Aldehyde Perfusion Research Articles

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

Apocrine sweat glands in the circumanal glands of the dog.

Apocrine sweat glands in the circumanal glands of the dog are not connected morphologically with the lobules of the circumanal glands. However, an apparent functional association has been demonstrated and it is possible that the apocrine sweat glands might serve as excretory ducts for degenerated polyhedral cells of the circumanal glands. In this study, we examined the ultrastructure of the apocrine sweat glands in the circumanal glands of the dog in an effort to define more precisely the relationship between the apocrine sweat glands and the circumanal glands. Paraffin sections stained with azan and sections after immunohistochemical staining with antibodies against actin were examined by light microscopy. Samples fixed by aldehyde perfusion were examined with the electron microscope. Diameters of apocrine sweat glands and height of cells in the secretory epithelium varied considerably. Immunohistochemical staining for actin was weakly positive in the supranuclear regions of secretory cells and very intense in myoepithelial cells. In secretory cells, the endoplasmic reticulum was well-developed. Multivesicular bodies were abundant and were discharged into lumens. Apocrine secretion and exocytosis were observed at luminal surfaces of secretory cells. There were three types of large granule in the cytoplasm: giant mitochondria without cristae; membrane-enclosed globules with or without myelin-like contents; and electron-dense, homogeneous, globular structures. Luminal surfaces were always covered with microvilli, and extensive folding of the cell membrane was found in basal regions. Bundles of actin filaments were dispersed throughout the cytoplasm. In the lumens of apocrine tubules, we observed shed secretory cells with well-preserved normal fine structures. We also noted the differentiation of secretory cells that was due to cell renewal. Apocrine sweat glands in the circumanal glands of the dog appear to be more active than those on the general body surface in terms of apocrine secretion, exocytosis, and the release of multivesicular bodies. Shed secretory cells containing large granules, as well as degenerated polyhedral cells from the circumanal glands, might contribute, to some extent, to the subtle composition of sweat from these apocrine sweat glands.

Interactions between Vasopressin- and Gonadotropin-Releasing-Hormone-Containing Neuroendocrine Neurons in the Monkey Supraoptic Nucleus

Vasopressin (VP) is a hypophysiotropic hormone which is also implicated in the control of gonadotropin-releasing hormone (GnRH) secretion. We examined whether VP- and GnRH-immunoreactive (-IR) elements interact directly in the supraoptic nucleus (SON) of cynomolgus monkeys. Neuroendocrine (NEU) neurons in 4 juveniles were retrogradely labeled from the median eminence with wheat germ agglutinin apohorseradish peroxidase conjugated to gold before aldehyde perfusion. Frontal vibratome sections were immunostained for GnRH with peroxidase-antiperoxidase (PAP) and for VP with 5- or 15-nm gold. Many of the GnRH-IR and more than half the VP-IR cell bodies in the SON were NEU. VP-IR elements formed axodendritic and axosomatic symmetrical synapses with one another. In addition, VP-IR boutons also synapsed with NEU GnRH-IR neurons. Although GnRH axon terminals and dendrites contacted VP-IR dendrites and NEU cell bodies, we were unable to find convincing examples of GnRH/VP synapses through serial sections, perhaps due to the use of PAP-diaminobenzidine as the GnRH (afferent) immunolabel. In summary, our study demonstrates anatomical synapses between VP-IR and other VP and GnRH-IR neurons in the SON, in which postsynaptic VP or GnRH cell bodies were NEU. On the other hand, reciprocal GnRH/VP contacts but no true synapses were seen. However, the results suggest coordinated roles for VP and GnRH in NEU control of gonadotropin secretion. Whether VP itself and/or coexistent neuroeffectors act directly on NEU GnRH secretion remains to be determined. As such, VP neurons could help coordinate suppression of gonadotropins and augmentation of glucocorticoids during the stress response in primates.

Preparation of bone for high‐voltage electron microscopic radioautography

This paper presents a reliable technique for the preparation of radioautographic specimens for study by high-voltage electron microscopy. Tissues are fixed by ventricular perfusion of aldehydes, sectioned, collected on slides, and coated with nuclear track emulsion using a coating machine. Sections are developed after 17 weeks' exposure in either undiluted Microdol-X or diluted D-19 developer, fixed in 25% sodium thiosulfate, and stained through the emulsion with uranyl acetate in absolute methanol and aqueous lead citrate. The technique produces sections of even thickness and staining density with negligible numbers of background grains and non-specific labeling. In this report, the efficacy of this technique for the study of the periosteal surface of demineralized alveolar bone specimens is demonstrated.

Acute and chronic reactions of dental sensory nerve fibers to cavities and desiccation in rat molars.

We have studied the effects of air drying of exposed, acid etched dentin on the sensory innervation of rat molars. In the acute series of experiments, trigeminal nerve fibers were labeled by axonal transport of radioactive protein prior to the dentin exposure and desiccation, the anesthetized rats were fixed by aldehyde perfusion 10 min later, and the teeth were prepared for autoradiography. The results confirmed the hydrodynamic theory by showing outward movement of labeled nerve material in response to dentinal drilling and desiccation. It also showed that some odontoblasts could be separated from the dentinal nerve fibers. In the chronic series, teeth were injured 25 h, 5-7 days, or 21 days prior to fixation and nerves were labeled during the last 24 hours; the surviving vital nerve fibers were evident because of their axonal transport of the radioactive label. In that series, sensory nerve fibers were found to have been lost from areas with newly-formed reparative dentin, or from dentinal tubules that had lost their odontoblasts. In the teeth injured 25 h, 5-7 days, or 21 days earlier, an abnormal nonneuronal labeling occurred 0.2-0.3 mm into injured dentin. Our results are discussed in relation to the hydrodynamic theory, nerve-odontoblast interactions, differences between shallow and deep cavity injuries, altered nerve location in response to pulpal or dentinal injury, and characteristics of the pulp-dentin border.

Double myelination of axons in the sympathetic nervous system of the mouse. II. Mechanisms of formation.

The phenomenon termed 'double myelination', present in sympathetic nerve of normal adult rats and mice, comprises regions of a myelinated axon which are concentrically ensheathed by additional (outer) myelinating Schwann cells. Evidence has been presented that in some instances the outer Schwann cell fails to make contact with an axon, yet its myelin sheath characteristically remains ultrastructurally intact. The present study has sought to identify and analyse configurations intermediate between single and double myelination, in order to determine the mechanism(s) underlying the formation of double ensheathment. Superior cervical ganglia from normal male mice aged 12-24 months were prepared for electron microscopy by systemic aldehyde perfusion. Regions of interest were extensively serial-sectioned for detailed electron microscopical analysis and reconstruction. The earliest evidence for alteration to the expected intimate ensheathment of axons by myelinating Schwann cells involved invasion of supernumerary Schwann cells and their processes at the node of Ranvier, resulting in displacement of the paranodal pockets from axonal contact. Similar paranodal displacement occurred at heminodes as a result of lateral extension and invasion of processes from the adjacent Schwann cell (i.e. the cell investing the unmyelinated domain of the axon). Subsequently, processes of the invading cell extended progressively into internodal regions, located at all times between the plasma membranes of the axon and displaced Schwann cell. The cytoplasmic pockets at the remaining paranode were then subject to invasion. At various stages of displacement myelin formation commenced within the invading cell, representing the first acquisition of double myelin ensheathment in the development of the configuration. Involvement of haematogenous cells in displacement was not detected. There was also evidence consistent with paranodal displacement by adjacent pre-existing myelinating cells, but this additional mechanism appeared minor relative to the involvement of (initially) non-myelinating Schwann cells. We found no evidence for the alternative possibility that Schwann cells could synthesize a myelin sheath around a pre-existing myelinated axon de novo, independent of any direct axonal contact. These results are consistent with the well-established requirement for axonal contact by Schwann cells engaging in initial myelin formation, in the sense that the myelin sheath of the outer cell was synthesized prior to its displacement, and that a myelin sheath was not formed by the invading cell until it had invested the axon in a 1:1 relationship.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of morphine on rat kidney glomerular podocytes: A scanning electron microscopic study

Degenerative kidney changes are associated with heroin use in human addicts, but it is not known whether these changes result from exposure to the opioid or from contaminants in street heroin. In the present study, 4–6-month-old rats each received 1 subcutaneous pellet containing 75 mg of morphine or placebo, followed 3 days later by implantation of 2 additional morphine or placebo pellets. Seven days after implantation of the first pellet, the rats were killed by aldehyde perfusion. The right kidney was excised, and coronal slices were prepared for scanning electron microscopy. Micrographs were taken at 5000X and were scored on the presence of short or long microprojections (a score of ‘1’ indicating few and a score of ‘4’ indicating many). Morphine significantly altered the frequencies of scores for long microprojections, suggesting that morphine treatment increased the number of microprojections on glomerular podocytes. No changes in filtration slits, pedicels, or blebbing (foval enlargements) were noted. The data support the view that kidney degeneration associated with opioid abuse reflects effects of opioids per se, and they are consistent with micro projection changes as a function of altered intracellular cyclic AMP levels.

A method for microscopic studies of cerebral angioarchitecture and vascular- parenchymal relationships, based on the demonstration of ·paravascular’ fluid pathways in the mammalian central nervous system

A new method is described for morphological studies of blood vessels and related cellular elements in the mammalian central nervous system (CNS). The tracer protein, horseradish peroxidase (HRP), in solution, is infused intraventricularly or intracisternally in anesthetized animals over 5–10 min. During this period, HRP in the subarachnoid space enters the perivascular spaces around penetrating arterioles and rapidly permeates the gliovascular basal laminae surrounding capillaries. After fixation by intravascular perfusion of aldehydes, brain sections are incubated with the highly sensitive chromogen, tetramethylbenzidine. Intraparenchymal blood vessels throughout the CNS are vividly demonstrated for light microscopy by HRP reaction product in their perivascular spaces or basal laminae. Correlative ultrastructural investigations of specific blood vessels and related parenchymal elements can be conducted using adjacent sections.

The distribution of filipin-sterol complexes in photoreceptor synaptic membranes.

The polyene antibiotic filipin, which binds to membrane sterols, has been used to investigate the relative distribution of cholesterol at photoreceptor synaptic junctions in the chick retina. Following anesthesia and aldehyde perfusion fixation, the retina is removed and immersed in fixative solution containing the filipin for 36-48 hours. The retinas are then processed for freeze-fracture. Electron microscopy of freeze-fracture replicas demonstrates that the filipin-sterol complexes are not evident between intramembrane particles of the presynaptic and postsynaptic particle arrays that are present at ribbon and basal junctions. In contrast, the synaptic vesicle fusion zone of ribbon junctions contains large numbers of filipin-sterol complexes which are observed merging with the free margins of the presynaptic particle array. There is a scarcity of such complexes, however, around the free margins of basal junction presynaptic particle arrays. These latter sites do not contain a vesicle fusion zone. Particle-poor areas of membrane that surround postsynaptic particles arrays of ribbon and basal junctions also do not contain filipin binding sites. The nonsynaptic membrane of photoreceptor terminals contains large numbers of filipin-sterol complexes, less tightly packed than in the synaptic vesicle fusion zone. Coated vesicle fusion sites in the presynaptic membrane contain groups of intramembrane particles but the filipin-sterol complexes are excluded from these sites. The observations suggest that synaptic membrane domains which interact with cytoskeletal components, such as clathrin and pre- and postsynaptic densities contain less cholesterol than other domains such as the synaptic vesicle fusion zones.

Afferent and efferent pathways of the vibrissal region of primary motor cortex in the mouse.

The afferent and efferent connections of the vibrissal representation within the mouse primary motor cortex (MsI) were identified by using the retrograde transport of horseradish peroxidase (HRP) and the anterograde transport of tritiated amino acids injected into MsI. Following aldehyde perfusion brains were frozen-sectioned at 40 microns and reacted for HRP using the 3-3' diaminobenzidine-cobalt chloride technique of Adams ('77). Alternate HRP reacted sections were processed for autoradiography. HRP-filled pyramidal cell somata and concentrations of developed silver grains above background levels were observed in both the vibrissal area of primary somatosensory cortex (SmI) cortex (i.e., the posteromedial barrel subfield; PMBSF cortex) and in the face region of SmII (area 40). In both regions labeled somata occurred predominantly in cortical layers II-III and V. Autoradiographic label was superimposed over the regions containing labeled somata but exhibited a less distinct laminar organization. A dense reciprocal projection connected the injection site with the homotopic area in contralateral MsI; somata occurred for the most part in layers III and V. Developed silver grains were uniformly dispersed over the area containing labeled cell bodies. HRP-labeled pyramidal somata were noted in contralateral PMBSF cortex, but no silver grains occurred in this region. Reciprocal projections linked MsI cortex with the ipsilateral thalamic nuclei: ventralis pars lateralis (VL) and centralis pars lateralis (CL) and with the zona incerta (ZI). Labeled cell bodies and developed silver grains were more dense in VL than in CL. The ipsilateral striatum and thalamic reticular nucleus (NRT) received afferents from the motor cortex but did not project to it. Thus, the vibrissal area of primary motor cortex is connected with a number of cortical and subcortical structures, each of which has been shown to play a role in motor performance. Identification of the afferent and efferent pathways of MsI cortex will now enable further investigation of the ultrastructural and synaptic organization of the vibrissal area of MsI.

'Free' postsynaptic-like densities in normal adult brain: their occurrence, distribution, structure and association with subsurface cisterns.

Samples of cerebral cortex (parietal and occipital) and thalamic nuclei (ventrobasal, posterolateral, dorsal lateral geniculate) from normal, adult, aldehyde perfusion fixed mice and rats were examined by electron microscopy for the presence of free postsynaptic-like densities (FPSDs). FPSDs are plaques of intracellular paramembranous electron-dense material, ultrastructurally indistinguishable from postsynaptic densities, but not aligned with a presynaptic specialization. In a systematic survey of the neuropil around 6000 neuronal perikarya, 250 FPSDs were encountered. Almost all of these were within dendritic spines and shafts and about 90% of them were apposed by a neuronal perikaryon, the remainder by a dendritic shaft. In every case a subsurface cistern (SSC) was present in the cell body or dendrite apposed to the FPSD, and was flattened along the extent of the FPSD. In none of the material were the FPSDs associated, even remotely, with degenerating elements, suggesting that they are not formed by degeneration of presynaptic boutons. The incidence of FPSD-SSC complexes was higher in thalamus than in cerebral cortex which, together with previous observations indicating their absence from normal cerebellar cortex, suggests significant regional variations in distribution. It is suggested that FPSDs might represent synaptic precursors perhaps induced to form as a response to loss (possibly age-dependent loss) of synaptic contacts on a neuron and that the SSCs are somehow involved in maintaining the FPSDs and/or preparing them for innervation by adjacent axon terminals to form new synaptic contacts.

Fine structure of myelinated mechanical nociceptor endings in cat hairy skin.

High-threshold mechanoreceptors (mechanical nociceptors) with myelinated axons were electrophysiologically identified in hairy skin of the cat as described by Burgess and Perl ('67). Such elements possess receptive fields consisting of a number of punctate areas from which maximal firing can be elicited by intense (skin-damaging) mechanical stimuli. The spots of the receptive field are separated from each other by unresponsive regions, i.e., by skin areas from which responses cannot be evoked by stimuli effective at the spots. Fine steel pins were inserted to bracket closely a number of the spotlike responsive areas for each of several units. After aldehyde perfusion of the animal, osmification of the tissue and embedding in plastic, the marked skin zones were examined in semithin and ultrathin sections at the light and electron microscopic level. Near each delineated area, a thinly myelinated axon was found that could be traced to the papillary layer where it loses its myelin sheath. Unmyelinated axons accompanied by thin Schwann cell processes were then traced and found to penetrate the epidermal basal lamina in one of the papillae. At the epidermal penetration site, the axons contained both clear round, and large, dense core vesicles; at this level, the surrounding Schwann cell cytoplasm exhibited numerous pinocytotic vesicles. The zone of penetration may constitute the receptive apparatus. Some of these axons have been traced within the basal epidermal layer where they become surrounded by keratinocytes, lose their Schwann sheath, and apparently terminate. This overall morphological pattern was consistently present in the demarked areas of focal responsiveness, and was rare in the surrounding skin; this and its difference from other cutaneous neural endings suggest that the intraepidermal axon-Schwann cel complex constitutes the receptive structure for myelinated mechanical nociceptors. It is suggested that such complexes are the sense organs responsible for initiating the sensation of pricking pain produced by localized mechanical injury of the skin.

Subacute reactions to intrathecal amipaque (metrizamide), conray and dimer X: a structural and ultrastructural study.

Adult New Zealand rabbits were injected intrathecally with 200 or 300 mgI/ml of metrizamide, meglumine iocarmate or meglumine iothalamate. They were placed in the Trendelenburg position for 20 min and allowed to survive for 1, 2 or 3 weeks. Sacrifice was by intraarterial perfusion of aldehydes under anesthesia followed by preparation of brain and major venous sinuses for light and electron microscopy. Major findings were subpial edema and inflammatory reaction of superficial brain, leptomeninges, subarachnoid space and arachnoid granulations. They were most severe after iothalamate and milder after metrizamide. The severity decreased with time. It is of significance that severe reactions occurred after 300 mgI/ml concentration of metrizamide.

Method for morphophysiological study of specific pial microvessels.

A method is described whereby specific segments of the pial vasculature of cats can be both physiologically and morphologically studied. This is accomplished by the insertion of a cranial window through which specific pial vessels can be visualized, illustrated, numbered in a simple sketch, and physiologically evaluated under both control and experimental conditions. Following these procedures, perfusion of aldehydes is initiated and the pia and associated vasculature are stripped from the cortical mantle. The thin velum of pia so obtained permits the direct visualization of the pial vasculature whose unchanged configuration can be matched to the appropriate previously sketched vessels whose individual physiological status is known. In turn these vessels are divided into alternate segments and are prepared for either scanning or transmission electron microscopy. The advantages of this methodology is exemplified by the fact that it provides the physiological status and morphological characteristics of the same microvascular segment. Although designed for the pial vasculature, this protocol may also prove of use in the study of other superficially coursing microvascular beds.

Light and electron microscopic structure of Golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure).

After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguished by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pices of tissue left in a 1% K2Cr2O7 solution for 13 h before impregnation in a 0.5% AgNO3 solution for 2 h. Thick sections (30-50 micron) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.

Some observations on the binding patterns of α-bungarotoxin in the central nervous system of the rat

Patterns of alpha-bungarotoxin (alphaBuTX) binding within the brain of the rat have been studied following one of the two procedures: (1) the intraventricular injection of 125I-labeled toxin followed by a survival period of 1--8 days before aldehyde perfusion, or (2) the incubation of fresh cryostat sections of brain tissue in dilute solutions of radioactive toxin. Appropriate controls with nicotine, curare, atropine and native alphaBuTX established that specific nature of the binding. The principal observations were that toxin binding sites are predominantly associated with central areas of the brain in direct receipt of sensory inputs (the main and accessory olfactory bulbs, superior colliculus, ventral lateral geniculate nucleus, cochlear nuclei, the substantia gelatinosa of the spinal cord and spinal trigeminal nucleus, the principal sensory nucleus of the trigeminal, and the dorsal column nuclei) and with limbic areas of the brain (hippocampus, amygdala, olfactory tubercle, medial mammillary nucleus, and the dorsal tegmental nucleus of Gudden). Toxin was not found to bind to cranial motor nuclei with the exception of the dorsal motor nucleus of the vagus and the nucleus ambiguus. The discrete distribution of clusters of silver grains within the granule layer of cerebellar folia I, IX, and X is described as well as the heavy labeling of the inferior and accessory olivary nuclei. In many areas of the brain silver grains were found to overlie cell bodies. It is suggested that this may reflect the presence of both membrane-bound toxin and internalized ligand following initial binding to a membrane receptor site. An attempt was made to correlate the localization of toxin binding sites with the terminal distributions of previously described cholinergic pathways. There appears to be a reasonably good agreement between the distribution of toxin receptors and proposed sites of cholinergic transmission within the hippocampus, interpeduncular nucleus and cerebellum. In most other cases however, lack of data precluded such correlations. The anatomical relationship of alphaBuTX binding activity and neurotransmitters other than acetylcholine is also considered.

Axosomatic synapses in the spinal cord of the shark

Previous published reports indicated that the shark central nervous system is atypical of other vertebrates with regard to its lack of internuncial neurons and the rare occurrence of axosomatic synapses. This study was undertaken to examine the general organization of the shark spinal cord and to examine the ventral horn with respect to the synaptic arrangement on motor horn neurons. Spinal cord segments from five juvenile Atlantic nurse sharks ( Ginglymostoma cirratum) were removed at the pectoral fin level after mixed aldehyde perfusion and were subsequently processed for light and electron microscopy. Light microscopy of the spinal cord in transverse section revealed an oval cord with relatively discrete dorsal and ventral gray horns connected only by finger-like projections of gray matter. The absence of a definitive intermediate gray zone was the most distinctive structural peculiarity observed. The ventral motor neurons exhibited characteristic neuronal cyto-architecture, and electron microscopy revealed abundant synapses on both ventral horn cell perikarya and primary dendrites. These observations indicate that the shark spinal cord is prototypic of “higher” vertebrate species.

The effect of aldehyde fixation on selected substrates for energy metabolism and amino acids in mouse brain.

The effect of aldehyde fixation on concentrations of low molecular weight constituents was determined by comparing amounts of selected intermediates in brains of mice exposed to aldehyde fixative solutions with those perfused with phosphate buffer solution alone. Aldehyde perfusion resulted in excellent preservation of cerebral cortex ultrastructure in the presence of dramatic declines in adenosine triphosphate, phosphocreatine, glucose and glucose-6-phosphate that occureed before exposure of the tissue to aldehyde fixatives. Decreases in hexose were accompanied by approximately a 4-fold increase in lactate and a 2-fold increase in pyruvate. Glycogen levels decreased by about 60% during the initial operative procedure but remained constant after aldehyde fixation. Glycogen content declined approximately 90% in tissues that were not treated with aldehyde. Concentrations of aspartate and glutamate changed only slightly during the initial period (1-5 min) and remained constant for at least 90 min in cerebral cortices fixed with aldehydes. Alanine levels increased in both fixed and unfixed tissue; however, this increase was much smaller in tissues exposed promptly to aldehydes. Total ninhydrin-positive material in perchloric acid extracts of brain decreased in mice exposed to aldehyde solutions but increased in tissues that were not. These results indicated that several amino acids may be measured reliably in tissues preserved for light and electron microscopy. In addition, determination of glutamate: alanine ratios in tissues perfused with aldehydes may provide an indication of the timing of fixation.

Microperoxidase uptake into the rat choroid plexus epithelium

Uptake, transport, and diffusion of the heme-peptide tracer microperoxidase (MP; MW 1900) in the rat choroid plexus were studied. MP was perfused ventriculo—cisternally or intravenously. The plexuses were fixed by ventriculo—cisternal perfusion of aldehydes, and tissue sections were incubated in a diaminobenzidine—H 2 O 2 medium containing imidazole at pH 8.8. After intravenous administration MP was found in the connective tissue of the plexus. Basement membranes were heavily labeled, while intercellular spaces between epithelial cells exhibited only a little MP. Some micropinocytic uptake of MP into vacuoles and lysosomes of the epithelial cells occurred. After ventriculo-cisternal perfusion of MP, it was micropinocytozed and incorporated into numerous vacuoles, multivesicular bodies, and dense bodies. Labeling of elongated cisterns (ER cisterns, or cisterns formed by fusion of endocytic vesicles) also occurred. Apical tight junctions between choroid plexus epithelial cells were never penetrated by MP. The intercellular spaces between epithelial cells, the connective tissue between epithelium and endothelium, and the blood vessels were, in general, devoid of MP, although diffusion of MP between ependymal cells into the extracellular space of the neuropil close to the base of the plexus was pronounced, as evaluated from the density of reaction product. No MP could be located in the kidney tubules of rats exposed ventriculo—cisternally to MP. The observations indicate that the choroid plexus is involved in absorption of molecules—also smaller than proteins—from the cerebrospinal fluid (csf). No significant diffusion of MP via ependyma and neuropil (the “functional leak”) into the plexus occurred, and no support of a transepithelial vesicular csf-to-blood transport of MP was obtained. This discrepancy in relation to previous horseradish peroxidase experiments is discussed.

Horseradish peroxidase uptake into the rat choroid plexus epithelium, with special reference to the lysosomal system

Uptake of horseradish peroxidase (HRP), lysosome formation, and acid phosphatase (AcPhase) activity in the rat choroid plexus epithelium were studied. HRP was administered intravenously or intraventricularly for various times, and the choroid plexuses were fixed by ventriculo—cisternal perfusion of aldehydes. AcPhase reaction was performed both on control material and on choroid plexuses from the HRP experiments. The uptake of HRP from the cerebrospinal fluid (csf) side of the epithelium was, in general, more pronounced than that from the blood side. HRP became micropinocytozed and sequestered in a well-developed lysosomal apparatus situated in the apical half of the epithelial cells. The main pathway for endocytozed HRP was from micropinocytic vesicles to large endocytic vacuoles which may appear as multivesicular bodies. These eventually developed into dense bodies or dense multivesicular bodies, representing secondary lysosomes. HRP labeling was also seen in elongated tubular structures, which may be chains of fused micropinocytic vesicles or smooth endoplasmatic reticulum cisterns. AcPhase activity was located in distinct Golgi cisterns within the Golgi complexes. Small Golgi vesicles (primary lysosomes), some (dark) multivesicular bodies, and most dense bodies were also AcPhase positive. The AcPhase activity of HRP-exposed choroid plexus epithelium and of control specimens was largely the same. However, the number of AcPhase-positive dense bodies appeared to increase after prolonged administration of a large dose of HRP into the csf. The results provide evidence that the choroid plexus epithelium is able to remove protein from the ventricular system.