Evidence that small molecule enhancement of ß-hexosaminidase activity corrects the behavioral phenotype in Dutch APP(E693Q) mice through reduction of ganglioside-bound Aß.

Certain mutant Alzheimer's amyloid-ß (Aß) peptides (that is, Dutch mutant APP(E693Q)) form complexes with gangliosides (GAß). These mutant Aß peptides may also undergo accelerated aggregation and accumulation upon exposure to GM2 and GM3. We hypothesized that increasing ß-hexosaminidase (ß-hex) activity would lead to a reduction in GM2 levels, which in turn, would cause a reduction in Aß aggregation and accumulation. The small molecule OT1001 is a ß-hex-targeted pharmacological chaperone with good bioavailability, blood-brain barrier penetration, high selectivity for ß-hex and low cytotoxicity. Dutch APP(E693Q) transgenic mice accumulate oligomeric Aß as they age, as well as Aß oligomer-dose-dependent anxiety and impaired novel object recognition (NOR). Treatment of Dutch APP(E693Q) mice with OT1001 caused a dose-dependent increase in brain ß-hex levels up to threefold over those observed at baseline. OT1001 treatment was associated with reduced anxiety, improved learning behavior in the NOR task and dramatically reduced GAß accumulation in the subiculum and perirhinal cortex, both of which are brain regions required for normal NOR. Pharmacological chaperones that increase ß-hex activity may be useful in reducing accumulation of certain mutant species of Aß and in preventing the associated behavioral pathology.

Molecular basis of growth cone adhesion: anchoring of adheron-containing filaments at adhesive loci.

Adhesive contacts made by filopodia of neuronal growth cones are essential for proper neurite elongation and may have a role in the formation of synaptic junctions. Previously we described the appearance of filamentous materials extending from growth cone surfaces that seem to be associated with the strongly adhesive behavior of filopodia (Tsui, H.-C., K. L. Lankford, and W. L. Klein. 1985. Proc. Natl. Acad. Sci. USA. 82:8256-8260). Here, we have used immunogold labeling to determine whether known adhesive molecules might be localized at points of adhesion and possibly be constituents of the filamentous material. Antibodies to an adhesive molecule (neural cell adhesion molecule [N-CAM]) and to an adhesive macromolecular complex of proteins and proteoglycans (adheron) were localized at the EM level in whole mounts of cultured avian retina cells. Labeling of fixed cells showed that N-CAM and adheron molecules were both present on growth cones and on filopodia. However, filamentous materials extending from the cell surface were labeled with anti-adheron but not with anti-N-CAM. If cells were labeled before fixation, patches of anti-N-CAM labeling occurred in random areas over the growth cones, but adheron antibodies concentrated at points of apparent adhesion. Particularly dense clustering of anti-adheron occurred at individual filopodial tips and at points of contact between pairs of filopodia. The different patterns of labeling imply that N-CAMS do not associate with the main antigenic components of adheron on the membrane surface. Most importantly, the data indicate the N-CAMs were mobile in the membrane but that constituents of adherons were anchored at adhesive loci. An appealing hypothesis is that molecules found in adheron preparations have an important role in establishing the adhesive junctions formed by growth cone filopodia.

Why Alzheimer's is a disease of memory: the attack on synapses by A beta oligomers (ADDLs).

Individuals with early-stage Alzheimer's disease (AD) suffer from profound failure to form new memories. A novel molecular mechanism with implications for therapeutics and diagnostics is now emerging in which the specificity of AD for memory derives from disruption of plasticity at synapses targeted by neurologically active A beta oligomers (1). We have named these oligomers "ADDLs" (for pathogenic A beta-Derived Diffusible Ligands). ADDLs constitute metastable alternatives to the disease-defining A beta fibrils deposited in amyloid plaques. In AD brain, ADDLs accumulate primarily as A beta 12mers (2) (approximately 54 kDa) and can be found in dot-like clusters distinct from senile plaques (3). Oligomers of equal mass have been reported to occur in tgmouse AD models where they emerge concomitantly with memory failure (4), consistent with ADDL inhibition of LTP (1). In cell biology studies, ADDLs act as pathogenic gain-of-function ligands that target particular synapses, binding to synaptic spines at or near NMDA receptors (5,6). Binding produces ectopic expression of the memory-linked immediate early gene Arc. Subsequent ADDL-induced abnormalities in spine morphology and synaptic receptor composition (7) are predicted consequences of Arc overexpression, a pathology associated with memory dysfunction in tg-Arc mice. Significantly, the attack on synapses provides a plausible mechanism unifying memory dysfunction with major features of AD neuropathology; recent findings show that ADDL binding instigates synapse loss, oxidative damage, and AD-type tau hyperphosphorylation. Acting as novel neurotoxins that putatively account for memory loss and neuropathology, ADDLs present significant targets for disease-modifying therapeutics in AD.

Targeting small Abeta oligomers: the solution to an Alzheimer's disease conundrum?

Amyloid beta (Abeta) is a small self-aggregating peptide produced at low levels by normal brain metabolism. In Alzheimer's disease (AD), self-aggregation of Abeta becomes rampant, manifested most strikingly as the amyloid fibrils of senile plaques. Because fibrils can kill neurons in culture, it has been argued that fibrils initiate the neurodegenerative cascades of AD. An emerging and different view, however, is that fibrils are not the only toxic form of Abeta, and perhaps not the neurotoxin that is most relevant to AD: small oligomers and protofibrils also have potent neurological activity. Immuno-neutralization of soluble Abeta-derived toxins might be the key to optimizing AD vaccines that are now on the horizon.

Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer's disease.

Pioneering work in the 1950s by Christian Anfinsen on the folding of ribonuclease has shown that the primary structure of a protein "encodes" all of the information necessary for a nascent polypeptide to fold into its native, physiologically active, three-dimensional conformation (for his classic review, see [Science 181 (1973) 223]). In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) appears to play a seminal role in neuronal injury and death. Recent data have suggested that the proximate effectors of neurotoxicity are oligomeric Abeta assemblies. A fundamental question, of relevance both to the development of therapeutic strategies for AD and to understanding basic laws of protein folding, is how Abeta assembly state correlates with biological activity. Evidence suggests, as argued by Anfinsen, that the formation of toxic Abeta structures is an intrinsic feature of the peptide's amino acid sequence-one requiring no post-translational modification or invocation of peptide-associated enzymatic activity.

Constitutive Alzheimer's-type tau epitopes in a neuritogenic rat CNS cell line.

Paired helical filaments (PHFs) of Alzheimer's disease (AD) largely comprise hyperphosphorylated forms of the cytoskeletal protein tau. AD-type tau phosphoepitopes, detected by various monoclonal antibodies, are absent from normal adult neurons, but recent studies have shown that their expression may contribute to neuritogenesis and axon differentiation in the developing nervous system. Therefore, we have examined a brain nerve cell line that is spontaneously neuritogenic for possible expression of AD-type tau epitopes. The neuritogenic rat brain cell line B103 was found to constitutively produce two-AD related epitopes of tau, detected by cellular immunofluorescence studies with the PHF-1 and Alz-50 monoclonal antibodies. Biochemical studies showed that the antibodies bound to proteins within the molecular, weight range expected for phosphorylated tau isoforms. Further verification was established by use of tau antisense oligomers, which eliminated cellular immunofluorescence due to the AD-related monoclonals and polyclonal anti-tau but did not eliminate fluorescence due to anti-tubulin. Cells treated with tau antisense were not neurite-free. Neurites that remained, however, were abnormal, generally short and wavy in appearance. Cellular distribution of the tau epitopes was found to be particularly interesting. Alz-50 recognized only cytoplasmic tau whereas PHF-1 recognized nuclear tau as well as cytoplasmic. Thus, the two epitopes, are morphologically segregated within the cell. Because subcellular segregation of tau is compromised in Alzheimer's disease, mechanisms that segregate AD-type phosphotau epitopes in B103 cells may have relevance to this neurodegenerative disorder.

Protein kinase C and F-actin are essential for stimulation of neuronal FAK tyrosine phosphorylation by G-proteins and amyloid beta protein.

Focal adhesion kinase (FAK) is a protein tyrosine kinase implicated in signal transduction pathways for integrins, neuropeptides, and lysophosphatidic acid. FAK, first discovered in non-neuronal cells, recently has been reported to occur in neurons, where its tyrosine phosphorylation is upregulated by fibronectin and by the Alzheimer's Abeta peptide. The current work has elucidated molecular events leading to tyrosine phosphorylation of FAK in the rat B103 CNS nerve cell line. Activation of receptor-coupled G-proteins by Mas-7 was found to evoke rapid upregulation of FAK tyrosine phosphorylation (Tyr(P)). Upregulation by Mas-7 was blocked by GF109203X, a potent inhibitor of protein kinase C (PKC). Phorbol ester also upregulated FAK-YP, verifying a role for PKC in the transduction cascade. Upregulation of FAK-YP by activation of G-proteins and PKC was dependent upon intact F-actin, as cytochalasin D abolished stimulation by Mas-7 and by phorbol ester. The relatively slow increase in FAK-YP evoked by chronic exposure to Abeta also was abolished by GF109203X and by cytochalasin D. The results show that tyrosine phosphorylation of FAK in neurons is regulated positively by PKC, functioning down-stream from G-proteins through an F-actin-dependent mechanism. The Alzheimer's Abeta peptide is capable of activating elements of this same signal transduction pathway, via membrane events that remain to be determined.

Alzheimer's-associated phospho-tau epitope in human neuroblastoma cell cultures: up-regulation by fibronectin and laminin.

Alzheimer's-afflicted neurons contain phosphorylated forms of tau that are not present in healthy adults. these can be recognized with great specificity by monoclonal antibodies such as paired helical filament-1 (PHF-1) [Greenberg S. G. and Davies P. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 5827-5831; Greenberg S. G. et al. (1992) J. biol. Chem. 267, 564-569]. The PHF-1 phospho-tau epitope is also present in immature neurons undergoing axodendritic differentiation [Pope W. B. et al. (1993) Expl Neurol. 120, 106-113]. Analogous to its presence in immature neurons, we report here that the PHF-1 tau epitope spontaneously occurs in the human neuroblastoma cell line SHSY5Y, where its level can be regulated by differentiation and by molecules found in the extracellular matrix. Confocal immunofluorescence studies showed PHF-1 epitope to be constitutively expressed in the somatic cytoplasm as well as in short neurites typical of undifferentiated SHSY5Y cells. Induction of differentiation with retinoic acid produced cells with a neuronal morphology and a redistribution of the expression of PHF-1 tau in the long neurites. Protracted exposure to retinoic acid decreased the levels of PHF-1 immunofluorescence without a loss of neurites, similar to the developmental down-regulation seen in situ. The effects of retinoic acid on PHF-1 immunofluorescence were modifiable by fibronectin, which can be released by some neuroblastoma cell lines [Ciccarone V. et al. (1989) Cancer Res. 49, 219-225; Yoshihara T. et al. (1992) Int. J. Cancer 51, 620-626]. Exogenous human fibronectin caused a marked up-regulation of PHF-1 immunofluorescence. Quantitative analysis of 15 multicellular areas, from six different cultures, per experimental condition showed a 16-fold increase compared to untreated controls. Up-regulation by fibronectin was also evident in undifferentiated cells. Cell counts indicated no proliferative effects of the fibronectin under the conditions used. Laminin also caused an increase of PHF-1 tau in retinoic acid-treated cells. Data obtained from immunoblots verified the results observed with immunofluorescence. The data show that the PHF-1 tau epitope is spontaneously expressed by non-degenerating human neuroblastoma cells, down-regulated by cellular differentiation, induced by retinoic acid and up-regulated by the extracellular matrix components fibronectin and laminin. One explanation of the data is that fibronectin maintains a population of SHSY5Y cells in a biochemical state of differentiation in which PHF-1 tau is expressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential regulation of muscarinic and nicotinic receptors by cholinergic stimulation in cultured avian retina cells.

Sustained cholinergic stimulation of retina cells grown in primary aggregate and monolayer cultures regulated the concentration of muscarinic but not nicotinic receptors. Muscarinic receptor sites, quantified by the binding of [3H]quinuclidinyl benzilate to membranes and the binding of [3H]N-methyl-scopolamine to intact cells, decreased up to 84% following long-term incubation of cultures in muscarinic agonists. This decrease was blocked by atropine and was not induced by chronic nicotine treatment. The rate of the muscarinic response was biphasic. A rapid binding decrease of 30% occurred within 15 min. The slower phase was half-maximal by 6 h and was complete by 24 h. Neither the fast nor the slow receptor loss was reversed by the guanine nucleotide GppNp. Three different depolarizing agents (gramicidin D, protoveratrine, and ouabain) blocked the cholinergic-induced receptor loss, but the hyperpolarizing ionophore valinomycin had no effect. In contrast to the muscarinic response, nicotinic receptor binding was not altered by chronic receptor stimulation. Exposure to receptor-saturating doses of carbamylcholine or nicotine for 48 h did not change [125I]alpha-bungarotoxin or [3H]bromoacetylcholine binding. Differential regulation of acetylcholine receptors is discussed in relation to the possible physiological role of receptor regulation by receptor activity.

Differential ontogenesis of D1 and D2 dopaminergic receptors in the chick embryo retina.

The differentiation of D1 and D2 dopamine receptors was investigated during the ontogenesis of the chick embryo retina. Our results reveal an interesting complexity in dopaminergic differentiation, with one major receptor system developing before synapses and another one developing after. The dopamine-dependent increase of chick retina cAMP level differentiates early during retina ontogeny. By the embryonic day 10-11 10(-4) M dopamine and ADTN elicit a 13-fold increase in cAMP content of the retina. However, [3H]spiperone (D2 ligand) binds very little to crude membrane preparation of retinas from embryos in the same developmental stage (12-13 fmol/mg protein). High specific binding of [3H]spiperone is only detected after the embryonic day 17-18, attaining 80 to 100 fmol of specific spiperone binding sites in the retinas from post-hatched animals. Apomorphine also promotes the accumulation of cAMP of retinas from early embryonic stages. However, it is only 20-30% as effective as ADTN or dopamine. In addition, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine effect remains practically constant throughout this period. Both dopamine and apomorphine are equally effective in eliciting cAMP accumulation of retinas from post-hatched animals. Moreover, apomorphine is a potent inhibitor of dopamine-induced cAMP level of the embryonic tissue. The results presented here indicate that D1 and D2 receptors differentiate independently from each other, and that apomorphine elevates retina cAMP levels via a subclass of D1 receptors that does not desensitize significantly during retina development.

Soluble proteins from rat olfactory bulb promote the survival and differentiation of cultured basal forebrain neurons.

A previous study of cholinergic development indicated a possible trophic relationship between the olfactory bulb and its afferents from the basal forebrain (Large et al., J. Neurochem., 46 (1986) 671-680). To examine this possibility further, cultured embryonic basal forebrain neurons from rat were used as a test system for trophic factor activity hypothesized to be present in olfactory bulb. Basal forebrain neurons grown in defined medium typically died within 2-3 days. However, survival and differentiation were strikingly enhanced by soluble extracts of olfactory bulb tissue. This trophic effect was noticeable with 2 micrograms/ml olfactory bulb protein, and plateaued at 100 micrograms/ml. The activity was heat- and trypsin-sensitive, non-dialyzable, stable in the cold, resistant to NGF antiserum, and approximately 100-150 kDa in size. Nerve growth factor, bovine serum albumin, laminin and extracts from heart did not mimic the activity. Long-term growth (21 days) in the presence of olfactory bulb proteins resulted in extensive neurite production, formation of thick neurite fascicles, and aggregation of cells. Some glia were present, as evidenced by the presence of glial fibrillary acidic protein, and large numbers of cells were positive for neuron-specific enolase and true acetylcholinesterase. Trophic activity was also present in medium conditioned by olfactory bulb slices, implying secretion of active factors.

L-glutamate evoked release of GABA from cultured avian retina cells does not require glutamate receptor activation.

gamma-Aminobutyric acid (GABA) and L-glutamate are the major inhibitory and excitatory transmitters in the central nervous system. Recent evidence has indicated that L-glutamate may stimulate GABA release by a novel exchange mechanism (Nascimento and De Mello, J. Neurochem., 1985, 45: 1820-1827). Here we provide strong support for this hypothesis by showing that the L-glutamate-evoked release of [3H]GABA from cultured avian retina cells is not dependent on the activation of excitatory amino acid receptors. Retina cells were found to incorporate [3H]GABA into a pool that was released when cultures were treated with L-glutamate (100 microM). This release was unaffected when calcium ions were removed, but was prevented when NaCl was replaced by LiCl. D-Aspartate, which in tracer experiments was shown to be taken into cells by the same carrier as L-glutamate, was also able to evoke release of [3H]GABA, with the same requirement for NaCl. In addition, L-glutamate and D-aspartate uptake by retina cells was inhibited in more then 80% when the uptake was measured in the presence of LiCl. As opposed to GABA, the release of acetylcholine (ACh) promoted by L-glutamate showed characteristics of classical mechanisms of neurotransmitter release. Glutamate-induced efflux of ACh was Ca2+-dependent and was not affected when NaCl was replaced by LiCl. Also, D-aspartate was ineffective in eliciting the release of ACh. Even at high concentrations, antagonists of excitatory amino acid receptors were unable to diminish the glutamate-evoked release of [3H]GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular mapping of m2 muscarinic receptors in rat olfactory bulb using an antiserum raised against a cytoplasmic loop peptide.

An antiserum that recognizes a sequence from the putative third cytoplasmic loop of the m2 subtype of muscarinic receptors (mAchR) has been raised and used to map the cellular distribution of this subtype in rat olfactory bulb. The antiserum was obtained by injecting BALB/C mice with a BSA-conjugated synthetic peptide whose sequence corresponded to amino acids 240-259 of the porcine cardiac m2 mAChR gene. Antibodies recognized the synthetic peptide in ELISA screening and labelled a single band corresponding to the peak of [3H]PrBCM-labelled heart mAchRs in immunoblots. Immunostaining of olfactory bulb, a region of the brain enriched in this muscarinic receptor subtype, showed that the antibodies labelled cell bodies and multiple dendritic processes. Broad fluorescent labelling throughout cell bodies was consistent with binding to the cytoplasmic face of the surface membrane, in support of the predicted cytoplasmic loop structure. m2-Positive cells throughout the bulb were sparsely distributed in different layers representing small subpopulations of the cells in each region: glomeruli, 6%; external plexiform layer, 16%; inner plexiform and granule cell layer, 3%. The results show that antibodies against specific sequences of different muscarinic receptor subtypes can be used to localize subtypes in situ, that the m2 subtype within the rat olfactory bulb is broadly distributed, and that the m2 subtype can occur postsynaptically in this central nervous system (CNS) region. The mapping of m2-positive cells in olfactory bulb may be of particular interest because loss of this subtype and degeneration of the olfactory system have been observed in Alzheimer's disease.

Biochemical differentiation of nascent neurite junctions: unilateral localization of adheron components.

Previous work showed that adhesive contacts made by growth cone filopodia involve extracellular filaments that can be labeled with an antiserum to adherons (adhesion-promoting complexes in conditioned medium). Here, adheron antigens were found to be differentially expressed by particular cell types and synaptic layers during chick retina development, and this differential expression at the cellular level was retained in culture. When applied to living cells, adheron antibodies characteristically patched at filopodial junctions. Monospecific antibodies to purpurin and a heparan sulphate proteoglycan (HSPG), two components of adherons, labeled a subpopulation of junctions. Most interestingly, anti-purpurin and anti-HSPG bound only to one end of adhesive filaments. Such localization to discrete microdomains provides support for a heterotypic adhesive mechanism in junction formation.

Ultrastructure of individual neurons isolated from avian retina: occurrence of microtubule loops in dendrites.

To investigate the cytoskeletal organization of neurons differentiating in vivo, we developed a procedure for isolating single arborized chick retina neurons, using papain and EGTA, and examining their structure in whole mounts. Ultrastructure of neurite tips and many regions along the neurite could be examined in detail in these preparations. Twenty to 25 nm linear elements which made tight 180 degree turns and returned to the original neurite were commonly observed in both detergent-extracted and intact whole mounts. The looped structures were identified as microtubules using antibodies to chick brain tubulin. Microtubule loops were prevalent in neurites at all ages examined, embryonic day 7-10 days post-hatch (E7-P10), but loops increased in frequency from being present in 24% of E7 neurites to 64% of E16 neurites. Often several neurites from the same cell contained microtubule loops, implying that at least some neurites with microtubule loops were dendrites.

Autoradiography of dendritic acetylcholine receptors: a method for study of isolated neurons of the adult central nervous system in the turtle.

Adult neurons were isolated from turtle retina in order to study the subcellular distribution of muscarinic acetylcholine receptors. Retinas were perfused in vitro with [3H]quinuclidinylbenzilate [3H]QNB) to label muscarinic receptors, and individual neurons with extensive dendritic arborization were obtained for autoradiography by use of papain and trituration. Papain caused no change in the number of muscarinic receptor sites nor in their affinity for [3H]QNB. Receptors, indicated by the positions of silver grains, were localized to dendritic processes.

A beta peptide enhances focal adhesion kinase/Fyn association in a rat CNS nerve cell line.

Neurotoxicity of the amyloid beta protein (A beta) is known to correlate with a selective change in protein tyrosine phosphorylation (Tyr(P)) of focal adhesion kinase (FAK) (Zhang et al., J. Biol. Chem., 269 (1994) 25247-25250). The current work has found that exposure of neuronal cells to A beta upregulates the stable association of FAK with Fyn, a neuronally-enriched protein tyrosine kinase of the Src-family. In cells incubated with aged A beta 1-42, the amount of immunoprecipitable FAK-Fyn complex increased approximately 280%. Equivalent results were obtained whether anti-FAK or anti-Fyn was used to precipitate the complex. Cells incubated with non-toxic A beta 17-42, which makes aggregates and attaches to cells but does not upregulate FAK Tyr(P), exhibited no increase in FAK-Fyn complex. Aberrant Fyn activity due to the A beta evoked association with FAK could play a role in neuronal degeneration and also cause anomalies in synaptic plasticity. These possibilities are of particular significance because of the reported increase in Fyn immunoreactivity in Alzheimer's-afflicted neurons (Shirazi and Wood, NeuroReport, 4 (1993) 435-437).

A transient embryonic dopamine receptor inhibits growth cone motility and neurite outgrowth in a subset of avian retina neurons.

To investigate the possible developmental significance of a transient dopamine receptor in the avian central nervous system, we examined the effects of dopamine on the morphology and motility of cultured retina neurons. Neurite arborization was significantly reduced by chronic dopamine stimulation. Using continuous video microscope monitoring, we observed that a subset of neurons responded to short-term dopamine with decreased filopodial activity and retracted neurites. The effects of dopamine could be blocked or reversed by haloperidol or SCH23390 and forskolin produced a response similar to dopamine, indicating the morphological changes were mediated by D1-receptor stimulation of adenylate cyclase.

Interaction of beta-amyloid peptides with integrins in a human nerve cell line.

beta-Amyloid accumulates as extracellular aggregates in Alzheimer's-afflicted brain tissue, but it also is secreted by healthy tissue, for reasons not yet established. One possibility is that beta-amyloid, which contains a sequence (RHDS) homologous to the cell-binding domain of fibronectin, may modulate integrin function, a possibility supported by previous data from non-neuronal cells (Ghiso et al., Biochem. J., 288 (1992) 1053-1059). The current work shows that functional interaction with beta-amyloid peptides is also supported by integrins in neuronal cells. Experiments used the SH-SY5Y human neuroblastoma cell line, which was shown to contain integrins that mediated cell adhesion to substratum-bound fibronectin. Adhesion to fibronectin was partially blocked by synthetic beta-amyloid peptides containing the RHDS sequence. beta-Amyloid sequences adsorbed to substratum themselves were found to mediate cell adhesion, although less effectively than fibronectin. Anti-integrin blocked the peptide-mediated adhesion, at doses commensurate with those blocking fibronectin-mediated adhesion. The data support the hypothesis that beta-amyloid peptides could physiologically, and perhaps pathogenically, modulate the activity of neuronal integrins, important cell surface receptors known to control protein kinase activities, Ca2+ levels, gene expression and organization of the cytoskeleton.

Delivery of membrane-impermeant fluorescent probes into living neural cell populations by lipotransfer.

Use of fluorescent probes to monitor f-actin in living cells typically relies on difficult microinjection procedures. The current work has developed cationic lipotransfer of membrane-impermeant probes as an alternative to microinjection. BODIPY FL-phallacidin, a fluorescent f-actin probe, was packaged into 40-50 nm cationic liposomes. Packaging, verified by gel filtration, enabled delivery of the probe into living nerve cells and provided an image of f-actin that was identical to that seen in fixed, permeabilized cells. Phallacidin alone did not enter living cells, nor was its uptake stimulated by the presence of empty liposomes. All predicted f-actin structures were fluorescent in living cells, indicating a high efficacy of delivery. Cationic lipotransfer of fluorescent probes was rapid, not disruptive to cells, and delivered a probe en masse to a large sample population. Lipotransfer appears to be a promising alternative to microinjection for introducing membrane-impermeant probes and reagents into living cells.