Coconut oil protects cortical neurons from amyloid beta toxicity by enhancing signaling of cell survival pathways.

Alzheimer's disease is a progressive neurodegenerative disease that has links with other conditions that can often be modified by dietary and life-style interventions. In particular, coconut oil has received attention as having potentially having benefits in lessening the cognitive deficits associated with Alzheimer's disease. In a recent report, we showed that neuron survival in cultures co-treated with coconut oil and Aß was rescued compared to cultures exposed only to Aß. Here we investigated treatment with Aß for 1, 6 or 24 h followed by addition of coconut oil for a further 24 h, or treatment with coconut oil for 24 h followed by Aß exposure for various periods. Neuronal survival and several cellular parameters (cleaved caspase 3, synaptophysin labeling and ROS) were assessed. In addition, the influence of these treatments on relevant signaling pathways was investigated with Western blotting. In terms of the treatment timing, our data indicated that coconut oil rescues cells pre-exposed to Aß for 1 or 6 h, but is less effective when the pre-exposure has been 24 h. However, pretreatment with coconut oil prior to Aß exposure showed the best outcomes. Treatment with octanoic or lauric acid also provided protection against Aß, but was not as effective as the complete oil. The coconut oil treatment reduced the number of cells with cleaved caspase and ROS labeling, as well as rescuing the loss of synaptophysin labeling observed with Aß treatment. Treatment with coconut oil, as well as octanoic, decanoic and lauric acids, resulted in a modest increase in ketone bodies compared to controls. The biochemical data suggest that Akt and ERK activation may contribute to the survival promoting influence of coconut oil. This was supported by observations that a PI3-Kinase inhibitor blocked the rescue effect of CoOil on Aß amyloid toxicity. Further studies into the mechanisms of action of coconut oil and its constituent medium chain fatty acids are warranted.

Endurance exercise regimens induce differential effects on brain-derived neurotrophic factor, synapsin-I and insulin-like growth factor I after focal ischemia.

The optimal amount of endurance exercise required to elevate proteins involved in neuroplasticity during stroke rehabilitation is not known. This study compared the effects of varying intensities and durations of endurance exercise using both motorized and voluntary running wheels after endothelin-I-induced focal ischemia in rats. Hippocampal levels of brain-derived neurotrophic factor, insulin-like growth factor I and synapsin-I were elevated in the ischemic hemisphere even in sedentary animals suggesting an intrinsic restorative response 2 weeks after ischemia. In the sensorimotor cortex and the hippocampus of the intact hemisphere, one episode of moderate walking exercise, but not more intense running, resulted in the greatest increases in levels of brain-derived neurotrophic factor and synapsin-I. Exercise did not increase brain-derived neurotrophic factor, insulin-like growth factor I or synapsin-I in the ischemic hemisphere. In voluntary running animals, both brain and serum insulin-like growth factor I appeared to be intensity dependent and were associated with decreasing serum levels of insulin-like growth factor I and increasing hippocampal levels of insulin-like growth factor I in the ischemic hemisphere. This supports the notion that exercise facilitates the movement of insulin-like growth factor I across the blood-brain barrier. Serum corticosterone levels were elevated by all exercise regimens and were highest in rats exposed to motorized running of greater speed or duration. The elevation of corticosterone did not seem to alter the expression of the proteins measured, however, graduated exercise protocols may be indicated early after stroke. These findings suggest that relatively modest exercise intervention can increase proteins involved in synaptic plasticity in areas of the brain that likely subserve motor relearning after stroke.

Stress-mediated signaling in PC12 cells - the role of the small heat shock protein, Hsp27, and Akt in protecting cells from heat stress and nerve growth factor withdrawal.

We have investigated the role of stress-activated signaling pathways and the small heat shock protein, Hsp27, in protecting PC12 cells from heat shock and nerve growth factor (NGF) withdrawal-induced apoptosis. PC12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to heat shock. This resulted in the rapid activation of Akt followed by p38 mitogen-activated protein kinase (MAPK) signaling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 was found to form an immunoprecipitable complex with Akt and p38 MAPK in both non-stimulated and heat shocked cells, although after heat shock there was a gradual dissociation of Akt and p38 from the Hsp27. Cells were differentiated with NGF and then subjected to NGF withdrawal, a treatment which results in substantial cell death over 24-72 h. Hsp27 was shown to be protective against this treatment, since HSPC cells which overexpress Hsp27 showed significantly less cell death than the parental PC12 cells. In addition, we observed that phosphorylation of Akt was maintained in HSPC cells subjected to heat shock and NGF withdrawal compared with the parental cells. Taken together, our results suggest that Hsp27 may protect Akt from dephosphorylation and may also act in stabilizing Akt.

Neurite growth promotion by nerve growth factor and insulin-like growth factor-1 in cultured adult sensory neurons: role of phosphoinositide 3-kinase and mitogen activated protein kinase.

Although neurons of the PNS no longer require neurotrophins such as Nerve Growth Factor (NGF) for their survival, such factors are involved in regulating axonal sprouting and regeneration after injury. In addition to the neurotrophin receptors, sensory neurons are reported to express IGF-1, EGF and FGF receptors. To investigate the influence of growth factors in addition to NGF, we examined the effects of IGF-1 EGF and FGF on neurite growth from adult rat dorsal root ganglion sensory neurons in both dissociated cultures and in compartmented cultures. As expected, NGF elicited robust neuritic growth in both the dissociated and compartmented cultures. The growth response to IGF-1 was similar, although there was minimal neurite growth in response to EGF or FGF. In addition, IGF-1 (but neither FGF nor EGF), when applied to cell bodies in compartmented cultures, potentiated the distal neurite growth into NGF-containing side compartments. This potentiation was not seen when these factors were provided along with NGF in the side compartments of compartmented cultures, or in the dissociated cultures. To determine the contribution of signaling intermediates downstream of receptor activation, we used inhibitors of the potential effectors and Western blotting. The PI 3-kinase inhibitor, LY294002 attenuated neurite growth evoked by NGF, IGF and EGF in dissociated cultures, although the MAP kinase kinase (MEK) inhibitor PD098059 diminished the growth in only IGF. Immunoprecipitation and Western blotting results demonstrated differential activation of MAPK, PI 3-kinase, PLCgamma1 and SNT by the different factors. Activation of PI 3-kinase and SNT by both NGF and IGF-1 correlated with their effects on neurite growth. These results support the hypothesis that the PI 3-kinase pathway plays an important role in neuritogenesis.

The anti-p75 antibody, MC192, and brain-derived neurotrophic factor inhibit nerve growth factor-dependent neurite growth from adult sensory neurons.

We have investigated nerve growth factor-dependent neurite growth from adult sensory neurons using the compartmented culture system. The requirement of both TrkA and the p75 neurotrophin receptors in neurite growth was examined using several experimental interventions. Inhibition of TrkA activation using K252a resulted in a total block of distal neurite extension into nerve growth factor-containing compartments. Brain-derived neurotrophic factor and the anti-p75 monoclonal antibody MC192 have been shown to interfere with the binding of nerve growth factor to p75. Brain-derived neurotrophic factor, which binds p75 but not TrkA, competes with nerve growth factorforp75, while the anti-p75 antibody MC192 has been shown to decrease the interaction of nerve growth factor with TrkA. The addition of brain-derived neurotophic factor to nerve growth factor-containing distal compartments inhibited, but did not totally block, distal neurite extension. MC192, on the other hand, totally inhibited nerve growth factor-dependent neurite growth. To test whether MC192 and brain-derived neurotrophic factor might be influencing Trk activation, TrkA phosphorylation was examined biochemically. Both compounds were found to attenuate nerve growth factor-induced Trk phosphorylation, although neither inhibited the activation completely. The possibility that MC192 or brain-derived neurotrophic factor might activate p75 signaling directly (and potentially antagonize TrkA signaling) was also investigated. This was assessed by quantitating the activation and nuclear translocation of the transcription factor NFkB using immunocytochemistry. Only treatment with the anti-p75 antibody MC192 resulted in prolonged and significant increase in the number of neurons displaying nuclear staining for NFkB. Our results demonstrate that both TrkA and p75 play a role in neurite growth response to nerve growth factor, and further suggest that any alteration in optimal TrkA-p75 interactions, or direct activation of p75 at the expense of TrkA, results in an inhibition of nerve growth factor-dependent neurite growth in adult sensory neurons.

The effects of NGF and sensory nerve stimulation on collateral sprouting and gene expression in adult sensory neurons.

Collateral sprouting of mature cutaneous nociceptive fibers is regulated by the availability of NGF, and the onset of this sprouting can be accelerated by electrical stimulation of the intact nerve. To investigate this influence of stimulation on NGF-induced sprouting, the thoracic dorsal cutaneous nerves of adult rats were exposed and those on the left side of the animals were electrically stimulated. NGF was then administered daily for 1-12 days. At 12 days poststimulation, the extent of nociceptive fibers sprouting was examined by an established behavioral mapping technique and was found to have occurred only in the NGF-treated animals. The dorsal root ganglia (DRGs) were sampled at various times throughout the experiment and processed for in situ hybridization to examine mRNA expression of the NGF receptors (p75 and trkA) and GAP-43. As well, expression of mRNAs for the neurotrophins NGF, BDNF, and NT-3 was examined. p75, trkA, and GAP-43 mRNAs were upregulated in DRGs from the NGF-treated, but not the control animals. The combination of stimulation plus NGF resulted in these increases being slightly higher than those in the absence of stimulation; however, stimulation alone had little effect on the mRNA expression. Examination of the neurotrophin mRNAs confirmed the absence of neuronal NGF and NT-3 expression and the presence of neuronal BDNF mRNA. The NGF treatment resulted in the upregulation of BDNF mRNA to peak levels within the first 2 days of treatment, although the electrical stimulation had little additional effect. These results demonstrate that exogenously supplied NGF itself can elicit sprouting from intact cutaneous nociceptive afferents and that electrical stimulation further influences the expression of mRNAs involved in the sprouting response. While the increases in NGF receptors and GAP-43 mRNA have been shown to be associated with collateral sprouting, the role of BDNF is not clear, but may be involved in altered sensory processing (i.e., hyperalgesia) that has been shown to occur subsequent to NGF administration.

Effects of the neurotrophins nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures.

We used compartmented cultures to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and BDNF on distal neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Neurons were plated in the center compartments of three-chambered dishes in the absence of neurotrophin, and neurite extension into the distal (side) compartments containing NGF, BDNF, or NT3 was quantitated. Initial proximal neurite growth did not require any of the neurotrophins, while subsequent elongation into distal compartments required NGF. After neurites had extended into NGF-containing distal compartments, removal of NGF by treatment with anti-NGF resulted in the cessation of growth with minimal neurite retraction. In contrast to the effects of NGF, no distal neurite elongation was observed into compartments with BDNF or NT3. To examine possible additive influences, neurite extension into compartments containing BDNF plus NGF or NT3 plus NGF was quantitated. There was no increased neurite extension into NGF plus NT3 compartments, while the combination of BDNF plus NGF resulted in an inhibition of neurite extension compared with NGF alone. We then investigated whether the regrowth of neurites that had originally grown into NGF subsequent to in vitro axotomy still required NGF. The results demonstrated that unlike adult sensory nerve regeneration in vivo, the in vitro regrowth did require NGF, and neither BDNF nor NT3 was able to substitute for NGF. Since the initial growth from neurons after dissociation (which is also a regenerative response) did not require NGF, it would appear that neuritic growth and regrowth of adult DRG neurons in vitro includes both NGF-independent and NGF-dependent components. The compartmented culture system provides a unique model to further study aspects of this differential regulation of neurite growth.

Anti-NGF treatment blocks the upregulation of NGF receptor mRNA expression associated with collateral sprouting of rat dorsal root ganglion neurons.

The collateral sprouting of intact cutaneous sensory neurons has been shown to be dependent upon the presence of nerve growth factor (NGF). We have examined NGF receptor (NGFR) mRNA expression in DRG neurons undergoing sprouting and in dorsal root ganglion (DRG) neurons whose sprouting had been prevented through exposure to anti-NGF antiserum during the course of the experiment. The results indicate that the low affinity p75 NGFR mRNA is increased by 4 days post-operatively in DRGs from control serum-treated animals, and that this increase is most pronounced in smaller neurons. In contrast, the expression of p75 mRNA was maintained at control levels in DRGs from anti-NGF-treated animals. Results for trkA expression indicate an increase in expression at days 4-6 post-op in both groups of animals, with the anti-NGF treatment having a delayed influence on mRNA levels. Examination of GAP-43 mRNA levels revealed an increased expression in sprouting DRG neurons, whereas this increase was not observed in DRGs from anti-NGF treated animals. Taken together, these results provide further evidence of NGF's role in the collateral sprouting of nociceptive neurons.

Expression of NGF receptor and GAP-43 mRNA in DRG neurons during collateral sprouting and regeneration of dorsal cutaneous nerves.

The collateral sprouting of intact sensory axons and the regeneration of damaged ones differ in a number of respects. Regeneration is triggered by axotomy-induced damage, probably involves the loss of a peripheral signal, and appears to occur independently of NGF, while collateral sprouting is evoked and sustained by an increase in a target-derived signal, namely NGF. New findings strengthen the distinction between these two phenomena. Nerve growth factor receptor (NGFR) mRNA is increased in undamaged DRG neurons whose axons are sprouting into denervated skin. This response is related to an increased availability of target-derived NGF, a proposal supported by a number of findings including increased NGF mRNA in the denervated target. In contrast, we observed little or no change in the NGFR mRNA levels in regenerating neurons, consistent with the observations that NGF does not play a role in this process. However, increases in neuronal GAP-43 mRNA are found during both regeneration and collateral sprouting, a result in keeping with the proposal that GAP-43 is primarily associated with nerve growth, and the observation that GAP-43 expression is not especially influenced by NGF.

Increased NGF mRNA expression in denervated rat skin.

Using a coupled reverse transcription and competitive polymerase chain reaction protocol, we have measured Nerve Growth Factor (NGF) mRNA expression in adult rat skin following its denervation. By 2-4 days, levels of NGF mRNA were increased approximately 5-10 fold over levels in innervated skin, remaining increased for up to at least 2 weeks. In situ hybridization carried out on skin samples revealed that the NGF message was expressed not only in the distal nerve pathways but also in non-nerve associated cells, such as dermal fibroblasts, and in the basal epidermal layer. Denervated skin evokes a NGF-dependent collateral sprouting from neighbouring intact sensory axons. The new findings indicate that an increased NGF production is associated with the increased availability of NGF in such skin, and that cutaneous nerves have a role in regulating this NGF production.

In situ hybridization and immunocytochemical localization of SERCA2 encoded Ca2+ pump in rabbit heart and stomach.

Heart tissue contains large amounts of the protein encoded by the Ca2+ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms by in situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca2+ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca2+ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.

Cloning and functional characterization of the rat glutamine synthetase gene.

Glutamine synthetase catalyzes the formation of glutamine from glutamate and ammonia. It plays a central role in both amino acid neurotransmitter metabolism and ammonia detoxification in the central nervous system. Glutamine synthetase expression is regulated in developmental, hormonal, and in tissue- and cell-specific manners. We have cloned a full-length cDNA coding for rat glutamine synthetase, and have found an AT-rich area of conservation in the 3' untranslated regions between rat, mouse, and chicken, which may play a part in the regulation of the stability of the glutamine synthetase message. We have also cloned and mapped the gene coding for rat glutamine synthetase, and identified, by sequence analysis, areas potentially important for the regulation of glutamine synthetase transcription. Transient transfection of a variety of cell lines with deletion constructs of the glutamine synthetase promoter driving a chloramphenicol acetyltransferase reporter gene functionally demonstrates regions of the promoter containing elements important for transcriptional regulation.

Characterization of human cDNA and genomic clones for glial fibrillary acidic protein.

Both a partial cDNA clone and a complete genomic clone have been isolated for human gfa, the gene encoding the major component of astrocyte intermediate filaments, glial fibrillary acidic protein (GFAP). The nucleotide sequence of the entire coding region and 102 bp of the 5' flanking DNA was determined. The mRNA start site was identified by primer extension and probe protection experiments, and a novel in vitro transcription and translation procedure was then used to establish that the first ATG in the mRNA initiates GFAP synthesis. The predicted amino-terminal sequence for human GFAP differs greatly from that previously deduced for mouse GFAP from its gene sequence, despite otherwise high homology. This discrepancy was resolved by determining that the published mouse genomic sequence has an incorrect additional base. The corrected sequence produces strong homology between human and mouse GFAP in their predicted amino acid sequences, and suggests that human and mouse GFAP initiate at homologous positions. The beginning sequence deduced here for both proteins is matched closely by that previously obtained for porcine GFAP by direct sequencing of its amino-terminal end. This supports the protein initiation sites proposed, and also indicates that GFAP is not processed at its amino-terminal end.

Neuron-glial interactions involved in the regulation of glutamine synthetase.

Cocultures of rat cortical astrocytes with cerebellar granule cell neurons, but not a variety of other cell types tested, resulted in an induction of glutamine synthetase (GS) mRNA over the basal levels expressed in pure astrocyte cultures. This induction involved both contact- and noncontact-mediated events and may be a result of astroglial differentiation promoted by interactions with the primary neurons. Astrocytes grown in the presence of the granule neurons (but not the other cell types tested) exhibited a more complex, process-bearing morphology typical of more differentiated cells. In addition, glial cell proliferation was inhibited not only by the presence of live granule cells, but also by fixed neurons and neuronal membranes. Under the same experimental conditions, GS mRNA was increased (two- to threefold) compared with the expression observed in pure astrocyte cultures. Because of the role of GS in glutamate metabolism and the influence of the glutamatergic granule neurons on glial GS mRNA levels, the effect of exogenous glutamate was examined. The addition of 100 microM glutamate to the culture medium resulted in an increase in GS mRNA in the astrocyte cultures similar to that observed in the cocultures, where the addition of glutamate did not further increase GS mRNA levels. These results provide further evidence for the importance of neuron-glial interactions in the regulation of glial gene expression.

The ontogeny and localization of glutamine synthetase gene expression in rat brain.

A 2.4 kb cDNA clone containing the coding sequence for glutamine synthetase (GS) was isolated from a rat brain cDNA library, and a probe constructed from this cDNA was utilized in Northern analysis of total RNA to study the tissue distribution and the ontogeny of GS mRNA expression in the rat brain from embryonic day 14 (E14) to adulthood. The levels of GS mRNA were highest in the brain, followed by kidney and liver. In the brain, the GS message was detected as early as E14, earlier than it can be detected by either enzymatic assays of GS activity or by immunocytochemical localization of GS. The relatively low levels of GS mRNA seen at E14 increase to a peak around the time of birth, and in the second postnatal week another rise in GS message occurs approaching adult levels by P15. Localization of GS to astrocytes in the brain was confirmed using both immunocytochemistry and in situ hybridization.

Stimulation-induced changes at crayfish (Procambarus clarkii) neuromuscular terminals.

The fine structure of neuromuscular terminals of the single excitor axon was examined in the limb stretcher muscle of the crayfish Procambarus clarkii. A morphometric comparison of the neuromuscular terminals of the left and right limbs of a control crayfish showed them to be similiar in qualitative as well as quantitative features. The excitor axon to the stretcher muscle of the right side was stimulated, by backfiring its branches in the adjacent opener muscle, at 20 Hz for 4-5 h per day over 4-5 days. The stretcher muscle on the left side was not stimulated and served as a control. Morphometric analysis of stimulated terminals revealed an increase in the number of dense bars and synaptic vesicles compared to their non-stimulated, contralateral counterparts. Since dense bars are regarded as active sites of transmitter release, changes in their number provide a morphological basis for synaptic plasticity.

Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles.

The regulation of glutamine synthetase expression by dexamethasone was studied in rat skeletal muscles. Daily administration of dexamethasone caused striking enhancement of glutamine synthetase activity in plantaris, soleus, and diaphragm muscles. Northern blot analysis revealed that the dexamethasone-mediated increase of glutamine synthetase activity was associated with dramatically increased levels of glutamine synthetase mRNA. Both glutamine synthetase activity and mRNA levels were significantly elevated in plantaris muscle at 0.5 mg.kg-1.day-1 of dexamethasone, a dose that approximates endogenous corticosteroid levels in animals under severe stress. Quantification of changes in glutamine synthetase mRNA on the basis of total mRNA (by oligo dT hybridization) also revealed a major increase in glutamine synthetase mRNA. Dexamethasone was without effect on beta-tubulin mRNA levels, indicating that glutamine synthetase induction is not part of a global response to glucocorticoids. Dexamethasone treatment resulted in only an approximately 15% increase in glutamine synthetase activity in heart; there was no change in glutamine synthetase mRNA level in this tissue. Thus glucocorticoids regulate glutamine synthetase gene expression in rat skeletal muscles.

Merkel cells and the mechanosensitivity of normal and regenerating nerves in Xenopus skin.

We have investigated some of the physiological, morphological and trophic characteristics of the Merkel cell-neurite complexes in the skin of Xenopus laevis. The Merkel cells, which are specialized sensory cells, occur in groups of 2-4 around the openings of the cutaneous gland ducts. A voltage-controlled mechanical stimulator was used to determine the distribution of mechanosensory thresholds across the skin; an analysis of the results revealed the presence of a single population of rapidly adapting, low threshold mechanoreceptors, whose locations coincided with those of the epidermal Merkel cell-neurite complexes. The possible role of the Merkel cell in the mechanosensory process, and its trophic interactions with the sensory nerve, were examined (i) by following the development of mechanosensitivity when sensory nerves regenerated into denervated, or newly regenerated, skin; (ii) by looking for possible correlations between the expression of physiological function and the appearance of morphological features characteristic of the Merkel cell-neurite complex; and (iii) by investigating the mechanosensitivity that remained after elimination of the Merkel cells. Not only did Merkel cells survive denervation without obvious changes in their fine structure, but they developed with normal morphology in new skin that had regenerated in nerve free limbs. Ingrowing sensory nerves contacted these Merkel cells, and eventually normal mechanosensory function was established; thus the Merkel cells act as targets for these nerves. The full recovery of the normal pattern of mechanosensitivity in the skin following nerve regeneration was correlated with the redevelopment of the specialized contacts between the nerve endings and Merkel cells, that eventually included reciprocal synapses. However, following the mechanical removal of the epidermis by enzymatic treatment, or the selective elimination of the Merkel cells by irradiation after they had taken up the fluorescent dye quinacrine, essentially normal mechanosensory responses could be initiated, though with somewhat increased thresholds. The results indicate that the Merkel cells are not involved in mechanosensory transduction; they do, however, act as targets for the growing nerves, thereby ensuring the appropriate distribution of low threshold mechanosensitivity, and they may have a role in enhancing and even inducing the excitability of the mechanosensitive nerve endings.

Merkel cell distribution in the epidermis as determined by quinacrine fluorescence.

The Merkel cell distribution in the basal epidermis of amphibian and mammalian skin was visualized in whole mounts by means of quinacrine fluorescence. In most cases only the isolated epidermis was viewed following dermal-epidermal separation. Tadpole tentacles contained numerous quinacrine fluorescent cells (QFC) 25-40 microns apart. Groups of 2-4 QFC were found around the gland openings in frog epidermis but not in salamander epidermis where the QFC were irregularly scattered 40-100 microns apart. In the rat, ring-like clusters of a few to 200 or more QFC were distributed across the basal epidermis of trunk skin (at touch domes or Haarscheiben), eyelid, ear, nose, and whisker pad. The ridged (glabrous) skin of the nose and footpad contained numerous QFC that appeared to follow the contours of the epidermal ridges. The isolated external root sheath of rat vibrissae contained an upper cylindrical cuff of several hundred QFC; enzymatic dissociation of these sheaths produced individually isolated as well as small clusters of fluorescent and non-fluorescent cells. Electron-microscopic examination of several of these cells confirmed that the fluorescent ones are Merkel cells, identified by the presence of characteristic dense-cored granules; in contrast, the non-fluorescent cells lack this ultrastructural feature.