranial fossa lesions in a period of 49 years (1948–1996). The majority (402) had tumours: cerebellar astrocytomas 149 (37.1%), medulloblastomas 139 (34.6%), brain stem gliomas 46 (11.4%), ependymomas 28 (7.0%), and others 40 (9.9%). Postoperative mortality was compared for the pre-CT era (1948–1977) and the CT era (1978–1996): astrocytomas (8.6%:4.7%), medulloblastomas (14.9%:2.9%), brain stem gliomas (21.7%:19.0%), ependymomas (18.2%:6.3%), and others (40.0%:7.4%). The initially high mortality was due to insufficient intracranial decompression, brain oedema and disturbances of cerebrospinal fluid circulation. Obstructive hydrocephalus was treated in 53 children with tumours and 25 with aqueduct stenoses, by Torkildsens drainage in 5.5%, and/or by catheterisation of aqueduct in 12.3%. The main postoperative complications of medial posterior fossa surgery in 429 children operated on were: pseudomeningocele (12.3%), active hydrocephalus (6.2%) and CSF leakage (4.6%). Only 8.2% had shunts placed for these complications. We presume that this low percentage of shunts used results from a frequent use of duraplasties and drains installed at the primary operation. The dura mater was initially (1948–1954) left open (50 cases), and later (1955–1958) also sutured (37 cases), and from 1958, onward, and especially from 1961, reconstructed by a medial approach by means of various grafts (377 cases). In all, duraplasty was performed in 81.6% of cases. The grafts used for dura mater reconstruction were prepared from autogeneic (1.6%), allogeneic (72.3%), xenogeneic (24.8%), or synthetic (1.3%) material. They were successful in 99.2% of cases (all materials). Our own suture technique for posterior fossa duraplasty is presented.