Modulation of Matrix Metalloproteinases Activity in the Ventral Horn of the Spinal Cord Re-stores Neuroglial Synaptic Homeostasis and Neurotrophic Support following Peripheral Nerve Injury

Modulation of extracellular matrix (ECM) remodeling after peripheral nerve injury (PNI) could represent a valid therapeutic strategy to prevent maladaptive synaptic plasticity in central nervous system (CNS). Inhibition of matrix metalloproteinases (MMPs) and maintaining a neurotrophic support could represent two approaches to prevent or reduce the maladaptive plastic changes in the ventral horn of spinal cord following PNI. The purpose of our study was to analyze changes in the ventral horn produced by gliopathy determined by the suffering of motor neurons following spared nerve injury (SNI) of the sciatic nerve and how the intrathecal (i.t.) administration of GM6001 (a MMPs inhibitor) or the NGF mimetic peptide BB14 modulate these events. Immunohistochemical analysis of spinal cord sections revealed that motor neuron disease following SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) response in the ventral horn of the spinal cord, indicative of reactive gliosis. These changes were paralleled by decreased glial aminoacid transporters (glutamate GLT1 and glycine GlyT1), increased levels of the neuronal glutamate transporter EAAC1, and a net increase of the Glutamate/GABA ratio, as measured by HPLC analysis. These molecular changes correlated to a significant reduction of mature NGF levels in the ventral horn. Continuous i.t. infusion of both GM6001 and BB14 reduced reactive astrogliosis, recovered the expression of neuronal and glial transporters, lowering the Glutamate/GABA ratio. Inhibition of MMPs by GM6001 significantly increased mature NGF levels, but it was absolutely ineffective in modifying the reactivity of microglia cells. Therefore, MMPs inhibition, although supplies neurotrophic support to ECM components and restores neuro-glial transporters expression, differently modulates astrocytic and microglial response after PNI.     

Spreading depression sends microglia on Lévy flights

Spreading depression (SD) is thought to cause migraine aura, and perhaps migraine, and includes a transient loss of synaptic activity preceded and followed by increased neuronal excitability. Activated microglia influence neuronal activity and play an important role in homeostatic synaptic scaling via release of cytokines. Furthermore, enhanced neuronal function activates microglia to not only secrete cytokines but also to increase the motility of their branches, with somata remaining stationary. While SD also increases the release of cytokines from microglia, the effects on microglial movement from its synaptic activity fluctuations are unknown. Accordingly, we used time-lapse imaging of rat hippocampal slice cultures to probe for microglial movement associated with SD. We observed that in uninjured brain whole microglial cells moved. The movements were well described by the type of Lévy flight known to be associated with an optimal search pattern. Hours after SD, when synaptic activity rose, microglial cell movement was significantly increased. To test how synaptic activity influenced microglial movement, we enhanced neuronal activity with chemical long-term potentiation or LPS and abolished it with TTX. We found that microglial movement was significantly decreased by enhanced neuronal activity and significantly increased by activity blockade. Finally, application of glutamate and ATP to mimic restoration of synaptic activity in the presence of TTX stopped microglial movement that was otherwise seen with TTX. Thus, synaptic activity retains microglial cells in place and an absence of synaptic activity sends them off to influence wider expanses of brain. Perhaps increased microglial movements after SD are a long-lasting, and thus maladaptive, response in which these cells increase neuronal activity via contact or paracrine signaling, which results in increased susceptibility of larger brain areas to SD. If true, then targeting mechanisms that retard activity-dependent microglial Lévy flights may be a novel means to reduce susceptibility to migraine.     

Proliferation of microglial cells induced by 1-methyl-4-phenylpyridinium in mesencephalic cultures results from an astrocyte-dependent mechanism: role of granulocyte macrophage colony-stimulating factor

There is evidence that an inflammatory microglial reaction participates in the pathophysiology of dopaminergic neuronal death in Parkinson's disease and in animal models of the disease. However, this phenomenon remains incompletely characterized. Using an in vitro model of neuronal/glial mesencephalic cultures, we show that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) stimulates the proliferation of microglial cells at concentrations that selectively reduce the survival of DA neurones. The mitogenic action of MPP+ was not the mere consequence of neuronal cell demise as the toxin produced the same effect in a model system of neuronal/glial cortical cultures, where target DA neurones are absent. Consistent with this observation, the proliferative effect of MPP+ was also detectable in neurone-free microglial/astroglial cultures. It disappeared, however, when MPP+ was added to pure microglial cell cultures suggesting that astrocytes played a key role in the mitogenic mechanism. Accordingly, the proliferation of microglial cells in response to MPP+ treatment was mimicked by granulocyte macrophage colony-stimulating factor (GM-CSF), a proinflammatory cytokine produced by astrocytes and was blocked by a neutralizing antibody to GM-CSF. Thus, we conclude that the microglial reaction observed following MPP+ exposure depends on astrocytic factors, e.g. GM-CSF, a finding that may have therapeutic implications.     

The chemokine CX3CL1 reduces migration and increases adhesion of neurons with mechanisms dependent on the beta1 integrin subunit

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the beta(1) and alpha(6) integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the beta(1) subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.     

Differential susceptibility of astrocytic and neuronal function to 3-chloropropanediol in the rat inferior colliculus

We have previously shown that systemic administration of S(+)3-chloropropanediol (3-CPD) produces a morphological loss of astrocytes in specific nuclei of the rodent brain that precedes loss of both neurones and endothelial tight junctions. Here, we have evaluated the differential susceptibility of neuronal and astrocytic function to 3-CPD, in order to see if this parallels the morphological selectivity. To do this, we have developed an in vivo method for monitoring astrocyte function over time by giving hourly 20-min bolus challenge exposures to ammonia via an implanted microdialysis probe and measuring the resulting transient increases in the extracellular glutamine : glutamate ratio. These challenge ammonia exposures evoked a stable response for at least 5 h when the probe was implanted in the rat inferior colliculus, but caused no behavioural response or morphological damage. Although 3-CPD produced a rapid and sustained abolition of the ammonia response within 2 h, the field potential response of inferior collicular neurones to sound fell significantly to 75.0 ± 3.9% pre-dose at up to 8 h but then fell markedly, reaching 20.5 ± 3.7% at 2 days. Blood flow in the inferior colliculus also showed only late changes, increasing substantially at 2 days. Astrocyte damage at the EM level was seen from 3 h, followed by loss of astrocytes from 18 h to a minimum of 7 ± 10% control at 3 days. The rapid abolition of the ammonia response suggests that in addition to selective astrocyte death, 3-CPD also produces an earlier impairment of astrocyte function that precedes loss of neuronal function. This initial functional selectivity of 3-CPD provides a potential investigative tool in neurochemical studies of astrocyte-neuronal interactions.     

Morphological embedding and phonetic reduction: the case of triconstituent compounds

In this paper we propose that the internal bracketing of a word with more than two morphemes is reflected in the phonetic implementation. We hypothesize that embedded forms show more phonetic reduction than forms at higher structural levels (‘Embedded Reduction Hypothesis’). This paper tests the prediction of the Embedded Reduction Hypothesis with triconstituent compounds. The analysis of the durational properties of almost 500 compound tokens shows that there is a lengthening effect on the non-embedded constituent, and a shortening effect on the adjacent embedded constituent. Yet, this predicted effect of embedding interacts with other lexical factors, above all the bigram frequency of the embedded compound. At a theoretical level, these effects mean that the durational properties of the cross-boundary constituents are indicative of the hierarchical structure and of the strength of the internal boundary of triconstituent compounds. Hence, morphological structure is reflected in the speech signal.     

Brain phagocytes may empty tissue debris into capillaries

The possibility that brain phagocytes may empty remnants of degenerated neurons into capillaries has been studied in frogs. Degeneration of nerve fibers was brought about by transectioning the optic tract, the tectothalamic and tectoisthmic tracts, the postoptic commissure or the radial nerve. To help identification of phagocytozed degenerated neuronal elements, the transected fibers were filled either with horseradish peroxidase (HRP) or cobaltous-lysine complex. The survival times were 3, 4, 7, 27, 47 and 70 days after the application of the markers. The HRP-labeled structures were identified in 60 μm thick sections using diaminobenzidine as chromogen, while cobalt was precipitated in the form of cobaltous sulfide. Small pieces of these sections were further processed for electron microscopy. In each area of the brain and spinal cord investigated, microglial cells and astrocytic processes containing fragments of degenerated neuronal elements could be seen close to capillaries. In some cases a microglial or astrocytic process pierced the capillary basal lamina and seemingly delivered inclusion bodies into the cytoplasm of capillary endothelial cells and pericytes. In the inclusion bodies, which were usually large vesicles, fragments of HRP or cobalt-labeled or unlabeled membranes with a foamy appearance, or condensed myelin lamellae could be observed. These vesicles protruded the luminal membrane of the endothelial cell that was disrupted in some cases suggesting that the content of the inclusion body was discharged into the lumen of the capillary. These results give support to Penfield's hypothesis (1925) that glial cells may empty phagocytozed materials into capillaries.     

Flexible polyimide microelectrode array for in vivo recordings and current source density analysis

This work presents implantable, flexible polymer-based probes with embedded microelectrodes for acute and chronic neural recordings in vivo, as tested on rodents. Acute recordings using this array were done in mice under urethane anesthesia and compared to those made using silicon-based probes manufactured at the Center for Neural Communication Technology, University of Michigan. The two electrode arrays yielded similar results. Recordings with chronically implanted polymer-based electrodes were performed for 60 days post-surgically in awake, behaving rats. The microelectrodes were used to monitor local field potentials and capture laminar differences in function of cortex and hippocampus, and produced response waveforms of undiminished amplitude and signal-to-noise ratios 8 weeks after chronic implantation. The polymer-based electrodes could also be connected to a lesion current to mark specific locations in the tissue. Current source density (CSD) analysis from the recordings depicted a source - sink-composition. Tissue response was assessed 8 weeks after insertion by immunochemical labeling with glial fibrillary acidic protein (GFAP) to identify astrocytes, and histological analysis showed minimal tissue reaction to the implanted structures.     

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1^GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.     

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (>3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.     

Multiple Implants Do Not Aggravate the Tissue Reaction in Rat Brain

Chronically implanted microelectrodes are an invaluable tool for neuroscientific research, allowing long term recordings in awake and behaving animals. It is known that all such electrodes will evoke a tissue reaction affected by its’ size, shape, surface structure, fixation mode and implantation method. However, the possible correlation between tissue reactions and the number of implanted electrodes is not clear. We implanted multiple wire bundles into the brain of rats and studied the correlation between the astrocytic and microglial reaction and the positioning of the electrode in relation to surrounding electrodes. We found that an electrode implanted in the middle of a row of implants is surrounded by a significantly smaller astrocytic scar than single ones. This possible interaction was only seen between implants within the same hemisphere, no interaction with the contralateral hemisphere was found. More importantly, we found no aggravation of tissue reactions as a result of a larger number of implants. These results highlight the possibility of implanting multiple electrodes without aggravating the glial scar surrounding each implant.     

Bidirectional Scaling of Astrocytic Metabotropic Glutamate Receptor Signaling following Long-Term Changes in Neuronal Firing Rates

Very little is known about the ability of astrocytic receptors to exhibit plasticity as a result of changes in neuronal activity. Here we provide evidence for bidirectional scaling of astrocytic group I metabotropic glutamate receptor signaling in acute mouse hippocampal slices following long-term changes in neuronal firing rates. Plasticity of astrocytic mGluRs was measured by recording spontaneous and evoked Ca²⁺ elevations in both astrocytic somata and processes. An exogenous astrocytic Gq G protein-coupled receptor was resistant to scaling, suggesting that the alterations in astrocyte Ca²⁺ signaling result from changes in activity of the surface mGluRs rather than a change in intracellular G protein signaling molecules. These findings suggest that astrocytes actively detect shifts in neuronal firing rates and adjust their receptor signaling accordingly. This type of long-term plasticity in astrocytes resembles neuronal homeostatic plasticity and might be important to ensure an optimal or expected level of input from neurons.     

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca²⁺ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca²⁺ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.     

Dissociation between hippocampal neuronal loss, astroglial and microglial reactivity after pharmacologically induced reverse glutamate transport

The inflammatory central nervous system response that involves activated microglia and reactive astrocytes may both heal and harm neurons, as inflammatory mediators lead to neuroprotection or excitation at one dose but to injury at a different concentration. To investigate these complex cellular interactions, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a selective substrate inhibitor of glutamate transport, was infused during 14 days in the rat hippocampus at three different rates: 5, 10 and 25 nmol/h. A microglial reaction appeared at the 5 nmol/h PDC rate in absence of astroglial reaction and neuronal loss. Microgliosis and neuronal death were observed at PDC 10 nmol/h in absence of astrogliosis and calcium precipitation, whereas all four aspects were present at the highest rate. This dissociation between neuronal loss and astroglial reactivity took place in presence of a permanent microglial reaction. These data suggest a specific response of microglia to PDC whose neuronal effects may differ with the infused dose.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.     

Activation of group II metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A,a peptide up-regulated in Alzheimer's disease

Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A-induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A-induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease.     

The participation of sialic acids in microglia-neuron interactions

Since it is known that sialic acid participates in neuronal plasticity, it is resonable to investigate its role in microglia-neuron interactions. In this study, we tested the effects of enzymatic removal of sialic acid on neurite and cell body density in microglia-neuron co-cultures. Additionaly, we analyzed the expression of Siglec-F protein, putative receptor for sialic acids, in microglial cells as well as its affinity to neurons. The results showed that removal of sialic acids affects neuronal integrity and changes microglial morphology. In presence of microglial cells, endoneuraminidase and α-neuraminidase significantly reduced neurite density (p<0.05). Endoneuraminidase (p<0.05) and α-neuraminidase (p>0.05) decreased the number of neuronal cell bodies in comparison to control co-cultures. Neuraminidases-treated neurons showed reduced binding of Siglec-F protein, which we found in microglial cells. Our results suggest that interactions between sialic acids and Siglec receptors may protect neuronal integrity during neurodegenerative processes.     

Triazoyl–phenyl linker system enhancing the aqueous solubility of a molecular probe and its efficiency in affinity labeling of a target protein for jasmonate glucoside

In methods employing molecular probes to explore the targets of bioactive small molecules, long or rigid linker moieties are thought to be critical factors for efficient tagging of target protein. We previously reported the synthesis of a jasmonate glucoside probe with a highly rigid linker consisting of a triazoyl–phenyl (TAzP) moiety, and this probe demonstrated effective target tagging. Here we compare the TAzP probe with other rigid or flexible probes with respect to target tagging efficiency, hydrophobic parameters, aqueous solubility, and dihedral angles around the biaryl linkage by a combination of empirical and calculation methods. The rigid biaryl linkage of the TAzP probe has a skewed conformation that influences its aqueous solubility. Such features that include rigidness and good aqueous solubility resulted in highly efficient target tagging. These findings provide a promising guideline toward designing of better linkers for improving molecular probe performance.     

Microglial AGE-Albumin Is Critical in Promoting Alcohol-Induced Neurodegeneration in Rats and Humans

Alcohol is a neurotoxic agent, since long-term heavy ingestion of alcohol can cause various neural diseases including fetal alcohol syndrome, cerebellar degeneracy and alcoholic dementia. However, the molecular mechanisms of alcohol-induced neurotoxicity are still poorly understood despite numerous studies. Thus, we hypothesized that activated microglial cells with elevated AGE-albumin levels play an important role in promoting alcohol-induced neurodegeneration. Our results revealed that microglial activation and neuronal damage were found in the hippocampus and entorhinal cortex following alcohol treatment in a rat model. Increased AGE-albumin synthesis and secretion were also observed in activated microglial cells after alcohol exposure. The expressed levels of receptor for AGE (RAGE)-positive neurons and RAGE-dependent neuronal death were markedly elevated by AGE-albumin through the mitogen activated protein kinase pathway. Treatment with soluble RAGE or AGE inhibitors significantly diminished neuronal damage in the animal model. Furthermore, the levels of activated microglial cells, AGE-albumin and neuronal loss were significantly elevated in human brains from alcoholic indivisuals compared to normal controls. Taken together, our data suggest that increased AGE-albumin from activated microglial cells induces neuronal death, and that efficient regulation of its synthesis and secretion is a therapeutic target for preventing alcohol-induced neurodegeneration.     

Immunogold determination of amylase concentrations in pancreatic subcellular compartments

We used the immunogold method on ultrathin cryosections to measure intracellular amylase (Am) concentrations in subcellular compartments of rat exocrine pancreatic cells. Previously, the quantitation procedure was characterized in a model system consisting of Am dispersed at known concentration in a matrix of gelatin. Variations in labeling efficiency, due to differences in matrix density, were equalized by embedding in 30% polyacrylamide (PAA). Here we applied these model conditions to rat pancreas and established intracellular Am concentrations [Am]. Specimen blocks were composed of tissue and a reference layer of gelatin mixed with a known Am concentration ([Am]r), both fixed in glutaraldehyde. Cryosections of the PAA embedded blocks were immunogold labeled for Am. The labeling density was measured in the reference layer (LDr) and in structures in exocrine cells that were involved in Am synthesis and transport (LDs). In each of these structures the Am concentration ([Am]s) was calculated from: [Am]s = [Am]r. LDs/LDr In this way we measured average concentrations ranging from 63 mg/ml in rough endoplasmic reticulum to 261 mg/ml in secretory granules. Concentration of Am appeared to occur mainly in the most cis- and the most trans-Golgi cisternae. To check whether sterical hindrance was an inherent bias to the [Am] measurements in compartments that contained high concentrations of the enzyme, the labeling efficiency for Am in intact isolated secretory granules in gelatin and embedded in PAA, was compared with the efficiency when the granules were lysed and approximately 50 times diluted in gelatin before PAA embedment. It appeared that Am was detected with similar efficiency under both conditions. This demonstrated that sterical hindrance did not cause errors in the measurements of cellular Am concentrations.