IDENTIFICATION OF SEROTONIN WITHIN VITAL-STAINED NEURONS FROM LEECH GANGLIA

Abstract— We have measured serotonin (5-HT) within large and small neurosomata which are vitally stained by Neutral Red dye. A micro-radioenzymatic technique which is sensitive to 50fmol of 5-HT was employed on intact ganglia, 75 μm Retzius Cells (RZ) and a 10 μm ventro-lateral cell (VL) taken from the leech Macrobdella decora. The stain does not affect the levels of 5-HT in either ganglia or RZ. The VL cell body contains 5-HT at concentrations of at least 100 m m . Microspectrofluorometry of all the ganglionic neurosomata which fluoresce following the Falck-Hillarp formaldehyde condensation reaction detected rapidly-fading emission peaks of 509–523 nanometers. We conclude that all seven fluorescent neurons in the leech ganglion very probably contain serotonin.     

Chromatographic and Histochemical Identification of Dopamine Within an Identified Neuron in the Leech Nervous System

Abstract: Each segmental ganglion of the leech nervous system has two pairs of lateral roots extending to the body wall and viscera. A cluster of about eight neuron cell bodies is located proximal to the first major branch of each anterior root and is termed the anterior root ganglion (ARG). Only one of these eight cells is vitally-stained by Neutral Red dye and fluoresces an intense blue-green following the glyoxylic acid histochemical condensation. The emission spectrum of this anterior root cell (AR) is stable under continuous UV illumination and is bimodal, with peaks at about 480 and 515 nm. This spectrum is indistinguishable from that of millimolar solutions of dopamine (DA) in gelatin droplets following glyoxylic acid histochemistry. We utilized high performance liquid chromatography and an amperometric detector to measure DA within the AR neurosomata at 1.01 pmol/cell. The AR cells in this study had an average diameter of 23 μm and therefore, the minimum intrasomatal concentration of DA is 160 m M , an unusually high level for any neu-rotransmitter. We measured DA in anterior axons at 0.83 pmol, in segmental ganglia at 1.07 pmol, and in longitudinal connectives at 0.16 pmol. Control neurosomata (Retzius cells) and axonal tracts which lack blue-green fluorescence (posterior and distal anterior roots), had no detectable DA (<0.06 pmol/sample). These data establish that the catecholamine DA is responsible for the fluorescence of the AR cell.     

CHEMICAL, ENZYMATIC AND ULTRASTRUCTURAL CHARACTERIZATION OF 5-HYDROXYTRYPTAMINE-CONTAINING NEURONS FROM THE GANGLIA OF APLYSIA CALIFORNICA AND TRITONIA DIOMEDIA

Abstract— Several identified neurons in Aplysia and Tritonia ganglia were shown to contain measurable quantities (4–6 pmol/cell body) of 5-hydroxytryptamine (5-HT). A metabolic correlate for the limited distribution of 5-HT among the neurons of Tritonia is provided by the finding that the enzyme, aromatic acid decarboxylase (AAD), is 500 times more active in nerve cells containing 5-HT than in neurons devoid of the amine. Although all Aplysia neurons have some AAD activity, 5-HT cell bodies in this species are 10-fold more active than cell bodies which do not contain 5-HT. The cytoplasm of 5-HT cell bodies in Aplysia and Tritonia characteristically contains granules that have minimum diameters of approx. 1000 Å and eccentric opaque cores. This type of granule was not found in somata which did not contain measurable 5-HT. These data illustrate the metabolic and morphological specialization in 5-HT-containing neurons of molluscs.     

Segmental specialization of neuronal connectivity in the leech

1. Every segmental ganglion of the leech Hirudo medicinalis contains two serotonergic Retzius cells. However, Retzius cells in the two segmental ganglia associated with reproductive function are morphologically distinct from Retzius cells elsewhere. This suggested that these Retzius cells might be physiologically distinct as well. 2. The degree of electrical coupling between Retzius cells distinguishes the reproductive Retzius cells; all Retzius cells are coupled in a non-rectifying manner, but reproductive Retzius cells are less strongly coupled. 3. Retzius cells in standard ganglia depolarize following swim motor pattern initiation or mechanosensory stimulation while Retzius cells in reproductive ganglia either do not respond or hyperpolarize. 4. In standard Retzius cells the depolarizing response caused by pressure mechanosensory neurons has fixed latency and one-to-one correspondence between the mechanosensory neuron action potentials and Retzius cell EPSPs. However, the latency is longer than for most known monosynaptic connections in the leech. 5. Raising the concentration of divalent cations in the bathing solution to increase thresholds abolishes the mechanosensory neuron-evoked EPSP in standard Retzius cells. This suggests that generation of action potentials in an interneuron is required for production of the EPSP, and therefore that the pathway from mechanosensory neuron to Retzius cell is polysynaptic. 6. P cells in reproductive segments have opposite effects on reproductive Retzius cells and standard Retzius cells in adjacent ganglia. Thus the difference in the pathway from P to Retzius is not localized specifically in the P cell, but elsewhere in the pathway, possibly in the type of receptor expressed by the Retzius cells.     

Leech Retzius cells and 5-hydroxytryptamine

A pair of giant Retzius (R) cells in each segmental ganglion of the leech contain 5-hydroxytryptamine (5-HT). They are the only 5-HT-containing neurones in the central nervous system to send branches to the periphery, yet many peripheral tissues (e.g. body wall muscles, heart, reproductive organs, nephridia and gut) possess 5-HT-like immunoreactive nerve fibres. 5-HT and/or R cell stimulation relax basal tension of body wall muscles and reduce their relaxation times following contraction, enhance pharyngeal movements and salivary gland secretion but inhibit muscle movements of the posterior gut regions and of the reproductive tract. It is suggested that R cells are multifunction neurones modulating activity of many tissues so that feeding behaviour of the leech is carried out as efficiently as possible.     

Chemical and ultrastructural identification of 5-hydroxytryptamine in an identified neuron

The two largest cells in a typical ganglion of the leech (Hirudo medicinalis) nervous system are the colossal cells of Retzius. These cells show a positive chromaffin reaction, and it has been suggested that they contain 5-hydroxytryptamine (5-HT). In this study, the presence of 5-HT in the colossal cells was confirmed by microspectrofluorometry and by thin-layer chromatography and spectrofluorometry of extracts of individually dissected and pooled colossal cell bodies. A single colossal cell body was found to contain, on the average, 3.8 x 10^-10 g (6mM) 5-HT. Electron microscopy shows that the colossal cells are distinguished by the presence of 1000 A granules with irregular, electron-opaque cores. Since the granules are distributed in the same pattern as the 5-HT fluorescence, we have suggested that they contain 5-HT. Furthermore, a chromaffin reaction modified for the electron microscope provides evidence that 5-HT is present in the granule cores. These data can now serve as a basis for further studies on the metabolism, distribution, and function of 5-HT in these identified neurons.     

Convergence of mechanosensory inputs onto neuromodulatory serotonergic neurons in the leech

By the frequency-dependent release of serotonin, Retzius neurons in the leech modulate diverse behavioral responses of the animal. However, little is known about how their firing pattern is produced. Here we have analyzed the effects of mechanical stimulation of the skin and intracellular stimulation of mechanosensory neurons on the electrical activity of Retzius neurons. We recorded the electrical activity of neurons in ganglia attached to their corresponding skin segment by segmental nerve roots, or in isolated ganglia. Mechanosensory stimulation of the skin induced excitatory synaptic potentials (EPSPs) and action potentials in both Retzius neurons in a ganglion. The frequency and duration of responses depended on the strength and duration of the skin stimulation. Retzius cells responded after T and P cells, but before N cells, and their sustained responses correlated with the activity of P cells. Trains of five impulses at 10 Hz in every individual T, P, or N cell in isolated ganglia produced EPSPs and action potentials in Retzius neurons. Responses to T cell stimulation appeared after the first impulse. In contrast, the responses to P or N cell stimulation appeared after two or more presynaptic impulses and facilitated afterward. The polysynaptic nature of all the synaptic inputs was shown by blocking them with a high calcium/magnesium external solution. The rise time distribution of EPSPs produced by the different mechanosensory neurons suggested that several interneurons participate in this pathway. Our results suggest that sensory stimulation provides a mechanism for regulating serotonin-mediated modulation in the leech.     

Parallel processing and selection of the responses to serotonin during reinnervation of an identified leech neuron

In an attempt to define the mechanism of synaptic specificity, we have been studying pairs of identified leech neurons isolated in tissue culture. The cultured neurons reform specific synapses when paired with appropriate partners in the absence of other cell types. In recent studies, we have examined in detail the reformation of a serotoninergic synapse between the Retzius cell and one of its targets, the pressure sensitive (P) cell. The P cell in vivo and its soma in vitro have two types of responses to serotonin (5-HT). From voltage clamp analysis of cultured P cells, we demonstrated the parallel activation of chloride (gCls) and monovalent cation (gCations) channels coupled to distinct receptor subtypes and gated by separate second messengers. Only gCls was activated by 5-HT released from the presynaptic Retzius cell both in vivo and in vitro. This demonstrates the remarkable specificity of the reformation of this synapse in culture since only the correct 5-HT receptor subtype is activated. An 80% reduction of gCations was observed in P cells that had failed to be innervated by Retzius cells in culture, suggesting that gCations may be lost prior to synapse formation. Retzius cells depleted of 5-HT also reduced gCations in the paired P cells and incubating single P cells in 5-HT did not reduce gCations. In addition, aldehyde-fixed Retzius cells were able to selectively reduce gCations when paired with P cells. We conclude that the loss of gCations was due to contact between the neurons. The early clearing of counter-effective receptor subtypes may be a prelude to synapse formation.     

Fluorescence marking of neuropile glial cells in the central nervous system of the leech Hirudo medicinalis

Summary Neuropile glial (NG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis L., were studied by histological and intracellular electrophysiological methods. Potential profiles of single leech ganglia were mapped by advancing an electrolyte-filled microelectrode into the ganglion as far as the NG cell. A small negative potential usually appeared during or immediately after penetration of the ganglion sheath. Most of the ganglia in the chain (ganglia 1–4 and 7–21) have Retzius-cell-bodies of normal size; in these, the potential associated with the ganglion sheath was followed by a jump to a more negative potential. Superimposed action potentials were associated with entry of the electrode into a Retzius cell. When the electrode tip passed out of the cell into the center of the ganglion, another potential change was observed, namely that to the membrane potential of the anterior NG cell. This membrane potential averaged -60.2 mV and ranged from -50 to -73 mV. In ganglia 5 and 6 the Retzius-cell-bodies are particularly small, and no changes of potential associated with these cells were observed; the first potential to appear after the electrode passed through the sheath of the ganglion was the membrane potential of the NG cell. Potential profiles like those of ganglia 5 and 6 are recorded in the posterior parts of all ganglia.Potential profiles of single leech ganglia were also recorded with microelectrodes filled with the fluorescent dye Procion Yellow M4-RAN. When the presumed membrane potential of an NG cell appeared, the dye was injected into the ganglion. Subsequent histological examination with the fluorescence microscope revealed that all of the dye was contained in NG cells.Supported by a Fellowship (Heisenberg-Stipendium, Schl 169/5) and grants (Schl 169/2, 4) to W.R.S. from the Deutsche ForschungsgemeinschaftThe authors thank Gisela Geiger for excellent assistance during this work     

Synapse formation and function: Insights from identified leech neurons in culture

Identified leech neurons in culture are providing novel insights to the signals underlying synapse formation and function. Identified neurons from the central nervous system of the leech can be removed individually and plated in culture, where they retain their characteristic physiological properties, grow neurites, and form specific synapses that are directly accessible by a variety of approaches. Synapses between cultured neurons can be chemical or electrical (either rectifying or not) or may not form, depending on the neuronal identities. Furthermore, the characteristics of these synapses depend on the regions of the cells that come into contact. The formation and physiology of synapses between the Retzius cell and its partners have been well characterized. Retzius cells form purely chemical, inhibitory synapses with pressuresensitive (P) cells where serotonin (5-HT) is the transmitter. Retzius cells synthesize 5-HT, which is stored in vesicles that recycle after 5-HT is secreted on stimulation. The release of 5-HT is quantal, calcium-dependent, and shows activity-dependent facilitation and depression. Anterograde and retrograde signals during synapse formation modify calcium currents, responses to 5-HT, and neurite outgrowth. The nature of these synaptogenic signals is being elucidated. For example, contact specifically with Retzius cells induces a localized selection of transmitter responses in postsynaptic P cells. This effect is signaled by tyrosine phosphorylation prior to synapse formation. © 1995 John Wiley & Sons, Inc.     

CHOLINE ACETYLTRANSFERASE, ACETYLCHOLINESTERASE AND AROMATIC l-AMINO ACID DECARBOXYLASE IN SINGLE IDENTIFIED NERVE CELL BODIES FROM SNAIL HELIX ASPERSA

—The distribution of choline acetyltransferase, aromatic l -amino acid decarboxylase and acetylcholinesterase in the nervous system of Helix aspersa has been studied using homogenates of whole ganglia, microdissection from freeze-dried sections and dissection of single neurons from fresh tissue. Choline acetyltransferase was found in both the cell body and neuropil layers of all the Helix ganglia. The enzyme was not specifically localized to any ganglion or region of ganglion. Between 10 and 30 per cent of the isolated single cell bodies contained the enzyme. The enzymic activity corresponded to 50–200 mmol ACh/1 cell bodies/h. Choline acetyltransferase is probably a specific marker for cholinergic cells in this species. Aromatic l -amino acid decarboxylase was more selectivity localized and its distribution corresponded well with that of monoamine containing cells as visualized by the fluorescence histochemical technique. A large proportion of cell bodies were localized in the boundary between the visceral and right parietal ganglia and in the pedal ganglion. The other ganglia contained few such cells. The activity of aromatic l -amino acid decarboxylase corresponded 10–50 mmol dopamine/1 cell bodies/h. A method was developed to measure the enzyme activity towards 5-hydroxytryptophan and DOPA in single cells simultaneously. The ratio between the activity towards both substrates did not vary significantly for the different cells. The enzyme is probably a specific marker for monoamine cells, but cannot be used to differentiate between the different monoamine cells. Acetylcholinesterase was uniformly distributed in the ganglia and was probably present in all nerve cells.     

Identification and quantitative measurements of biogenic monoamines in the central nervous tissue of some gastropods

Investigations were carried out to determine levels of biogenic monoamines in the central nervous tissue of seven species of terrestrial gastropods (taxonomic family: Helicidae) using high-performance liquid chromatography (HPLC) with electrochemical detection (ELCD). The central nervous tissue of all species examined contained assayable amounts of dopamine (DA) and serotonin (5-HT). Noradrenaline (NA) was occasionally present; adrenaline (A) could not be detected. The central ganglia of two Mediterranean species generally contain 3-4 times more 5-HT relative to DA than the ganglia of the other species which contain amounts of 5-HT only slightly higher than DA.     

Cell-specific contact selects transmitter responses in an identified leech neuron.

Serotonergic Retzius (R) neurons of the leech form a Cl-dependent synapse with pressure-sensitive (P) neurons both in vivo and in vitro. However, P cells show an extrasynaptic, cationic response to application of 5-hydroxytryptamine (5-HT) which is reduced upon contact between the neurons in culture. We have examined the cellular specificity of the selection of 5-HT responses in the P cell by pairing it in culture with a variety of identified neurons. Non-synaptic sensory cells, non-serotonergic pre- and postsynaptic partners and serotonergic neurons that do not form chemical synapses with the P cell failed to alter its responses to 5-HT. The selective reduction of the extrasynaptic response to 5-HT in the P cell therefore appears to be induced specifically by contact with its only known serotonergic partner during neuronal recognition leading to synapse formation.     

Chemical and thermal stimuli have short-lived effects on the retzius cell in the medicinal leech

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi-intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding.     

Quantitative effects of a neurotoxin upon serotonin levels within tissue compartments of the medicinal leech

The serotonin (5-hydroxytryptamine, 5-HT) content of tissue compartments in the medicinal leech, Hirudo medicinalis, was measured by means of high-pressure liquid chromatography coupled with electrochemical detection (HPLC-EC). Each segmental ganglion contains 21.3 +/- 2.9 (9) pmol 5-HT [X +/- SEM (N)]. The pharynx contains 7.1 +/- 1.1 (9) pmol 5-HT/mg wet weight; the salivary glands 3.2 +/- 0.9 (10), ventral body wall 2.0 +/- 0.2 (11), and vasofibrous tissue 1.2 +/- 0.2 (11). The blood of hungry leeches contains 8.7 +/- 1.9 (7) nM 5-HT while that of well-fed leeches is 2.2 +/- 0.4 (6) nM. Leeches were injected with the cytotoxic analog of serotonin, 5,7-dihydroxytryptamine (5,7-DHT) producing selective lesions of the peripherally projecting serotonin-containing neurons, and which in turn abolished their feeding behavior. The serotonin content of the pharynx and ganglia of these toxin-treated leeches were lowered significantly. The serotonin levels within the body wall and salivary glands were not altered significantly by the toxin treatment, but the levels within the vasofibrous tissue and blood were elevated substantially.     

Chemical and thermal stimuli have short‐lived effects on the Retzius cell in the medicinal leech

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi‐intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 304–311, 2000     

Development of dopamine-containing neurons and dopamine uptake in embryos of Hirudo medicinalis

The embryonic development of neurons which contain or take up dopamine was studied with glyoxylic acid histofluorescence in Hirudo medicinalis. Beginning at the time of the formation of the tail ganglion, one pair of dopamine-containing neurons was stained per segmental ganglion. The normal outgrowth of the cell bodies into the anterior roots was prevented in isolated and cultured chains of embryonic ganglia. Preincubation of intact embryos in dopamine led to the staining of additional neurons at certain developmental stages. These neurons presumably are the precursors of serotonin-containing cells, which have a temporary capability of taking up and storing dopamine.     

Steps in the development of chemical and electrical synapses by pairs of identified leech neurons in culture

Experiments have been made to follow the development of chemical and electrical transmission between pairs of leech neurons in culture. 1. The cell bodies of identified neurons were isolated from the CNS by suction after mild enzyme treatment, together with a length of the initial segment (or 'stump'). The neurons tested were Retzius cells (R), annulus erector motoneurons (AE), Anterior pagoda cells (AP) and pressure sensory cells (P). Pairs of cells were placed together in various configurations, with different sites on their surfaces making contact. 2. When pairs of Retzius cells were apposed with their stumps touching, serotonergic, chemically mediated synaptic transmission became apparent before electrical transmission. By 2.5 h impulses in either of the two Retzius cells produced hyperpolarizing inhibitory potentials in the other. These potentials were reversed by raised intracellular Cl and showed clear facilitation. The strength of chemical transmission between Retzius cells increased over the next 72 h. 3. After chemical transmission had been established, weak non-rectifying electrical transmission became apparent between Retzius cells at about 24-72 h. By 4 days coupling became stronger and tended to obscure chemically evoked synaptic potentials. 4. When pairs of Retzius cells were aligned in culture with the tip of one cell stump touching the soma of the other, chemical transmission also developed rapidly. Transmission was, however, in one direction, from stump to soma. At later stages non-rectifying electrical coupling developed as with stump-stump configuration. With the cell bodies of two Retzius cells apposed, electrical coupling developed after several days, before chemical transmission could be observed. 5. When Retzius and P cells were cultured with their stumps in contact, inhibitory chemical synaptic transmission developed within 24 h. Transmission was always in one direction, from Retzius to P cell. Electrical coupling of Retzius and P cells never occurred whatever the spatial relations of the cells to one another. 6. Annulus erector motoneurons, which contain ACh and a peptide resembling FMRFamide, first developed electrical coupling when the two stumps were in contact and then, later, bi-directional chemical transmission. Anterior Pagoda pairs placed stump-to-stump showed electrical connections. 7. Electronmicrographs revealed the presence of synaptic structures within 24 h after Retzius-Retzius, Retzius-P or AE-AE stumps were apposed. 8. The specificity of connections between cultured cells was similar to that observed in earlier experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

The identification of two inhibitory cells in each segmental ganglion of the leech and studies on the ionic mechanism of the inhibitory junctional potentials produced by these cells.

The present study identifies a pair of inhibitory cells that are located on each anterolateral margin of a leech segmental ganglion. These cells, which we label as cells 119, are electrically interconnected. These cells give rise to inhibitory junctional potentials (ijp's) in contralteral longitudinal body wall muscle cells. The latencies of the ijp's following spikes in cell 119 are variable. The ijp's are caused by transient increases in premeability to the Cl- ion. Previous studies demonstrated that 5-HT causes a hyperpolarization of body wall muscle cells by increasing the permeability of muscle membrane to the Cl- ion. Accordingly, 5-HT was searched for in the 119 cell bodies, but autoradiography, fine structure, and gas chromatography-mass spectrometry gave no indication that 5-HT was present in these cells. However, the variable latencies of the ijp's may indicate that there is a neuron interposed between cell 119 and the muscle cells. If this is the case, then the interposed neuron should be analyzed for 5-HT. Further experiments to locate the terminals of cells 119 and the cell bodies of the presumed interposed neurons are thus desirable.     

Hirudo medicinalis: A Platform for Investigating Genes in Neural Repair

We have used the nervous system of themedicinal leech as a preparation to study the molecular basis of neural repair. The leech central nervous system, unlikemammalian CNS, can regenerate to restore function, and contains identified nerve cells of known function and connectivity.We have constructed subtractive cDNAprobes from whole and regenerating ganglia of the ventral nerve cord and have used these to screen a serotonergic Retzius neuron library. This identifies genes that are regulated as a result of axotomy, and are expressed by the Retzius cell.This approach identifies many genes, both novel and known. Many of the known genes identified have homologues in vertebrates, including man. For example, genes encoding thioredoxin (TRX), Rough Endoplasmic Reticulum Protein 1 (RER-1) and ATP tsynthase are upregulated at 24 h postinjury in leech nerve cord.To investigate the functional role of regulated genes in neuron regrowthwe are using microinjection of antisense oligonucleotides in combination with horseradish peroxidase to knock down expression of a chosen gene and to assess regeneration in single neurons in 3-D ganglion culture. As an example of this approach we describe experiments to microinject antisense oligonucleotide to a leech isoform of the structural protein, Protein 4.1.Our approach thus identifies genes regulated at different times after injury thatmay underpin the intrinsic ability of leech neurons to survive damage, to initiate regrowth programs and to remake functional connections. It enables us to determine the time course of gene expression in the regenerating nerve cord, and to study the effects of gene knockdown in identified neurons regenerating in defined conditions in culture.