Transplantation and functional integration of an identified respiratory interneuron in lymnaea stagnalis

Neuron,

Vol. 8, 767-774,

April,

1992, Copyright

0 1992 by Cell

Press

Transplantation and Functional Integration of an=Identified Respiratory Interneuron in Lymnaea stagnalis N. I. Syed, R. 1. Ridgway, and A. G. M. Bulloch

K. Lukowiak,

Department of Medical Physiology and Neuroscience Research Group Faculty of Medicine University of Calgary Calgary, Alberta Canada T2N 4Nl

Summary The possibility that damaged neural circuitries can be repaired through grafting has raised questions regarding the cellular mechanisms required for functional integration of transplanted neurons. Invertebrate models offer the potential to examine such mechanisms at the resolution of single identified neurons within well-characterized neural networks. Here it is reported that a specific deficit in the respiratory behavior of a pulmonate molIusc, caused by the ablation of a solitary interneuron, can be restored by grafting an identical donor interneuron. The transplanted interneuron not only survives and extends neurites within the host nervous system, but under specific conditions forms synapses with appropriate target neurons and is physiologically integrated into the host’s circuitry, thereby restoring normal behavior. Introduction Damage to the nervous system through injury or disease may lead to permanent behavioral deficits if critical neurons die or if synaptic connections with target cells are destroyed. In the adult mammalian brain and spinal cord, where natural replacement of neurons seldom occurs, researchers have sought to facilitate behavioral recovery through surgical transplantation (Bjorklund and Stenevi, 1984; Dunnett, 1990; Bjorklund, 1991; Gage and Fisher, 1991; Fiandaca, 1991; Tessler, 1991). Such transplants are usually solid grafts or dissociated cell suspensions of embryonic neural tissues (Bjorklund and Stenevi, 1979; Perlow et al., 1979; Low et al., 1982; lsacson et al., 1984; Lindvall et al., 1990) or other tissues of neuroectodermal derivation (e.g., adrenal medulla) (Freed et al., 1981; Backlund, 1985; Bohn et al., 1987). These operations have met with variable success depending on the nature of the graft, the species examined, and the rigor of tests applied to determine the degree of functional restoration. An important consideration is the mechanisms by which neural transplants exert their functional effects. Grafts may serve as a source of neurotrophic factors, may diffusely release neurohormones or transmitters, or may act as bridges to assist the regeneration of host neurons (Dunnett and Bjiirklund, 1987; Gage and Buzsaki, 1989; Bjorklund, 1991). Least com-

mon arecases in which transplanted neurons reestablish original connectivity by being incorporated into the neural circuitry of the host, as exemplified in the functional recovery of “point-to-point” systems such as cerebellar and hippocampal circuitries (Segal et al., 1988; Sotelo and Alvarado-Mallart, 1991). A number of attempts at transplanting annelid, arthropod, or molluscan ganglia (Guthrie and Banks, 1969; Murphy and Kater, 1978; Moffett and Austin, 1981; Page, 1982; Vining and Drewes, 1985; Gomot et al., 1990), as well as individual leech neurons (Zhang and Nicholls, 1983; Zhang, 1989), have been successful. These examples suggested to us that neuronal transplantation in the invertebrates might be of potential benefit in examining the mechanisms underlying functional restoration of neural circuits. Given their simpler nervous systems and the limited behavioral repertoires of invertebrates, it is perhaps surprising that invertebrates have not been exploited more as models for neuronal transplantation. For the present study, we have taken advantage of three significant attributes of the molluscan nervous system: first, a demonstrated ability of axotomized adult neurons to regenerate and reestablish specific synaptic connections with target cells either in vivo (Murphy and Kater, 1978, 1980; Benjamin and Allison, 1987) or in vitro (Camardo et al., 1983; Hadleyet al., 1985); second, the presence of substrate-bound and soluble factors needed for neurite outgrowth of transplanted neurons (Wonget al., 1981; Mattson and Kater, 1988; Miller and Hadley, 1991; Ridgway et al., 1991); and finally, the presence of large, individually identifiable neurons whose functional role(s) within a specific neural network is known and can be directly correlated with observable behaviors (Benjamin et al., 1987). The freshwater pond snail Lymnaea stagnalis respires primarily through its lung, but significant gas exchange (up to 40%) can also take place across its skin (Jones, 1961). These snails can be forced to rely on pulmonary (aerial) ventilation by displacement of pond water dissolved oxygen with nitrogen. Pulmonary ventilation is accomplished by the alternate opening (expiratory) and closing (inspiratory) movements of the respiratory orifice, the pneumostome (Syed et al., 1991). Rhythmic respiratory movements in Lymnaea originate from a central pattern generator (CPG), a small networkof interneurons within the central ganglionic ring. The neural circuit underlying the respiratory CPG has been identified and characterized in vivo (Syed et al., 1991; Syed and Winlow, 1991a) and has recently been reconstructed in vitro (Syed et al., 1990). An identified peptidergic interneuron, visceral dorsal 4 (V.D.4) was found to be necessary for the respiratory rhythm (and associated movements) to occur. V.D.4 is involved in the inspiratory phase of respiration via its excitation of pneumostome closure motor neurons, identified within the visceral K (V.K)

NWKI” 768

cell cluster (Syed et al., 1991; Syed and Winlow, 1991 b). Concomitantly, V.D.4 inhibits motor neurons that open the pneumostome and those that compress the mantle, identified within the visceral J (V.J) cell cluster and right parietal A (R.P.A) group neurons, respectively, which are involved in the expiratory phase of respiration (Syed et al., 1991; Syed and Winlow, 1991b). In the present study we directly confirm the necessity of V.D.4 in the respiratory behavior of Lymnaea by removing it from the visceral ganglion. We further show that the subsequent transplantation of a donor V.D.4 results in the restoration of respiratory behavior. We also demonstrate that this restoration of neural circuitry and the behavior by a transplanted V.D.4 can be achieved only in the absence of host V.D.4. Results

Is V.D.4

4 -2 6

Days

7

i 14

b

21 -2%

post-surgery

Figure 1. Location of Neurons V.D.4. Removalnransplantation tory Behavior

Necessary for Respiratory

Behavior?

In the first series of experiments V.D.4 was removed from the visceral ganglia of anesthetized snails. This was achieved by adapting our technique for isolating neurons for cell culture (Ridgway et al., 1991). After surgery, the snails were allowed to recover under either normal or oxygen-deprived experimental conditions. All of the snails maintained in oxygen-deprived water died within 28 days following surgery; 50% had died within 7-10 days (Figure IB). Snails from which V.D.4 was removed exhibited no aerial respiratory movements regardless of the oxygenation state of the water (Figure IC). This contrasted with sham-operated animals (Figure IC) and those from which other identified neurons (e.g., from the visceral F cluster or right parietal B group, n = 11 in 9 preparations; data not shown) had been removed. In these cases there was

Used in This Study and Effects of on Animal Survival and Respira-

(A) Schematic diagram of the central ring ganglia of L. stagnalis showing the location of identified neurons used in this study. Ganglia are numbered as follows: left and right cerebral ganglia (1 and 2); left and right pedal ganglia (3 and 4); left and right pleural ganglia (5 and 6); left and right parietal ganglia (7 and 8); unpaired visceral ganglion (9). Identified neurons areV.D.4, aV.J cell, a V.K cell, an R.P.A group neuron, and right parietal dorsal 1 (R.P.D.l). (B) Effect of removing V.D.4 on animal survival. The percentage of snails surviving after V.D.4 was removed (closed circles) is compared with that in sham-operated control snails (open circles), which received only a skin incision. For this experiment, snails were maintained in artificial pond water in which nitrogen was bubbled for 6 hr to displace dissolved oxygen; this environmental condition forces Lymnaea to rely on pulmonary respiration, with little or no contribution from the skin. (At start of experiment: SHAM, n = 15; -V.D.4, n = 15.) (C) Loss of pulmonary respiratory behavior in snails after V.D.4 was removed. The percentage of snails exhibiting respiratory movements (e.g., opening and closing of the pneumostome) was observed intermittently or videotaped over a period of -100 hr beginning 48 hr after surgery in sham-operated (SHAM) snails

and in snails in which V.D.4 was removed (-V.D.4). These visual observations were confirmed by intracellular electrophysiological recordings made from respiratory interneurons and motor neurons, which revealed no neural activity associated with respiration. (SHAM, n = 25; -V.D.4, n = 31.) (D) Effect of V.D.4 transplantation on survival of snails in which the hostV.D.4 had been removed IO-14days earlier. The percentage of snails surviving after receiving a transplanted V.D.4 (+V.D.4) is compared with sham-operated (SHAM) controls that had undergonesurgerytwice:first (at timezero)when hostV.D.4 interneurons were removed from test snails and second (at IO14days) when donorV.D.4 interneurons were transplanted into test snails. Survival was monitored for 6 weeks after the second (transplant) surgery. Snails were maintained in oxygen-deprived pond water throughout the experimental period. (SHAM, n = IO; +V.D.4, n = 7.) (E) Restoration of pulmonary respiratory behavior following V.D.4 transplantation into snails from which the host V.D.4 had been removed lo-14 days earlier. The percentage of snails exhibiting pulmonary respiratory movements was observed over a period of 6 weeks beginning 48 hr after the second (transplant) surgery. Animals receiving a transplanted V.D.4 (+V.D.4 ) are compared with doubly sham-operated (SHAM) controls. Restoration of behavior, including coordination of locomotory, wholebody withdrawal, and pneumostome movements, was first observed 7-10 days after V.D.4 transplantation. (SHAM, n = 9; +V.D.4, n = 7.)

Trancplantatmn 769

of a Lymnaea

Interneuron

A

0 0 V.J

?

V.K

?

.

TRANSPLANTED

-0

v.D.4

.

.:

D V.K

V.K ----..

V.J V.J

R.P.A

Figure

i3.P.A

2. Transplantation,

Regeneration,

and

Functional

Integration

of V.D.4

(A) Diagram of the strategy employed in experiments in which the host V.D.4 was removed from the animal. The normal synaptic connections of V.D.4 on three follower cells are shown on the left side of the diagram. Closed and open symbols represent inhibitory and excitatory connections, respectively. The ability of a transplanted V.D.4 to restore these connections was tested (right side of diagram). (Band C) Morphology of V.D.4 interneurons transplanted into host visceral ganglia at 6 hr LB) and 24 hr (C) after transplantation. Note the neuritic outgrowth from the soma of both of these cells and the extension of neurites from the visceral ganglion into and beyond the adjacent parietal ganglia in (C). Bars, 188 Pm. (D) Synaptic connections of a host (normal) V.D.4 on three follower cells involved in Lymnaea respiratory behavior. Simultaneous intracellular recordings were made from V.D.4 and three follower cells in isolated central ring ganglia preparations. Stimulation of V.D.4 by depolarizing current injection (at arrows) inhibited an R.P.A group neuron and a V.J cell while exciting a V.K cell. (E) Restoration of synaptic connections between a transplanted V.D.4 and three follower cells 24 hr after V.D.4 transplantation. In the absence of a host V.D.4, a transplanted V.D.4 reestablished appropriate synapses with follower R.P.A, V.J, and V.K cells (compare with [D]). Intracellular electrophysiological recordingswereobtained by impaling neuronswith glass microelectrodes asdescribed previously (Syed and Winslow, 1989).To ruleoutthe possibility of polysynaptic connections, all preparationswere superfused with salinecontaining 6x Ca2+, 6x MgZ’

no obvious periments pulmonary

effect on respiratory strongly support the respiration.

Does a Transplanted Functional Circuitry? In the

second

series

V.D.4 of

behavior. necessity

These of V.D.4

exfor

Restore the experiments

V.D.4

was

re-

moved, and after IO-14 days of recovery, the animals underwent a second operation in which a V.D.4 from another snail was transplanted into the host visceral ganglion. All snails receiving a transplanted V.D.4 survived (Figure ID) and exhibited respiratory movements that were indistinguishable from doubly shamoperated snails (Figure IE). Thus, the transplantation

TRANSPLANTED IN

ORGAN

CULTURE

I

IOrnV

-.

L

40mV

-J 1s

of a donor V.D.4 resulted in the functional recovery of respiratory behavior. Since the observed behavioral recovery might be due to compensatory mechanisms rather than integration of the donor V.D.4 into the respiratory circuitry, we examined whether the transplanted neurons had extended neurites and formed synapses on host neurons (Figure 2A). For these experiments, neurons were transplanted both in vivo as before and into central ganglionic rings maintained in organ culture. The extent of neurite outgrowth achieved by transplanted V.D.4 interneurons was revealed by intracellular staining with the fluorescent dye Lucifer yellow. Within 6 hr of transplantation, V.D.4 had begun to extend neurites into the host visceral ganglion (Figure 28). By 24 hr, neurites extended beyond the visceral ganglion and into the parietal and pleural ganglia (Figure 2C), partially restoring the normal morphology of V.D.4 within the central ganglionic ring. Snails exhibiting full behavioral recovery after V.D.4 transplantation were dissected, and their central ganglia were removed for electrophysiological study. Simultaneous intracellular recordings were made from V.D.4 and three of its target respiratory motor neurons (i.e., V.K cluster, V.J cluster, and R.P.A group neurons). In all instances, the transplanted V.D.4 was found to have reestablished synapses with the appropriate follower cells (Figure 2E), similar to those observed in acutely dissected preparations (Figure 2D). The ability of V.D.4 to establish correct synapses was indistinguishable whether the transplant was performed in

Table 1. Synaptic V.K Ceils

Connections

Between

Transplanted In Vivo

Normal Amplitude 0-N

Latency (ms)

4.6 f

116 * 10 4.6 f 0.5

0.6

Neuron

V.D.4

and

Transplanted In Organ Culture

Amplitude

Latency

Amplitude

Latency

(mV)

(ms)

(mV)

(ms)

114 f 9 4.7 f 0.5

115 + 12

Synaptic connections between V.D.4 and one group of its follower cells (V.K cells) were tested as shown in Figure 3 (since neuron V.D.4 fires regenerative bursts of action potentials, single action potentials are difficult to induce). The amplitude and latency of the first excitatory postsynaptic potential in each train were quantitated on a storage oscilloscope. For each condition, n = 8.

Figure 3. Comparison of Synaptic tions of Normal and Transplanted Neurons with V.K Cells

ConnecV.D.4

In each record a burst of action potentials was evoked in V.D.4 by a depolarizing current ramp. This current injection induced apparent monosynaptic, I:1 excitatory postsynaptic potentials of constant latency in the V.K cell. These preparations were maintained in 6x Ca’+, 6x Mg” saline (see also Table 1).

vivo (n = 6) or into ganglia maintained in organ culture (n = 8). To test further the specificity of these synaptic connections, we attempted to replace the host V.D.4, which is thought to use the tetrapeptide FMRFamide as a transmitter, with other neurons of the same phenotype (e.g., visceral F, visceral yellow, and right parietal B group neurons, total n = 17; see Benjamin et al., 1988). Although these neurons exhibited neurite outgrowth when transplanted into organ cultured ganglia, they did not form the appropriate synaptic connections with respiratory motor neurons even though ample time (IO-15 days) was provided (Murphy et al., 1985; Moffett and Ridgway, 1988). Thus, in the absence of a host V.D.4, only a transplanted V.D.4 can fully restore the functional circuitry. The synaptic connections from transplanted V.D.4 interneurons compared favorably with those of normal V.D.4 interneurons (Figure 3). Specifically, the latency, amplitude, and variability of synaptic potentials of V.D.4 in normal preparations were comparable to those observed after transplantation both in vivo and in organ culture (Table 1). All of these measurements were made in 6x Ca*+, 6x Mg2’ saline, suggesting that intervening neurons linked by chemical synapses are not involved. These observations suggest that V.D.4 synapses directly upon postsynaptic target neurons, although the possibility of electrically coupled and/or nonspiking intervening neurons cannot be eliminated. Does a Transplanted V.D.4 form Specific Synapses in the Presence of the Host V.D.4? Next we tested the ability of a transplanted V.D.4 to reestablish its connections with target cells in the presence of the host V.D.4. In these experiments the host V.D.4 was not removed; rather, an additional V.D.4 (isolated from another snail) was transplanted into the host visceral ganglion in organ culture (Figure 4A). Although such transplanted V.D.4 interneurons exhibited neurite outgrowth (Figure 4B), they made only inappropriate synaptic connections. For example, an excitatory chemical connection was always observed (n = 6) between the transplanted V.D.4 and an identified neuron (right parietal dorsal 1) not associated with respiratory behavior (Figure 4C). Additionally, inappropriate inhibitory synapses were always observed on the right parietal C cluster (Benjamin and

Transplantation 771

of a Lymnaea

Interneuron

Winlow, 1981), neurons of unknown function (n = 7; data not shown). In the same animals, the host V.D.4 was found to maintain its normal connections with appropriate target cells. Thus, in the presence of an intact host V.D.4, a transplanted V.D.4 does not synapse with appropriate targets (which are innervated by the host V.D.4), but can establish connections with inappropriate targets.

-x,

JlOm”

TRANSPLANTED V.D.4

\

KiT . . -.

4

Does a Regenerated Host V.D.4 Compete with the Transplanted V.D.4? Finally, we tested the possibility that two V.D.4 interneurons, one host and one transplanted, might compete for appropriate targets given the right conditions (Figure SA). In these experiments, which were performed in organ culture, the host V.D.4 was not removed, but its synaptic connections with follower cells in the right parietal ganglion (e.g., R.P.A group neurons) were severed by crushing the connective between this ganglion and the visceral ganglion (Figure SA). A donor V.D.4 was then transplanted into the right parietal ganglion. Both the host V.D.4 (one axon crushed) and the transplanted V.D.4 were allowed to regenerate. After 24 hr each V.D.4 had sprouted across the crush site and entered the adjacent ganglion (Figure SB). To examine the synaptic connections formed following regeneration, simultaneous intracellular recordings were made from the host V.D.4, the transplanted V.D.4, and two appropriate target cells (V.K cluster and R.P.A group neurons). The host V.D.4 was found to have maintained its connections with visceral ganglion follower cells (e.g., V.K), but had not reestablished synaptic contact with right parietal ganglion follower cells (e.g., R.P.A; Figure SC). The transplanted V.D.4, on the other hand, had established synaptic contact with the R.P.A group neuron, but not with the V.K cluster neuron. In no instances (n = 5) were target cells found to be innervated by both the host and the transplanted V.D.4 interneurons. These experiments suggest that a transplanted V.D.4 will establish appropriateconnections if such target cellsare first deprived of synaptic input by the host V.D.4. It appears that, given equal opportunity, two V.D.4 interneurons will compete for the same target, with the

‘i-2DmV 38

Figure4. Failure priate Connections Host V.D.4

of a Transplanted with Follower

V.D.4 to Establish Cells in the Presence

Approof the

(A) Diagram of the experimental strategy employed. Shown on the left side of the diagram are the normal connections of V.D.4 with its follower R.P.A, V.K, and V.] cells. Closed and open symbols represent inhibitory and excitatory connections, respectively. This experiment was designed to test the ability of a transplanted V.D.4 (right side of diagram) to establish either appropriate connections (with R.P.A, V.K, orV.J cells) or inappropriate connections with other cells(X) in the presence of the host V.D.4. (B) Morphology of host (H) and transplanted 0 V.D.4 interneurons within the visceral ganglion 24 hr after transplantation. Note the two primary axonal projections characteristic of the host

V.D.4 (see Figure 5A for a schematic depicting the host V.D.4). Most of the fine processes represent neurite outgrowth by the transplanted V.D.4 soma. Bar, 100 Bm. (C) In the presence of the host V.D.4, a transplanted V.D.4 estahlished connections only with inappropriate target cells. Simultaneous intracellular recordings were made from the host V.D.4 (bottom trace), an appropriate target V.K cell (second from bottom trace), a transplanted V.D.4 (third from bottom trace), and an inappropriate target cell: R.P.D.l (top trace). Stimulation of the host V.D.4 by depolarizing current injection (at closed arrow) showed the presence of its appropriate (excitatory) connection with a V.K cell, but not with the inappropriate R.P.D.l cell. The reciprocal situation was observed when the transplanted V.D.4 was stimulated (at open arrow). Note the absence of synaptic connections between the host and transplanted V.D.4 interneurons.

?.~

. . .. ..

HOST

first cell establishing TRANSPLANTED

Host ,*71a”ted V.D.4

ki

-7t’ Vi&era1 Ganglion

R. Parietal Ganglion

to

make contact functional

preventing connections.

the

other

from

Discussion We have shown that a transplanted interneuron (V.D.4) can regenerate and make appropriate synaptic connections that restore functional respiratory circuitryinapulmonatesnail. Furthermore,onlyan identical donor cell can functionally replace an ablated V.D.4. Although neurites of a transplanted V.D.4 can find their way to appropriate targets, they still may not establish synapses if these targets are already innervated by the host V.D.4. Removal of the host V.D.4 inputs prior to transplantation, however, allows the donor V.D.4 to compete for appropriate targets, thus facilitating functional integration of the graft. Our results complement transplantation experiments in the mammalian nervous system. For example, entorhinal cortex neurons innervate the host hippocampus only after its deafferentation by a host entorhinal lesion (Gibbs and Cotman, 1987; Zhou and Raisman, 1989). Furthermore, if axons growing into the hippocampus have a choice of terminal fields, they exhibit a preference for zones previously denervated by fibers of identical transmitter phenotype (Bjorklund and Stenevi, 1984). In contrast to mammalian models, however, an advantage of our new molluscan model is the ability to use strict physiological criteria (via intracellular recordings) rather than histological evidence to test the specificity and pharmacology of synapses formed by transplanted neurons. Transplantation of invertebrate ganglia to the body cavity of host animals has been routinely employed for studies of neuronal regeneration in molluscs (Murphy and Kater, 1978; Moffett and Austin, 1981; Comot et al., 1990), annelids (Page, 1982; Vining and Drewes, 1985), and arthropods (Cuthrie and Banks, 1969). The more demanding approach of single neuron transplantation, however, had previously been reported only in the leech (Zhang and Nicholls, 1983; Zhang, 1989). The latter studies showed that an effector neu-

V.K

HOST V.D.4 J20mv

Figure 5. Competition-Dependent nections between a Regenerating V.D.4

Specificity of Synaptic ConHost V.D.4and aTransplanted

(A) Diagram of the experimental strategy employed. The host V.D.4 was not removed, but its primary axonal projection to the right parietal ganglion was severed by crushing the connective between these two ganglia. This procedure leaves the connections between the host V.D.4 and its follower cells in the visceral ganglion (e.g., V.J and V.K cells) intact, but severs connections with follower cells located in the right parietal ganglion (e.g., R.P.A group neurons). A donor V.D.4 was then transplanted into

the right parietal ganglion. Both the host and the transplanted V.D.4 interneurons were allowed to regenerate. The ability of the two V.D.4 interneurons to compete for common appropriate target cells or to establish new connections with inappropriate cells (X) was then tested. (B) Morphology of an axotomized host (H) V.D.4 (within the visceral ganglion) and of a V.D.4 transplanted into the right parietal ganglion (T) 24 hr after axotomy/transplantation; within this period, each neuron sent out neurites that traversed the crush site (asterisk) and entered the adjacent ganglion. Bar, 50 urn. (C) Simultaneous intracellular recordings were made from the host (axotomized)V.D.4,afollowerV.Kcell,atransplanted V.D.4, and a follower R.P.A group neuron. Stimulation of the host V.D.4 by depolarizing current injection (at the closed arrow) excited the V.K cell but had no effect on the R.P.A group neuron (normally an inhibitory connection). The reciprocal situation was observed when the transplanted V.D.4 was stimulated (at the open arrow). Furthermore, no synaptic connections were observed between the two V.D.4 interneurons.

Transplantanon 773

of a Lymnaea

Interneuron

ron (a Retzius cell) could be transplanted into host ganglia, where it survived, extended processes, and formed synaptic connections with host neurons. Our study, however, shows that an interneuron can be transplanted and can restore a behavioral deficit caused by removal of the host interneuron. The results of this study provide a direct demonstration of the necessity of V.D.4 in respiration. These observations complement in vivo studies showing that hyperpolarization of V.D.4 causes cessation of the rhythmic output of the respiratory CPG (Syed and Winlow, 1991b). Furthermore, we recently showed that neuron V.D.4 is an essential component of the CPG by reconstruction of this three neuron network in vitro (Syed et al., 1990). The transplantation experiments reported here not only confirm the behavioral role of neuron V.D.4, but also provide a new system for examining the cellular mechanisms by which transplanted neurons become physiologically integrated into host nervous systems. Experimental

Procedures

Animals A recently established laboratory stock of L. stagnalis derived from that of the Department of Biology at the Free University of Amsterdam was used. Animals were maintained as described by Ridgway et al. (1991). Snails with a shell length of 15-20 mm (approximate age 2-4 months) were used both as donors and as host animals. Neuron Transplantation For cell removal and transplantation, the snails were anesthetized with 2% halothane and immobilized on a platform. A small dorsal incision was made in the head skin of recipient animals to expose the central ganglionic ring, which was then stabilized on a wax-covered spatula. A crystal of protease (type XIV; Sigma) was focally applied (for 20 s) to the perineurium of the visceral ganglion. The preparations were immediately washed with normal saline, and fine forceps were then used to tear a small hole in the perineurium. When only cell removal was desired, V.D.4 was isolated by gentle suction applied through a fire-polished glass micropipette. The skin incision was then sealed with cyanoacrylate glue (Eastman). Animals were kept under anesthesia for at least 6 hr before being allowed to recover. In transplantation experiments, V.D.4 was isolated from donor snails as described previously (Ridgway et al., 1991). These donor interneurons were transferred in a glass micropipetteand then gently inserted into the host animal’s visceral ganglion or right parietal ganglion. The animals were then allowed to recover as described above. Organ Culture For some experiments, the recipient central ganglionic excised under sterile conditions from the animal and the silicone rubber base of a 35 mm petri dish. Donor were transplanted as described above, and the recipient were organ cultured (see Berdan et al., 1987). Since transplantation studies obtained in vivo (n = 6) were guishable from those performed in organ culture (n have combined both data sets for the present study.

ring was pinned to neurons ganglia results of indistin= 8), we

Electrophysiology For morphological studies, V.D.4 was injected with the fluorescent dye Lucifer yellow and preparations were prepared according to the methods described earlier (Syed and Winlow, 1989). Conventional electrophysiological techniques were used to make intracellular recordings. Briefly, preparations were bathed in saline buffered to pH 7.9 with HEPES (Benjamin and Winlow,1981).Toruleoutthepossibilityof polysynapticconnec-

tions, preparations were superfused with 6x Ca”, 6x Mg” saline (Syed and Winlow, IVVla). The glass microelectrodes fresistance IO-20 MD) were filled with a saturated solution of KzSO+ Intracellular signalswere recorded on chart paper using a Gould four-channel pen recorder. Acknowledgments We thank Dr. Richard Hawkes, Brent Reynolds, and Dr. Sam Weiss for critical review of the manuscript, Janet Ma for typing the manuscript, and Carry Hauser for technical assistance. This work was supported by NSERC and MRC (Canada). A. C. M. B., and R. L. R. are Alberta Heritage Foundation for Medical Research Scholarship and Fellowship awardees, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

November

14, 1991; revised

January

23, 1992.

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