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Gene duplication in the genome of parasitic Giardia lamblia

Background Giardia are a group of widespread intestinal protozoan parasites in a number of vertebrates. Much evidence from G. lamblia indicated they might be the most primitive extant eukaryotes. When and how such a group of the earliest branching unicellular eukaryotes developed the ability to successfully parasitize the latest branching higher eukaryotes (vertebrates) is an intriguing question. Gene duplication has long been thought to be the most common mechanism in the production of primary resources for the origin of evolutionary novelties. In order to parse the evolutionary trajectory of Giardia parasitic lifestyle, here we carried out a genome-wide analysis about gene duplication patterns in G. lamblia. Results Although genomic comparison showed that in G. lamblia the contents of many fundamental biologic pathways are simplified and the whole genome is very compact, in our study 40% of its genes were identified as duplicated genes. Evolutionary distance analyses of these duplicated genes indicated two rounds of large scale duplication events had occurred in G. lamblia genome. Functional annotation of them further showed that the majority of recent duplicated genes are VSPs (Variant-specific Surface Proteins), which are essential for the successful parasitic life of Giardia in hosts. Based on evolutionary comparison with their hosts, it was found that the rapid expansion of VSPs in G. lamblia is consistent with the evolutionary radiation of placental mammals. Conclusions Based on the genome-wide analysis of duplicated genes in G. lamblia, we found that gene duplication was essential for the origin and evolution of Giardia parasitic lifestyle. The recent expansion of VSPs uniquely occurring in G. lamblia is consistent with the increment of its hosts. Therefore we proposed a hypothesis that the increment of Giradia hosts might be the driving force for the rapid expansion of VSPs.

Background
Giardia are a group of flagellated unicellular protists which are the most common infective parasites of a number of vertebrates. For example, G. lamblia is a common human parasite. In the United States, about 20,000 cases of giardisis are reported each year [1]. Aside from being a prevalent pathogen, in the last two decades G. lamblia has caught a lot of attentions, as being the most primitive eukaryotes [2]. Phylogenetic and cellular evidence indicate that this organism might branch away from the ancestor of extant eukaryotes around the endosymbiotic origin of mitochondria in eukaryotes [2][3][4][5]. Therefore before the emergence of multicellular animals, G. lamblia may have survived freely in the world for several hundred million years [6]. It suggested that later on it developed the ability to successfully parasitize vertebrates, as it is now recognized as one of the most prevalent intestinal parasites in a variety of vertebrates from amphibians to mammals [7]. An intriguing question is how this ancient eukaryote became an obligate parasite in the later multicellular animals. The draft genome sequence of G. lamblia provides us an opportunity to uncover what genomic features resulted in its parasitic lifestyle [8]. Comprehension of how the parasitic ability developed would not only be of evolutionary biological significance, but also shed light on the mechanism of giardisis.
Genetic novelties emerge in organisms by creation of new genes through three major mechanisms: de novo creation, lateral gene transfer and gene duplication [9,10]. The Origin of new genes de novo in G. lamblia is impossible to detect because of the deficiency of close relatives in the lineage. Lateral gene transfer (LGT), which is a predominant force of acquisition of new genes in many microorganisms [11], may play an important role for the adaptive evolution of G. lamblia in animal intestines because half of the 15 LGT genes identified are associated with its surveillance in an anaerobic environment [12]. However, gene duplication, which has long been thought to be the primary mechanism in producing resources for the origin of evolutionary novelties, has not yet been thoroughly studied in G. lamblia. The most obvious contribution of gene duplication to organisms is that it provides genetic material to generate neo-function or sub-function while maintaining the original function of duplicated genes [9,13]. Moreover, the generation of duplicated genes can increase genetic robustness within cellular networks [14]. The dynamic evolution of duplicated genes inflects adaptive evolution of organisms under varying environments [15,16]. Therefore there is no doubt that gene duplication is extremely pervasive, conducting function in almost all organisms from prokaryotes to eukaryotes [9,13].
Previously, many studies on fungi, plants and animals have shown that gene duplication contributes novelties for their adaptive evolution [17][18][19][20][21]. In order to investigate the impact of gene duplication on the parasitic lifestyle of G. lamblia, we surveyed and depicted the evolutionary relationships of all the duplicated genes in its genome. Our results showed that two rounds of large scale duplication events took place in the evolutionary process of G. lamblia. Furthermore, most of the recent duplicated genes in the second round duplication events are VSP genes, which are essential for the parasitic properties of G. lamblia that utilizes them to evade the host's immune response [22]. Largely expanded VSP genes are helpful to parasitize a variety of hosts, because they allow G. lamblia to enact a more complex regulation of VSPs in different hosts and at different times [23][24][25].

Identification of Gene duplication events in G. lamblia
Gene duplication and subsequent divergence of the duplicated copies provide opportunities to generate neofunction for adaptive evolution of organisms in varying environments. Identification of duplicated genes is valuable to understand the adaptive evolution of organisms. In order to identify all putative gene duplication events in G. lamblia, a very ancient organism which have survived in the world for several billion years [6], a global survey of protein similarities was conducted using the BLASTP program with loose parameters (E-value < 10 -4 ) [26]. After all-against-all alignments for the entirety of proteins in G. lamblia, we detected 2,403 duplication genes which cover about 40% of the total proteins in G. lamblia. Duplicated genes usually can be classified into tandem, segmental and dispersed duplicates. According to the location of duplicated genes in the assembled contigs, we found only 23 tandem duplicated genes whose original gene and duplicated copy are tandemly located on the same contig. Additionally eight genes were involved in segmental duplication events, which resulted in two or more duplicated genes located in the same contig (Additional file 1: The list of 2,403 duplicated genes). These results imply that the majority of duplication events happened dispersedly.
In order to further analyze the evolutionary scenario of these duplication events, subsequently, a cluster analysis was done based on sequence divergence of the duplicated genes. Briefly, we constructed a matrix with 2,403 rows and columns where each represented a duplicated gene. The amino acid similarities for each gene pair in the row and column were then extracted from the BLASTP results. Based on this matrix, a hierarchical clustering was conducted by the program AGNES (agglomerative hierarchical clustering algorithms) where proteins are clustered closer if their protein sequences have higher similarity (Materials and Methods) [27]. Interestingly, as shown in figure 1, roughly 30% of the duplicated genes were classified into two large duplicated groups: 235 genes in Group I and 500 genes in Group II. The rest of the 1,668 genes formed many small gene groups. A control dataset which contains these 1,668 genes (Group III) was also constructed, in order to see if the evolutionary pattern of the two large groups is different from these small groups. Comparison of average similarities among genes in the three groups showed that the average similarity for genes from Group I (35.26 ± 0.09) is significantly higher than those from Group II (31.32 ± 0.02) and III (29.03 ± 0.18). This implies perhaps the majority of duplication events in Group I took place more recently than those in Groups II and III did.

Two rounds of large scale duplication events happened in G. lamblia
In order to further clarify the evolutionary order of the duplication events in each group, we defined the best hit for each duplicated gene as its direct parental gene (Materials and Methods). Due to a large divergence in sequence, it is impossible to detect the parental genes for some ancient duplicated genes. Fortunately, we identified 1,907 pairs of parent-daughter relationships among all duplicated genes in G. lamblia, but the rest of the 496 duplicated genes lacked detectable parental genes. To gauge the evolutionary distances for each parent-daughter pair, we used non-synonymous distance (dN) and synonymous distance (dS) [28,29] (See Additional file 1 for the list of dN and dS value for all duplicated pairs). As shown in figure 2A, most genes in Group I were created very recently compared with the genes in Group II and III. For example 56% of the genes in Group I have dS smaller than 1, whereas the genes with such a low dS in Group II and III are only 11% and 17%, respectively. Thus, more than half of the duplicated genes in Group I were originated recently. Since the larger dS values for most duplicated genes in Group II and Group III might result from reverse mutations at synonymous sites, the evolutionary distances for a large number of duplicated genes in Group II and III cann't only be based on dS. As a result, we turned to dN.
Although positive selection would accelerate nonsynonymous mutation rate in a short period, in the long term, especially for duplicated genes in Group II and III with such a large divergence, dN is possible to use as an index of evolutionary time. Therefore we used dN to characterize the duplication events within the three groups. Unexpectedly, the distribution of dN values for the totality of duplicated genes clearly shows that there were two rounds of gene duplication events in the genome of G. lamblia ( Figure 2B): the first round of duplication events that were mainly enriched in Groups II and III happened earlier, while the second round of events focusing on Group I occurred very recently. Larger dN values in Groups II and III maybe the result of functional relaxation of the genes within both groups. However functional relaxation of genes in Groups II and III is not enough to explain why the dN values in both groups are higher than those in Group I, due to no significant difference between their dN/dS ratio (P-value of t-test is 0.5 between dN/dS from Group I and II and 0.15 between Group I and III). Based on the distribution of dN values, we arbitrarily defined two types of duplicated genes in each group: recent duplicated gene (RDG) with dN < 0.5 and ancient duplicated gene (ADG) with dN > 0.5. Thus 68.5% of the genes in Group I belongs to RDG. This proportion is much higher than those in Group II (13.4%) and III (16.5%). Therefore no matter what methods we utilize, duplicated genes in Group I seem much younger than those in Groups II and III.
Recently duplicated genes in G. lamblia are significantly biased towards VSP genes The current adaptation of an organism relies on recent genomic contents. We are interested in testing if the recently duplicated genes in Group I are related to the parasitic life in G. lamblia. In order to do this, we first annotated functional domains for duplicated genes in each group using the Pfam database (Materials and methods). For the two large duplicated gene groups, 86% of the duplicated genes in Group I were annotated as VSP function, while in Group II 72% of the genes were annotated as Ank function and 27% as Pkinase (See Additional file 1 for functional domain annotation). As we mentioned above, more recent duplication genes are rich in Group I than those in Group II and III. By counting RDG and ADG in each group, we found that in Group II more than 80% of Ank genes and Pkinase belong to ADG, while 74% VSP genes belong to RDG in Group I.
Secondly, in order to study the functional distribution of duplicated genes in detail we clustered all proteins from G. lamblia into different gene families by Tribe-MCL [30]. We checked the proportion of RDG and ADG in each gene family to see if functional bias occurred between RDG and ADG. As shown in figure 3, functional bias between RDG and ADG is obvious in most of the gene families (here only families with more than 4 members were listed, Additional file 2 listed the ratio of RDG and ADG in each gene family). Many gene families with important functions had been duplicated long before such as: the Ank domain which possibly plays roles for localization in cells, the Pkinase which may be functional in signal transduction and protein degradation. Interestingly, two types of motor proteins (Kinesin and Dynein) were also significantly enriched in the ADG. It may be possible that such an enrichment of motor proteins were related to the earlier adaptation of G. lamblia, which moved in water by its flagella [31,32]. In addition, six out of the nine gene families enriched in RDG were annotated as hypothesis proteins and the Group I

Group II
Group III 0% 100% Figure 1 Cluster analysis of duplicated genes. The similarities of duplicated genes were denoted with different colors. As the bottom bar showed, the black means the similarity value is 0% and the white means the similarity value is 100%. Group I and Group II were defined based on the cluster results. As a control for comparing analysis, the rest proteins were put together as Group III.
additional three families were Ank, Copine and VSP. Although Ank function is also enriched in RDG, there are only four members in the family, as well as in Copine. It was noticed that six gene families were annotated as hypothesis proteins. Further functional studies of these families would provide more valuable insights into the recently adaptive evolution of G. liamblia. Most of the VSPs were created very recently, which is consentient with our results above. Many studies have shown that VSPs play a profound role in antigenic variation of G. lamblia, which expresses only one of the VSP genes at a particular time and spontaneously switches to varying VSP genes every 6-13 generations [23]. Therefore numerous duplications of VSPs are indispensable for G. lamblia to be able to infect a variety of hosts.
The rapid expansion of VSPs in G. lamblia may be consistent with the evolutionary radiation of placental mammals Evolutionary arms-race is an important driving force for the adaptive evolution [33][34][35]. The most important thing for a successful parasite in this race is to develop a mechanism to allow antigenic variation to escape arrest from the host immune system. VSPs are an essential gene family in G. lamblia to carry out antigenic variation. Therefore, we asked if the rapid expansion of VSPs in G. lamblia is associated with the evolution of its hosts. Comparison of the evolution rate of VSPs in G. lamblia with the divergence of its hosts may provide us some valuable insights. As the host range of Giardia extends from amphibians to mammals, we identified orthologous relationships for all proteins from human to mouse, platypus and fish by InParanoid [36]. We constructed a phylogenetic tree based on the VSP homologs (Materia and Methods. Additional file 3: the phylogenetic tree). Based on amino acid similarities, we found that about 60% of VSPs have higher amino acid similarities than the VSP homologs in human and platypus, while 78% of VSPs have higher amino acid similarities between VSP homologs in human and fish. Many recent duplicated genes underwent positive selection, which would accelerate the evolutionary rate of proteins [37]. dS is presumably considered to be neutral during evolutionary process [37,38]. Thus, we compared the dS values of VSPs with the synonymous substitution rates of orthologous genes from fish to human. As shown in figure 4, about half of the VSPs have smaller dS values than the average dS values of orthologs between human and mouse, and approximately 80% of such duplicated genes have a dS value smaller than the average dS between human and platypus. Although the evolutionary rates in unicellular organisms is more rapid than multi-cellular organisms, for the reason that unicellular organisms usually have shorter generation time, it would be conservative to conclude that VSPs were rapidly expanded in G. lamblia after the period in which platypus separated from the ancestor of human and mouse. This time scale is almost consistent with the evolutionary radiation of placental mammals [39][40][41]. Therefore, it is available to propose a hypothesis that the increment of Giardia hosts is the driving force for the rapid expansion of VSPs.

Discussion
Gene duplication is one of important mechanisms to provide neo-function for adaptive evolution. Systematic surveys of gene duplication in the intestinal parasite G. lamblia provided much more information than what we had previously expected. Although many biological machines in G. lamblia are considerably compact [8], more than 40% of the genes in G. lamblia were identified as duplicated genes in our analysis. This proportion of duplicated genes is similar with those in fly and yeast using the same parameters for BLASTP [42]. Interestingly, we found that a large number of duplicated genes were focused within two large duplicated groups. Further analysis of the two large duplicated groups indicated that there were two rounds of large scale gene duplication events in the evolutionary process of G. lamblia. Based on dN values of identified duplication genes, the first round of duplication events happened a long time ago. Due to the extended time of divergence among these duplicated genes, dS and even dN values would be saturated by mutations. Therefore, perhaps the accumulation of ancient duplication genes might result from a saturation of mutations at non-synonymous sites. However, the dS for most of the genes from the second round duplication events have a relatively small dS value (< 1), which could fall short of the saturation of mutations [29] (Additional file 4: the distribution of dS for the ancient and recent duplicated genes). Gene conversion would also result in very high similarities among duplicated genes [37]. Nevertheless, based on current knowledge, the mechanism for gene conversion is still unresolved in G. lamblia [43]. Thus, these recent duplication events might be authentic and correspond to the adaptive evolution in G. lamblia.
Antigenic variation is an essential mechanism allowing parasitic pathogens to escape from arrest of the host immune system. G. lamblia performs this variation by changing the expression of its VSP genes in a variety of hosts at different time points [23,24,[44][45][46]. Our results showed that 74% of the genes in the second round of duplication are VSP genes. Furthermore, in comparison with other parasitic protists, we found that VSP genes expanded independently in G. lamblia genome (Additional file 5: gene number in each family in the five studies parasitic protists), even when considering its very close relative Spironucleus salmonicida [40]. A probable explanation for the recently rapid expansion of VSP genes in the genome of G. lamblia is that the dramatic expansion was driven by the selection of evasion from the host immune systems. Since most of the hosts for G. lamblia are animals, we analyzed the possible relationships between the evolution of VSPs and the evolutionary rates for orthologous genes from fish to human. Given the shorter generation time in unicellular organisms compared to the multi-cellular organisms, we inferred that at least VSP genes became expanded in G. lamblia after the separation of platypus from the ancestor of placental mammals. At the same time species in mammals also expanded after the divergence from platypus [39][40][41]. Our results indicate an interesting co-evolution pattern for the parasitic G. lamblia in mammals' evolutionary processes. Further, analysis at the genomic level would shed more lights on the understanding of the co-evolution between the parasite and hosts. The percentages of RDG and ADG in each gene family ADG RDG Figure 3 Rates of RDG and ADG in functional categories. The proportions of RDG and ADG in each family with more than 4 duplicated genes in G. lamblia were displayed. X axis means functional categories and Y axis denotes the proportion.

Conclusion
Gene duplication always plays a pivotal role for the adaptive evolution of organisms under changing environments. Although G. lamblia is one of the most primitive eukaryotes, the origin of its parasitic lifestyle is not as long as it's surveillance. Global identification of duplicated genes in the genome of G. lamblia indicated that gene duplication was essential for the origin and evolution of its parasitic lifestyle. Our results advocated that the recent expansion of VSPs uniquely took place in G. lamblia.
Comparison of the evolution of VSPs with the divergence of its hosts indicated that the rapid expansion of VSPs is consistent with the increment of its hosts. Therefore we proposed a hypothesis that the increment of Giardia hosts is the driving force for the rapid expansion of VSPs.

Duplicated genes in G. lamblia
In order to detect all possible duplicated genes in G. lamblia, all-against-all blast search for 5,986 studied proteins in G. lamblia were done by BLASTP program with a loose parameter E-value < 10 -4 . 2,403 proteins which have significant hits with another protein were defined as duplicated genes. The rest (3,583 genes) are single genes. Protein identities between each duplicated pair were used to do cluster analysis by agglomerative hierarchical clustering algorithms (AGNES) in R cluster package. Briefly, a symmetric matrix with 2,403 rows (each one represents a duplicated gene) and 2,403 columns was constructed. And then the amino acid similarities for each gene pair in the row and column were extracted from the BLASTP results. Based on this matrix, we used average method in AGNES to do cluster. Proteins will be clustered closer if they have higher protein similarities. Group I and Group II were defined based on the cluster results. Apart from Group I and II, the rest of the proteins were put together as Group III.

Evolutionary distance of duplicated genes
In order to estimate evolutionary distance of duplicated genes, we used a reverse-searching method to identify a putative parental gene for each duplicated gene. Briefly, based on BLASTP results, the duplicated pairs with the highest similarity were selected from all of duplicated pairs. For the two copies in each duplicated pair, if the first copy has higher similarities with other genes than the second copy, the first copy would be defined as parental gene for the second copy. After this, the defined daughter gene was removed from all duplicated pairs. Then we iterated this process until we can not find daughter gene at all. Finally, we identified 1,906 duplicated pairs with parental-daughter relationships. The software YN00 in the package PAML was used to estimate the synonymous distance (dS) and non-synonymous distance (dN) for each of the duplicated pairs with parental-daughter relationships [29].

Functional domain annotation and Gene family identification
Functional domains for all of the duplicated genes have been detected based on the Pfam database. The sequences for duplicated genes were used as queries to search the Pfam_fs database by the hmmpfam program in the HMMER package 2.3.2 [50]. The cut-off for the search was chosen at E-value < 0.1 according to the advice of the author for HMMER. Finally 1,767 genes were annotated as containing the known functional domain, 636 genes do not contain the known functional domain and were annotated as Hypothesis proteins. In order to study gene family expansion in G. lamblia, Tribe-MCL was used to do family classification among G. lamblia and other four parasitic protists (Cryptosporidium parvum, Entamoeba histolytica, Leishmania major and Plasmodium falciparum).

Comparative evolution analysis of VSPs
Initially using human genes as queries, we identified all orthologous relationships of genes in human with genes in mouse, platypus and zebrafish. Synonymous substitution rates of one-to-one orthologs were used to estimate divergence among these species. The software YN00 in the package PAML was used to estimate the synonymous distance (dS) for all orthologous pairs [29]. The best hit of VSPs in human, mouse, platypus and zebrafish were identified as VSPs homologs. Then the VSPs gene and its homologs were used to construct the phylogenetic tree. All protein sequences were aligned by MUSCLE [51]. The tree was constructed by Clustalw2 after alignment [52].


extfieldsofstudy: Medicine Biology

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Dynamics of peptidergic secretory granule transport are regulated by neuronal stimulation

Background Peptidergic neurons store and secrete the contents of large dense core vesicles (LDCVs) from axon terminals and from dendrites. Secretion of peptides requires a highly regulated exocytotic mechanism, plus coordinated synthesis and transport of LDCVs to their sites of release. Although these trafficking events are critical to function, little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated. Sensory neurons also package opiate receptors in peptide-containing LDCVs, which is thought to be important in pain sensation. Since peptide granules cannot be refilled locally after their contents are secreted, it is particularly important to understand how neurons support regulated release of peptides. Results A vector encoding soluble peptidylglycine α-hydroxylating monooxygenase fused to green fluorescent protein was constructed to address these questions in cultured primary peptidergic neurons of the trigeminal ganglion using time lapse confocal microscopy. The time course of release differs with secretagogue; the secretory response to depolarization with K+ is rapid and terminates within 15 minutes, while phorbol ester stimulation of secretion is maintained over a longer period. The data demonstrate fundamental differences between LDCV dynamics in axons and growth cones under basal conditions. Conclusions Under basal conditions, LDCVs move faster away from the soma than toward the soma, but fewer LDCVs travel anterograde than retrograde. Stimulation decreased average anterograde velocity and increases granule pausing. Data from antibody uptake, quantification of enzyme secretion and appearance of pHluorin fluorescence demonstrate distributed release of peptides all along the axon, not just at terminals.

Background
Communication within the nervous system relies on the stimulus-dependent release of neurotransmitters from vesicles. Neurons contain two vesicle classes competent for regulated exocytosis: small synaptic vesicles (SSVs) and large dense core vesicles (LDCVs, also referred to as secretory granules). Different stimuli regulate secretion from SSVs and LDCVs. Unlike neuropeptide-containing LDCVs, SSVs contain low molecular weight neurotransmitters synthesized in the cytosol [1]. Newly synthesized membrane proteins of SSVs are transported separately to the nerve terminal where they are incorporated into SSVs. Biogenesis and recycling of SSVs at the nerve terminal are closely coupled, since formation may arise from a presynaptic endosomal compartment or directly from the plasma membrane, controlling the ability of neurons to react rapidly to stimulation and sustain neurotransmitter release [2].
In contrast to synaptic vesicles, LDCVs contain peptides synthesized as inactive precursors into the endoplasmic reticulum and transported to the trans-Golgi network, to be packaged into immature LDCVs and transported down the axon [3]. These LDCVs undergo a series of maturation steps, transforming them into regulated exocytotic carriers. This biosynthetic pathway precludes local refilling, but LDCVs may engage in incomplete discharge of their contents [4]. LDCVs also deliver integral membrane proteins such as opiate receptors and ion channels to the cell surface in a regulated manner [5][6][7][8][9].
Little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated in neurons. Most previous studies have utilized neuroendocrine cells such as PC12 or chromaffin cells, which preludes the analysis of events that occur during transport along long axons [4,10] Comparing the short-range motions of LDCVs in developing hippocampal neurons to their motions in neuroendocrine cells has demonstrated differences; the immobile fraction of LDCVs in neurons is smaller, and mobile LDCVs participate in exocytosis [11], while chromaffin LDCVs are usually immobile for several seconds before secretion [4].
Several lines of evidence suggest that LDCV mobility is responsive to secretagogue stimulation. In neuronally derived NS20Y cells, LDCV velocity is increased by forskolin but not by high K + [12]. At the Drosophila neuromuscular junction, mobilization of synaptic peptidergic LDCVs occurs following a depolarizing stimulus independent of axonal transport motors or F-actin polymerization [13]. Moreover, after synaptic peptide content has been depleted, neuropeptide stores are replenished in en passant terminals using retrogradely transiting LDCVs, not anterograde LDCVs [14].
The trigeminal ganglion contains primary afferent neurons which relay sensory information from craniofacial tissues. These neurons have axons but not dendrites. In vivo, 40-50% of these neurons contain calcitonin gene related peptide (CGRP); a subpopulation (20%) of these CGRP-positive neurons also express substance P [15]. These peptides have been implicated in the transmission of nociceptive information [16]. When dissociated trigeminal ganglion neurons are cultured in the presence of nerve growth factor (NGF), the majority show a peptidergic phenotype. NGF both supports survival of these neurons [17] and causes an increase in peptide expression [18].
LDCV dynamics in neurons have been described as either diffusive or directed, with mobilities that vary over a wide range [11]. However, these studies have not differentiated between LDCVs in transit versus those at exocytotic sites. Using primary trigeminal ganglion neurons as a model peptidergic system, we first sought to describe the dynamic behavior of LDCVs under basal conditions. We compared the behavior of LDCVs in growth cones to those in the distal portion of axons adjacent to the growth cone. We evaluated the effects of two different secretagogues on transport dynamics, including both cumulative and instantaneous parameters. Secretion was assessed by expressing a fluorescently tagged LDCV enzyme.
The PCR product and the pEGFP-N2 vector were both digested with EcoRI and NotI; the pHluorin insert was ligated into the vector, replacing EGFP (now referred to as p.pHluorin-N2). The reading frame was then corrected using the Stratagene QuickChange method of site-directed deletion mutagenesis with the sense primer 5'-CTGCTAGGAGAAAGGGAAGAA-TTCATGAGCGGAAG-3' and antisense primer 5'-CTTCCGCTCATGAATTCTTCCCTTTCTCCTAG-CAG-3'. Next, PHM was removed from the pBluescript PAM-1 vector by digestion with XmnI and HindIII. The PHM fragment was inserted into the p.pHluorin-N2 vector digested with EcoRI, blunted with Klenow, followed by HindIII digestion, yielding PAM-1 residues 1-407 fused in reading frame with pHluorin. The construct was verified by DNA sequencing.

Cell culture and transfections
Trigeminal ganglia were dissected from P3-P5 Sprague Dawley rat pups, digested sequentially in collagenase and trypsin, and mechanically triturated to create a single cell suspension. Cells were passed through a 70 μm nylon cell strainer, and resuspended in 3 ml L15 containing 1 mg/ml fatty acid free bovine serum albumin (BSA). This suspension was layered on top of 6 ml L15 containing 10 mg/ml BSA, centrifuged at 100 × g for 3 minutes, and the supernatant removed. This density gradient and centrifugation procedure was repeated to enrich cultures for neurons. The final cell pellet was either resuspended in L-15 growth medium (containing 10% fetal calf serum, 50 ng/ml nerve growth factor [NGF, BD Biosciences], 50 mM glucose, 250 μM ascorbate, and 8 mM glutathione) or in Rat Neuron Nucleofector solution (Amaxa). For nucleofection, the cell suspension was added to 10-12 μg DNA (for approximately 24-28 ganglia) and program G-13 was used with the Nucleofector II. Cells were resuspended in growth medium, and plated onto collagen coated 0.17 mm glass coverslips or 4 well plastic culture dishes.

Live cell imaging
Live neurons were imaged in L-15 without phenol red and maintained at 30°C with a Bioptechs DeltaT environmental control system. Imaging was performed on a Perkin Elmer Ultraview spinning disk confocal microscope, with image acquisition by ImageSuite software. Selective photobleaching was done using a Photonics Micropoint Mosaic digital diaphragm system. Z-stacks of 5 μm thickness (0.3 μm intervals) were acquired continuously (1.7 sec/stack, 2 × 2 binning) for 5 minutes under basal conditions, during stimulation with 1 μM phorbol 12-myristate 13-acetate (PMA) or with 50 mM KCl. This mode of imaging allowed tracking LDCVs for greater distances along the axons, moving up and down with respect to the tissue culture dish. It does place an upper limit of~0.4 μm/sec on LDCV velocity for the tracking software to recognize the same LDCV (4 pixels in 1.7 sec). Visual inspection would have identified LDCVs moving faster than 0.5-1 μm/sec and none were seen.
To image exocytosis of PHM-pHluorin, transfected neurons were maintained at 35°C with a Zeiss stage heater, and lines scans were continuously recorded in the presence of secretagogue in line scanning mode with a Zeiss LSM 510 confocal microscope. The line selected for imaging was oriented so that it ran through the center of both an axon and a growth cone. The focal plane chosen for imaging was at the interface of the cell and coverslip. For these experiments, neurons were co-transfected with PHM-pHluorin (8 μg DNA) and pDsRed-Monomer (1 μg, Clontech) to facilitate identification of transfected cells; expression of PHM-pHluorin was verified at the conclusion of the experiment via addition of 5 mM NH 4 Cl to the medium.

Image analysis
Maximum-intensity projections were generated from zstacks and used for motion tracking analysis. An automated workfile was created to identify and track individual LDCVs in sequential frames using the Simple PCI motion tracking module (Compix). Identification involved passing images through a smoothing filter and setting an intensity threshold. Selected objects that appeared to be touching each other were separated, and inclusion criteria for area and roundness set identically for all image sequences. Centroid X and Y positions were calculated by the software to produce coordinates for each object in every image frame in which it was identified. Any object tracked for less than 7 frames was excluded from further analysis. Tracks were initially verified manually by checking that the center of gravity as defined by the software corresponded to the same LDCV at each timepoint throughout its trajectory. Velocity, direction, linearity, and processivity were then calculated for each LDCV being tracked. Velocity, direction and linearity for a whole run (several time points) were taken from the Simple PCI analysis. For instantaneous velocity and direction, the X-Y coordinates for each time point were used to calculate distance traveled in the time bin (hence instantaneous velocity) and the X and Y movements were used to calculate direction (0-360°). The definition of anterograde/retrograde was made using images of the axon or ending being studied.

Antibody internalization
Neurons expressing PAM-GFP were incubated with PHM antibody (JH1761, 1:100) [19] for 15 minutes in basal medium or during stimulation with 1 mM PMA or 50 mM KCl. Cells were rinsed, fixed in 4% formaldehyde, and processed for immunocytochemistry as describe above.

Secretion experiments and enzyme assays
Cells were rinsed 3 times with pre-warmed L-15, collections were made of basal secretion, followed by stimulation with 1 μM PMA or 50 mM KCl in L-15 (5, 15, or 30 minutes each) made by mixing L-15 with isotonic 150 mM KCl. When indicated, temperature was used as a variable and cells were kept at either room temperature or above 30°C for the duration of the experiment. Medium was centrifuged to remove non-adherent cells, and protease inhibitors added. Cells were harvested in 20 mM Na-N-Tris [hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES), 10 mM mannitol, and 1% Triton X-100, pH 7.4 (TMT) with protease inhibitors [19], extracts were frozen and thawed three times and centrifuged to remove debris. Cell extracts and media samples were analyzed for PHM secretion by enzyme assay using 125 I labeled α-N-acetyl-Tyr-Val-Gly as substrate [20]. Samples were assayed in triplicate, and reactions were carried out for 3 hours.

Use of PHM-GFP to monitor trigeminal ganglion neuron peptidergic LDCVs
In order to investigate the dynamic behavior of LDCVs in living neurons, we utilized a primary culture system of trigeminal ganglion neurons ( Figure 1). In our culture conditions, immunocytochemical analysis showed that greater than 95% of β III tubulin-positive neurons expressed either CGRP [21] or substance P or both ( Figure 1). Since nociceptive neurons can be identified by their small soma size (diameter < 25 μm), all neurons analyzed met this criterion.
The soluble secretory protein PHM-GFP is efficiently targeted to LDCVs in endocrine cells [19] and was therefore expressed in trigeminal ganglion neurons. The fusion protein localized to puncta distributed throughout the cell soma and axonal processes. Simultaneous visualization of endogenous CGRP ( Figure 2A) or substance P (not shown) revealed substantial colocalization. LDCVs accumulated at a higher density in growth cones ( Figure 2B, green), which are the site at which regulated release is generally studied. Growth cones contained a large amount of filamentous actin ( Figure 2B, red), while microtubules were more prevalent in axons ( Figure 2B, blue).
Localization of PHM-GFP to the regulated secretory pathway was confirmed by quantifying basal and stimulated secretion. We chose two secretagogues with different mechanisms of action for our studies. An increase in KCl depolarizes the cells, potentially triggering action potential initiation, and opens voltage-gated calcium channels to increase secretion in a Ca ++ -dependent manner. Application of phorbol myristate acetate (PMA) activates Protein Kinase C, which phosphorylates various components of the SNARE complex as well as ion channels, causing secretion [22]. In order to select an appropriate temperature for our imaging experiments, we evaluated the effect of temperature on basal and stimulated secretion. Basal secretion of PHM-GFP was similar at 23°C (room temperature) and at 30°C ( Figure 2C). Basal secretion from AtT-20 cells releases processed products from LDCVs, but is not dependent on calcium Figure 1 Trigeminal ganglion neurons are peptidergic. Trigeminal ganglia from P3-P5 rat pups were dissociated and maintained in culture for 2 days. Cells were fixed and processed for immunocytochemistry using antibodies to CGRP (green), substance P (red), and βIII tubulin (blue).  [23][24][25]. Although PMA challenge at room temperature doubled the secretion rate, raising the temperature to 30°C resulted in a further three-fold increase in PHM-GFP secretion, to 8% of cell content in 30 minutes. A similar effect of temperature on stimulated secretion was observed when pituitary cells were evaluated (Eipper & Mains, unpublished). Since regulated secretion was greatly diminished at room temperature, all of our LDCV tracking data were from cultures maintained at or slightly above 30°C.
To select the time period over which LDCV motion was evaluated, we quantified secretion of PHM-GFP for 5 to 30 minutes following application of basal medium or medium containing KCl or phorbol ester at 37°C ( Figure 2D). Stimulated secretion was calculated by subtracting basal from total secretion (blue minus red = gray line). Both PMA and KCl stimulated secretion of PHM within 5 minutes. PMA-stimulated secretion persisted for the duration of the experiment, while KCl-stimulated secretion occurred primarily within 5 minutes and was completed by 15 minutes. After 30 minutes at 37°C, both PMA and KCl had stimulated the secretion of about 15% of the total cell content of PHM-GFP, a larger percentage than observed at 30°C. Based on these experiments, LDCV movement was evaluated in neurons maintained at or above 30°C and data were accumulated within the first 5 minutes after secretagogue application.

LDCV dynamics in axons and growth cones differ under basal conditions
The dynamics of LDCVs were first characterized under basal conditions. Dissociated trigeminal ganglion neurons were transfected with vector encoding PHM-GFP, and imaging experiments performed 24-48 hours later. This timing was chosen because process outgrowth occurred rapidly and growth cones were abundant. The short time made it possible to identify the cell soma associated with the growth cone chosen for imaging and distinguish anterograde from retrograde trafficking. Growth cones of small diameter neurons containing fluorescent LDCVs were identified, and the entire growth cone and the axonal segment immediately preceding it were imaged. Selective photobleaching of axonal segments was performed to facilitate LDCV tracking. Z-stacks were acquired continuously and maximumintensity projections were generated for tracking analysis. Trajectories were generated for all tracked LDCVs based on the coordinates at each point in time; sample trajectories for two LDCVs that displayed different types of behavior are shown ( Figure 3A).
LDCV #219 moved in a rapid and directed manner without changing direction, while LDCV #235 demonstrated slow, dithering motion, with frequent changes in direction. The tracking protocol was validated by manually comparing the trajectories with the actual LDCV motion in several time lapse image sets. In order to distinguish directed from diffusive motion, we used the method of Steyer and Almers to calculate diffusion constants (D) [26] for these vesicles. For the rapidly moving LDCV (#219), D is greater than 1000 × 10 -4 μm 2 /sec; for the dithering LDCV (#235), D = 95 × 10 -4 μm 2 /sec. For LDCVs diffusing freely in the cytosol, D = 10 × 10 -4 μm 2 /sec [26]. Polystyrene beads the size of LDCVs have comparably small diffusion coefficients when studied in viscous media [27,28]. Thus the majority of the movements we are observing are not due to diffusion.
Velocity, linearity, and direction were quantified for basal analysis of LDCVs in axons compared to growth cones. Velocity data of identified LDCV trajectories in growth cones and distal portions of axons were pooled from 6 cells each. Calculation of velocity was done as an average for the entire trajectory of each LDCV analyzed. The distribution of binned data is shown in the histogram ( Figure 3B), with the cumulative plot ( Figure 3C) emphasizing the differences between the two regions. For a given speed on the x-axis of the cumulative velocity plot, the associated fraction on the y-axis gives the fraction of LDCVs moving at, or below, that speed. Under basal conditions, axons contained a subpopulation of LDCVs traveling with greater velocities than those observed in growth cones (p < 0.05, n = 227; marked by asterisks). These LDCVs moved with speeds greater than 0.1 μm/sec. Statistical significance was assessed using the Kolmogorov-Smirnov test [29]. LDCVs moving faster than 0.4 μm/sec would be underestimated due to the time required for the z-stack imaging, but such fast LDCVs were not detected by visual inspection.
LDCV trajectories were further analyzed to determine the distribution of linearity. Linearity was calculated by comparing how close the actual path was to a straight line representing the distance between the first and last point, so that a maximum linearity score is 1.00. As expected, the cumulative plot showed that LDCV tracks in axons were more linear than LDCV tracks in growth cones (p < 0.025, n = 227; marked by bracket) ( Figure 3D).
The net direction of LDCV movement was determined based on the average angle of each trajectory. A score of 0 was assigned for a net angle representing retrograde movement (toward the cell body), and a score of 1 for movement in the anterograde direction (directed toward the ending). Cumulative directionality scores for LDCVs in axons and growth cones did not differ significantly ( Figure 3E). In growth cones, LDCVs moved in both directions with approximately equal frequencies. To our surprise, retrograde transport was slightly favored in the axon.

An intact actin cytoskeleton is required for efficient LDCV transport
The lack of directionality in growth cones as compared to axons could be related to detachment of motor proteins upon entry into growth cones, and to a prevalence of actin over microtubules. We tested whether an intact cytoskeleton were required for efficient LDCV transport by pharmacological disruption of filamentous actin with cytochalasin B. Filamentous actin was visualized using a fluorescent phalloidin conjugate, verifying that cytochalasin treatment resulted in the rearrangement of F-actin into puncta ( Figure 4A, red); microtubules were unaffected ( Figure 4A, green). The effect of cytochalasin treatment on LDCV dynamics was assessed; both cumulative velocity (p < 0.05) ( Figure 4B) and cumulative linearity (p < 0.005) ( Figure 4C) were reduced following Factin depolymerization. Cytochalasin treatment did not have an effect on trajectory direction (not shown). The response observed may reflect a role for LDCV-associated filamentous actin in normal transport or an inhibitory effect of depolymerized actin, and further strengthens our conclusion that the movements observed are not diffusive.

Effects of secretagogue stimulation on cumulative trajectory parameters
In addition to stimulating exocytosis, we hypothesized that secretagogue might regulate LDCV transport, perhaps by increasing directed transport to sites of exocytosis and/or initiating signaling events required to retrieve membrane components for recycling. Analysis of LDCV trafficking in axons during exposure to PMA or KCl was performed as described for basal conditions ( Figure 3); data for each treatment group were pooled from 6 cells each. The distribution of cumulative velocities is shown for LDCVs during basal conditions and during stimulation with PMA and KCl ( Figure 5A). Figure 5 shows that 100% of LDCVs move at speeds of 0.4 μm/sec or slower; from the plot, about half the LDCVs move at 0.025 μm/sec or slower, which may represent simple diffusion (D = 6 × 10 -4 μm 2 /sec). Neither drug had a significant effect on overall LDCV velocity. When linearity was analyzed ( Figure 5B), both PMA (p < 0.01, n = 223) and KCl (p < 0.005) were seen to reduce the fraction of LDCVs exhibiting low linearity of motion.

Effects of secretagogue stimulation on instantaneous trajectory parameters
Since the rapidly changing behavior of LDCVs was not reflected by cumulative measurements, we next assessed the effects of secretagogue on instantaneous velocity and direction. To do this, axons from three neurons were analyzed under basal conditions and after stimulation with KCl or PMA; velocity and direction were calculated for each time point (1.7 sec/stack) in the trajectory using individual LDCV coordinates. The histogram in Figure 6A shows the distribution of instantaneous velocities for each condition; within each group there was no statistically significant difference between axons. LDCVs moving in the retrograde direction were assigned a negative sign, while those moving in the anterograde direction were assigned a positive sign. LDCVs moving less than one pixel in 1.7 sec (<0.08 μm/sec) were categorized as paused.
The overall effect of secretagogue on instantaneous direction is displayed as an average for each treatment condition ( Figure 6B). Under basal conditions, a larger fraction of LDCVs moved in the retrograde direction than in the anterograde direction and less than 10% of the LDCVs were classified as paused. Stimulation with either secretagogue increased the fraction of LDCVs categorized as paused, perhaps corresponding to an "immobile" fraction of LDCVs eligible for release. Stimulation with KCl also resulted in a shift from retrograde to anterograde transiting LDCVs ( Figure 6B). Since LDCVs were tracked for 5 min or less, secretion was not sufficient to bias calculations of directionality ( Figure 2).
Next, we analyzed the relationship between direction and velocity for each time interval ( Figure 6C). LDCVs are transported away from the cell soma at a significantly faster rate than those traveling towards the cell soma under basal conditions. For those LDCVs undergoing retrograde transit, KCl increased instantaneous velocity. PMA was without effect on retrograde velocity, but reduced the average instantaneous velocity of anterograde LDCVs to half of that observed under basal conditions ( Figure 6C). In contrast to its effect of LDCVs undergoing retrograde transport, KCl diminished the velocity of LDCVs traveling anterograde.

PMA stimulation increases LDCV pausing
To be fully or partially secreted, a LDCV must pause or stop, at least momentarily [26,30]. We therefore looked more closely at the pausing behavior of individual LDCVs. The effect of secretagogue on the percentage of time any given LDCV spent paused vs. moving was determined; data were plotted both as a histogram (Figure 7) and as a cumulative plot (not shown). The individual coordinates of LDCVs at each timepoint in the trajectory were used for this calculation. If the coordinates remained identical for two consecutive timepoints, the LDCV was counted as "paused." Under basal conditions, approximately 30% of the LDCVs moved continuously, with no pauses observed. Exposure to PMA decreased the fraction of LDCVs that moved continuously three-fold, to 10% (p < 0.005). In contrast, KCl was without significant effect on the percentage of time a LDCV spent paused. This result is consistent with the ability of PMA to restrict the mobility of LDCVs, which in turn correlates with an increase in priming [31].

LDCV fusion events occur along axons and in growth cones
More frequent stopping of LDCVs in axons is suggestive of docking and fusion events occurring along the length of the axon. To assess this possibility we used an Figure 4 An intact actin cytoskeleton is required for efficient LDCV transport. Trigeminal ganglion neurons were treated with 5 μM cytochalasin B for 1 hour and then fixed and processed for immunocytochemistry (A). Filamentous actin was visualized by staining with a TRITCphalloidin conjugate (red) and microtubules were stained with an antibody to βIII tubulin (green). Inset: phalloidin staining is shown at higher magnification to illustrate puncta that appeared in cytochalasin-treated neurons. Neurons expressing PHM-GFP were pretreated with cytochalasin B in the same manner before live imaging; cumulative velocity (B) and cumulative linearity (C) were plotted as in Figure 3.
antibody to the luminal domain of a LDCV membrane enzyme, PAM. Internalized antibody was subsequently visualized in fixed cells with a Cy3 conjugated secondary antibody. To increase the signal obtained, PAM-GFP was expressed in trigeminal neurons; PAM-GFP was visible in tubulovesicular structures in the cell soma and in vesicular structures throughout axonal processes (Figure 8, left). Under basal conditions, antibody uptake was barely detectable (not shown). When PMA was added to the cultures, uptake of PAM ectodomain antibody was readily assessed; internalized PAM antibody was present in vesicular structures throughout the cell soma (Figure 8, top), along axonal processes and at growth cones (Figure 8, bottom). Internalized antibody (red) was seen in a subset of the PAM-GFP LDCVs (yellow arrows), while other PAM-GFP LDCVs did not contain detectable antibody (green arrows). Uptake was also stimulated by KCl (not shown).
Since this approach did not permit direct visualization of fusion events in real time, we constructed a vector encoding PHM fused to pHluorin, a pH sensitive mutant of GFP [32]. Neurons were co-transfected with vectors encoding PHM-pHluorin and DsRed to enable identification of transfected cells ( Figure 9A). Under basal conditions, the fluorescence of PHM-pHluorin is quenched by the acidic interior of the LDCVs, and is apparent only upon alkalinization ( Figure 9B, green, right). We reasoned that LDCV release would be detectable as PHM-pHluorin was released from LDCVs that fused with the plasma membrane [32]. Diffusion of the secreted PHM-pHluorin would be expected to produce a transient increase in fluorescence at release sites.
In order to detect release of PHM-pHluorin, a horizontal line was placed through the center of the axon and growth cone ( Figure 9A, right), and was continuously scanned during secretagogue stimulation. The transient appearance of green fluorescent streaks presumably correlates with individual sites of exocytosis, since the fluorescence should be visible only when the pHluorin is near neutral pH, during LDCV fusion and secretion; sample recordings during PMA stimulation are shown ( Figure 9C). Each of these events occurred in a different location. The event shown in the left panel occurred in the growth cone between 1 and 2 seconds into the recording, while the event in the center panel occurred in the axon over a 2 second period later in the session. Following image acquisition, NH 4 Cl was added to the medium in order to visualize the total LDCV population containing PHM-pHluorin ( Figure 9C, right). The line scan data acquired after addition of NH 4 Cl showed stabilization of PHM-pHluorin fluorescence over time, as expected; LDCVs were more prevalent in growth cones than in the axon. Based on both antibody uptake experiments and direct visualization of secretion, LDCVs are released from axons and growth cones. In five neurons recorded in detail using line scans, the vast majority of the fluorescence events (presumptive secretion) occurred in axons, not in endings/growth cones.

Discussion
Advantages of the trigeminal ganglion culture system for studying LDCV mobility Our analysis of LDCV dynamics took a novel approach by utilizing primary neurons specialized to produce high levels of neuropeptides. Secretion of peptides such as CGRP and substance P from dorsal root and trigeminal ganglion neurons is an essential component of the transmission of nociceptive stimuli and contributes to peripheral inflammatory responses. In addition, these LDCVs deliver opiate receptors to the cell surface and may be involved in opiate receptor internalization as well [5]. Unlike neuroendocrine cell lines, sensory neurons such as trigeminal ganglion neurons must be equipped to transport LDCVs over long distances to reach their target tissues, both centrally and peripherally. The speed with which these neurons extend long, straight processes makes them especially useful for in vitro trafficking studies.
LDCV mobility is essential for sustaining the secretory response at distant sites of release. New LDCVs must come from the cell soma, the only site at which preproneuropeptides are synthesized and packaged into LDCVs. Since trigeminal ganglion neurons released 15% of their cell content during a 30 minute exposure to PMA or KCl, we hypothesized that these secretagogues would alter LDCV transport. We first compared LDCV transport in axons and growth cones. Differences were expected since long distance movements must occur in axons and secretion is generally assumed to occur at terminals.
Under basal conditions, LDCVs in axons moved with greater average velocities than LDCVs in growth cones. LDCVs moving with average velocities greater than 0.1 μm/second were seen only in axons; these rapidly moving LDCVs accounted for only~20% of the total LDCVs observed. Trajectories were more linear in axons than in growth cones, reflecting the influence of axonal Figure 6 Secretagogues differentially affect instantaneous velocity and direction. Instantaneous velocity and direction were calculated for tracked LDCVs in neurons kept under basal conditions or exposed to PMA or KCl for 5 min. LDCVs moving in the retrograde direction were assigned a negative sign, and those moving in the anterograde direction received a positive sign (A); the distribution of binned data is shown for representative cells from each treatment group. The fraction of LDCVs stopped or moving in a given direction (B) is plotted as an average; the relationship between direction and velocity was also analyzed (C). One-way ANOVA with Bonferroni post-test (SPSS package): *, p < 0.02; **, p < 0.01. geometry. We focused our analysis on the distal segments of axons, the region immediately preceding a growth cone. To our surprise, under basal conditions, LDCVs in this region were slightly more likely to move back toward the cell soma than into the adjacent growth cone.

Differential regulation of dynamics by secretagogues
The most significant effect of secretagogue on average LDCV dynamics was its effect on trajectory linearity. Both PMA and KCl stimulation resulted in more linear LDCV paths in axons, suggestive of motor proteins staying attached longer. The average velocity of transported LDCVs was unaffected by stimulation with KCl or PMA, consistent with the lack of effect of KCl on orphanin FQ containing LDCV velocity in NS20Y cells [12]. Average LDCV velocities in trigeminal ganglion neurons and NS20Y cells were in the same range. Average LDCV translocation rates for orphanin-GFP LDCVs in NS20Y cells were increased by forskolin, which increases intracellular cAMP by activating adenylate cyclase [12]. In PC12 cells, Ba 2+ stimulation increased the mobility of the subset of atrial natriuretic factor-GFP LDCVs located immediately adjacent to the plasma membrane, suggesting that the releasable pool might be expanded by making more LDCVs available to refill it [33]. Mobilization of LDCVs containing ANF-GFP, as detected by fluorescence recovery after photobleaching, was observed following depolarization, while no recovery of bouton fluorescence was observed without stimulation [13]. This liberation was proposed to facilitate replacement of secreted LDCVs, perhaps by allowing directed movement of mobilized LDCVs to other terminals to be recruited for exocytosis. In chromaffin cells, PMA limited the mobility of LDCVs in the evanescent field, and long-range motions were abolished [31]. These authors concluded that PMA increased priming, but not docking, of LDCVs; a similar phenotype was observed by overexpressing the priming factor Munc13-1 [31]. Using wide-field imaging, Shakiryanova et al. correlated a decrease in retrograde transport with an increase in synaptic neuropeptide stores at the Drosophila neuromuscular junction following depletion [14]. They concluded that nerve terminals do not wait passively for new LDCVs to be synthesized and transported from the soma; rather, the "mismatch" between arrival and replacement that occurs under basal conditions is the source of replenishment for the releasable pool during activity [14].
Our spinning-disk confocal microscopy approach allows us to look over a prolonged observation time and to track LDCVs that move up and down with respect to the dish surface, as they traverse the axon. Our average LDCV velocity measurements included 7 to over 30 images (10 to 50 seconds of tracking). The fact that LDCVs paused and then began to move again is not reflected in these average velocity measurements. Since LDCVs must stop at least momentarily before they can be secreted, we quantified the movements made by each LDCV from one image to the image taken 1.7 seconds later; these are designated instantaneous velocities and include all LDCVs anywhere within the region of interest. LDCVs whose centers remained in the same pixel were scored as paused. Secretion of LDCV content would result in diminution or complete loss of signal and termination of that track. Both partial secretion of a docked LDCV, which has been shown to occur [13,14], and loss of motor proteins would be scored as pausing. Although it has been proposed that stopping or pausing before secretion is very brief and might be missed [34], most investigators argue that LDCVs stop for more than 2 sec before secretion, with the majority of LDCVs stopped for over 15 sec before secretion [30].  When we analyzed the effects of PMA and KCl on instantaneous direction and velocity, significant differences were observed. KCl both increased the fraction of LDCVs undergoing anterograde transport and decreased the fraction undergoing retrograde transport. PMA did not alter the anterograde/retrograde distribution of LDCVs. Interestingly, KCl but not PMA increased instantaneous velocity in the retrograde direction, while both KCl and PMA decreased instantaneous velocity in the anterograde direction. Neuropeptides stored in the en passant terminals of Drosophila motorneurons are replenished via capture of retrogradely transiting LDCVs, not by arrival of LDCVs from the soma [14]. These differences in the effects of KCl and PMA on LDCV directionality and velocity suggest differences in the downstream targets of the signaling pathways triggered by these two secretagogues. These observations also suggest that LDCV transport is less like a unidirectional voyage, and more like sightseeing or commuting.
Most LDCVs were found to travel less than 0.2 μm/ sec. Fast axonal transport is typically defined as microtubule-kinesin-dynein dependent movement at 20 mm/d or faster (0.2-4.5 μm/sec), and slower transport within axons has long been recognized to be bidirectional [35]. We found that LDCV movements faster than 0.05 μm/ sec were eliminated by the actin depolymerizing drug cytochalasin ( Figure 4). There is quite a large range of top speeds reported for LDCV movements, from 0.1 to over 2.0 μm/sec [11,14,36]. Some studies reporting faster velocities used differential interference contrast optics, noting that the fastest objects could be mitochondria, lysosomes or phagosomes while stationary or slowly moving objects would not be tracked at all [37]. LDCV speeds and microtubule sliding velocities are usually similar, ranging from 0.1-0.3 μm/sec [38][39][40]. Any LDCV moving more than 4 pixels (0.68 μm) in 1.7 sec would be considered lost (or secreted) by the tracking program (>0.4 μm/sec). Since secretion events for peptides typically take 1-2 seconds (Figure 9 and literature), the tracking software would define that as the end of a track. Given the large number of LDCVs in the axons, the small size of the LDCVs, and the extent to which the axons move in the Z-axis toward and away from the focal plane of the dish surface, we could not resolve LDCVs adequately by epifluorescence and needed the confocal quality of the images.

Nonsynaptic localization of release sites in neurons
The vast majority of previous studies of LDCV dynamics and exocytosis utilized neuroendocrine cell lines as model systems, and nearly all were performed at room temperature. This has included chromaffin cells, which are unpolarized [26,31,36,41], and PC12 cells, which may be differentiated by NGF to extend short neuronal-like processes which accumulate LDCVs at their endings [33]. More recent work used developing hippocampal neurons to analyze motions of LDCVs containing tPA-EGFP; these were found to be highly mobile in growth cones and participate in exocytosis at this location with a slow onset after stimulation [11]. Further studies demonstrated that this fusion protein was localized to dendrites and dendritic spines [42]. LDCVs in spines were found to be immobile under both basal and stimulated conditions, and partial release of the tPA-GFP fusion protein from these LDCVs occurred over an extended period of time. One possibility is that retention of LDCVs and their peptide contents within spines may be a way to regulate the quantity of neuropeptide released, and also provide a potential mechanism to prevent depletion of peptide transmitters at postsynaptic sites [42]. In hippocampal neuronal cultures, secretion from cell bodies can be 50-times higher than from axons, based on total internal reflection microscopy [43].
Our findings that LDCVs containing PAM-GFP or PHM-pHluorin undergo fusion along axons as well as at growth cone/endings in trigeminal ganglion neurons are consistent with a role for non-synaptic peptide release. In fact, a plausible interpretation of Figures 2 and 8 together (8% secretion in 15 min yet a majority of LDCVs showing internalized antibody) is that partial release of LDCV content is more common than full fusion; with full fusion and 8% secretion, one would expect to see only a small number of yellow LDCVs after 15 min of antibody uptake. This may be particularly relevant to the normal physiology of the dorsal root and trigeminal ganglia, in which peptides such as CGRP and substance P participate in both transmission of sensory information from the periphery [44], as well as in peripheral effector functions such as neurogenic vasodilation [45]. The peripheral axons of the sensory neurons do not make synaptic contacts, yet secretion of peptides is known to contribute to local inflammatory processes. Based on our cell culture data showing 15% of PHM-GFP released in 30 min, plus PAM antibody internalization all along the axon at a level comparable to the growth cone, it seems apparent that exocytosis must occur along the length of the axon as well, perhaps serving an autoregulatory or feedback function. Whether this occurs in vivo has yet to be determined. It will be interesting in the future to correlate activity-dependent LDCV dynamics and secretion with physiological functions.

Conclusions
In the peptidergic neurons of the trigeminal ganglion, LDCVs are transported away from the cell soma (anterograde) at a significantly faster rate than those traveling towards the cell soma (retrograde) under basal conditions. Anterograde LDCV velocity is slowed following depolarization by KCl or stimulation with PMA; both methods of stimulation increase granule pausing and LDCV exocytosis. Consistent with this, secretagogue stimulates the uptake of antibody that recognizes the luminal domain of a LDCV membrane protein and also stimulates the distributed appearance of a pHluorintagged LDCV content protein along the axon.