The Journal of Neuroscience, January 1993, 13(l): 13-28 Enhanced ACh Sensitivity Is Accompanied by Changes in ACh Receptor Channel Properties and Segregation of ACh Receptor Subtypes on Sympathetic Neurons during Innervation in viva Brenda L. Mossa and Lorna W. Role Department of Anatomy and Cell Biology, Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, New York 10032 Although presynaptic input can influence the number and [Key words: sympathetic neurons, Oil, cytochrome oxi- distribution of ACh receptors (AChRs) on muscle, the role dase, ACh sensitivity, ACh receptor, neuronal bungarotoxin] of cellular interactions in the development of transmitter sen- sitivity in neurons is less clear. To determine whether pre- The expressiono f transmitter sensitivity is fundamental to syn- synaptic input modifies neuronal AChR channel function and aptogenesisS. tudies of the formation of the neuromusculars yn- distribution, we must first ascertain the profile of changes apse have shown that the incoming motor nerve plays an im- in receptor properties relative to the timing of synapse for- portant role in the development of postsynaptic transmitter mation. We have examined the temporal aspects of syn- sensitivity. Innervation of embryonic skeletal muscle induces aptogenesis in the lumbar sympathetic ganglia of the em- an increasein ACh sensitivity at the site ofnerve-muscle contact bryonic chick in anatomical experiments with anterograde by regulating ACh receptor (AChR) number and distribution 1,l ‘-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine per- (reviewed in Schuetze and Role, 1987). The postsynaptic re- chlorate labeling of presynaptic inputs and cytochrome ox- sponsei s further modified by subsequentc hangesi n the func- idase histochemistry. Biophysical studies of sympathetic tional properties of the AChR channels:t he single-channecl on- neurons, within hours of removal from animals at different ductance increasesa nd the apparent mean open time decreases stages relative to synapse formation, show that both the (reviewed in Schuetze and Role, 1987; Brehm and Henderson, properties and distribution of AChR channels are modified 1988). concurrent with a significant increase in presynaptic input Presynaptic input can alsoi nfluence the distribution and func- to the neurons. The most striking change in AChR channel tional properties of AChRs in neurons. To date, most studies distribution is revealed by patching multiple sites on the evaluating the role of presynaptic input in the regulation of surface of individual neurons. Following innervation in viva, neuronal AChRs have examined mature neurons, rather than many neurons express only one of the four AChR channel studying AChR expression during synaptogenesis.S tudies of subtypes and the AChRs are clustered in discrete, high- mature neurons,e mploying toxin binding (Marshall, 1981 ; Lor- activity patches. Furthermore, when neurons at this stage ing and Zigmond 1987) or monoclonal antibodies to receptor express more than one AChR channel subtype, the different subunits (Jacob et al., 1984; Sargent and Pang, 1989; Jacob, classes are often spatially segregated from one another on 1991) to reveal the distribution of AChRs, indicate that the the cell surface. This contrasts with patches from neurons AChRs are clustered at the synapses.F urthermore, studieso f removed earlier on, which have lower overall activity, often the denervation and subsequentc ross-reinnervation of mature comprised of multiple channel subtypes. Comparison of the sympathetic neurons in frog indicate that the incoming nerve AChR properties of acutely dispersed neurons to those of influences AChR channel kinetics (Marshall, 1985). However, neurons maintained in vitro indicates that most features of denervation of mature neurons apparently does not alter the AChR channels are conserved despite their removal from number or distribution of AChRs as initially suggested(K uffler presynaptic and other in viwo influences. These findings are et al., 1971 ; Dunn and Marshall, 1985; Jacob and Berg, 1988; consistent with inductive interactions between pre- and but seeS argenta nd Pang, 1988). postsynaptic neurons playing an important regulatory role There is relatively little known about the initial development in transmitter receptor expression. and subsequentc hangesi n AChRs during synapsef ormation on neurons. Recent work on chick ciliary ganglion in vivo by Received Nov. 22, 1991; revised June 19, 1992; accepted June 29, 1992. Jacob (1991) shows a tight temporal relationship between the We thank Dr. A. J. Silverman, Dr. J. Dodd, and A. Dolorico for help with the appearance of synapsesa nd the expressiono f surface AChRs DiI experiments, Dr. G. M. Mawe for participating in initial cytochrome oxidase detected by monoclonal antibody 35. In addition, recent studies experiments, and Dr. R. Loring for his generous gift of neuronal bungarotoxin. We also thank Drs. L. Chasin, S. DeRiemer, D. Kelley, and S. Siegelbaum for of central neurons indicate upregulation of receptor subunit their critical reading of an earlier version of the manuscript. This work was sup- mRNA concurrent with the expressiono f presynaptic ChAT in ported by NIH Award NS22061 (L.W.R.). the chicken lateral spiriform nucleus in vivo (Daubas et al., Correspondence should be addressed to Loma W. Role, Department of Anat- omy and Cell Biology, Center for Neurobiology and Behavior, 630 West 168 1990). Street, Room 12-404, Columbia University P and S, New York, NY 10032. Several studiesh ave examined the development of ACh sen- &Present address: Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794. sitivity of neuronsm aintained in vitro in the presenceo r absence Copyright 0 1993 Society for Neuroscience 0270-6474/93/l 300 13-16$05.00/O of presynaptic input (Role, 1988; Gardette et al., 1991; Hume 14 Moss‘and Role * Regulation of Neuronal AChRs during Innervation and Honig, 199 1). These studies indicate that presynaptic input the surface of individual cells. If these hot spots of AChR chan- enhances the ACh sensitivity of postsynaptic neurons fairly rap- nel subtypes reflect individual synaptic sites, this organization idly. The upregulation of the ACh sensitivity of neurons by the of AChR subtypes might encode critical differences in the effi- incoming nerve is mimicked by a soluble factor(s) derived from cacy of transmission at distinct inputs. presynaptic neurons and is apparently due to an increase in the rate of appearance of new AChRs on the cell surface (Role, 1988; Gardette et al., 199 1). A limitation of these studies, how- Materials and Methods ever, is that they assess changes in ACh sensitivity relative to DiZ labeling. For anterograde labeling of the projections from pregan- the onset of innervation in vitro. These changes may be super- glionic neurons to sympathetic ganglia, the spinal cord and sympathetic chains were dissected out as a single block of tissue from both ED 11 imposed on perturbations in neuronal function that take place and ED 17 chickens. The chest cavity was opened from the ventral due to removal of the neurons from other regulatory influences surface, and transverse cuts were made at the cervical and sacral levels. to which they are subject in vivo. TWO longitudinal cuts were then made lateral to each chain. The tissue Our first goal was to examine whether innervation of chicken was removed and fixed for I hr in 4% paraformaldehyde in 0.1 M sodium lumbar sympathetic neurons in vivo alters their ACh sensitivity. phosphate buffer (pH 7.3, room temperature). The tissue was then rinsed in three changes of 0.1 M phosphate buffer and stored at 4°C in buffer Previous electron microscopic data had indicated that by 1 d with 10 PM azide. posthatch, approximately 25% of the adult number of synaptic In avians, the preganglionic neurons innervating the lumbar sym- profiles are present (Hruschak et al., 1982). To identify when pathetic chain ganglia are located in the dorsal (rather than interme- presynaptic fibers project to the lumbar sympathetic ganglia diolateral) spinal cord (Levi-Montalcini, 1950; Yip, 1986, 1990). To during embryogenesis, we injected l,l’-dioctadecyl-3,3,3’,3’-te- inject DiI into the preganglionic nucleus, a longitudinal cut was made along the dorsal surface of the vertebral column exposing the spinal tramethylindocarbocyanine perchlorate (DiI) into the pregan- cord. To increase the efficiency of DiI labeling, a micropipette (tip glionic nucleus to label presynaptic input orthogradely. To eval- diameter approximately 10 Frn) was first used to perforate the dorsal uate whether the arrival of presynaptic fibers was associated surface of the spinal cord. A second micropipette filled with DiI (Mo- with functional innervation of the neurons, we assessed their lecular Probes) dissolved in Triton X-100 was used to inject the DiI solution into the spinal cord (cervical to sacral) dorsal and lateral to the electrical activity by cytochrome oxidase (CO) histochemistry lution into the spinal cord (cervical to sacral) dorsal and lateral to the (Mawe et al., 1990). Both techniques indicate a significant in- central canal (schematic Fig. IA). To prevent nonspecific labeling due crease in innervation of lumbar ganglia between embryonic day to dispersion of DiI in the buffer, the injected area was repeatedly blotted (ED) 11 and ED 17. To assess changes in ACh sensitivity during with filter paper. Fluorescence micrographs of an ED 1 I spinal cord, innervation in vivo, we recorded from neurons immediately after which was embedded and vibratome sectioned (see below) immediately following DiI injection, indicated that the region of the preganglionic dispersal (without enzyme treatment) and compared peak ACh- nucleus was completely labeled (Fig. IB). For anterograde diffusion of evoked macroscopic currents at each embryonic age. These stud- the DiI, the tissue was stored in 0.1 M phosphate buffer with azide in ies reveal a 4.4-fold increase in ACh sensitivity during inner- the dark at 37°C for 4-8 weeks following injection. vation in vivo. DiI-labeled lumbar sympathetic chains (LI-L5) were dissected from the fixed tissue block, incubated at 37°C in 6% gelatin for 10 min and Neuronal AChRs, like muscle AChRs, may be further mod- 12% gelatin for 5 min, and embedded in egg yolk:gelatin (12%) at 1: 1. ified by changes in their functional properties during the de- Egg-gel blocks were stored in 4% paraformaldehyde (in 0.1 M sodium velopment of presynaptic input. Previous studies have shown phosphate buffer) for 2-3 d in the dark at 4°C until sufficiently firm for that neurons removed at different developmental stages and sectioning. Vibratome sections (20-30 pm thick, 15-25 serial longitu- maintained in vitro express AChR channels with distinct bio- dinal sections per chain) were cut and mounted onto gelatin-subbed slides with Gel/Mount (Biomeda). To visualize neuronal cell bodies, physical characteristics (Moss et al., 1989). These studies do not sections adjacent to those examined for DiI labeling were stained with resolve, however, whether these changes in AChR channels might cresyl violet. Sections were viewed with an epifluorescence microscow be related to innervation or whether the biophysical properties equipped with a rhodamine filter set. Photographs were taken with- a of AChR channels are faithfully retained by neurons removed 40 x Plan Neofluar objective on Ilford HP5 (800 ASA) black-and-white film. from their in vivo setting and maintained in vitro. Our next goal Cytochrome oxidase (CO) histochemistry. Lumbar sympathetic chains was to determine whether changes in the distribution and func- were dissected from ED 11 and ED 18 chickens, fixed in 4% parafor- tional behavior of ACh-activated channels take place during maldehvde (in 0.1 M nhosohate buffer. DH 7.3) for 1 hr at room tem- innervation in vivo. We find that the observed increase in ACh perature, rinsed in phbsph’ate buffer, anh immkrsed in 30% sucrose in sensitivity in acutely dispersed neurons may be due, in part, to 0. I M phosphate buffer overnight. The tissue was frozen in OCT com- pound (TissueTek), and 1O -1 5 pm cryostat sections were collected onto changes in AChR channel properties. Comparison of ACh-ac- gelatin-subbed slides. tivated single-channel currents in neurons acutely dispersed on Sections from ED 1 I and ED 18 animals were processed concurrently ED 1 1 to those in neurons isolated on ED 17 revealed multiple for CO histochemistry. The sections were first rinsed in 0.1 M Tris HCI AChR subtypes and changes in the properties of these channels (pH 7.4) for 5 min. To intensify the diaminobenzidine (DAB) reaction, sections were preincubated in 2% CoCI, (in 0.1 M Tris HCI) for 5 min. that are consistent with the net increase in ACh-induced con- The sections were then rinsed for 15 min in Tris HCI buffer, followed ductance. In addition, analysis of the neuronal bungarotoxin by a 5 min rinse in 0.1 M sodium phosphate buffer. CO activity was (NBT) sensitivity of the AChRs revealed differences that further assayed by incubating the sections in a solution of cytochrome C (0.3 distinguish the AChR subtypes from one another. mg/ml; type III, Sigma) and DAB (0.5 m&ml) in 0.1 M sodium phos- phate buffer at 37°C until reaction product was apparent (approximately The most striking change in AChR channels following in- 30-60 min). The reaction was stopped by rinsing in phosphate buffer nervation was revealed by experiments where the AChR channel and the sections were dehydrated in ethanol, cleared in xylene, and distribution of individual neurons was “mapped” by recording coverslipped with Permount. AChR channel activity at multiple sites over the cell surface. We quantified the density of CO reaction product using computer- Regions of high channel activity (or “hot spots”) are found on assisted video-microdensitimetry with software obtained from Imaging Research. Toronto. Canada (see Mawe et al.. 1990). Brieflv. bright-field a majority of the neurons at later stages, indicating clustering images of sections were captured and displayed on the Gideosystem, of functional AChR channels. Furthermore, the different AChR and the perimeters of individual neurons randomly selected from all channel subtypes are apparently segregated both on a cell-by- lumbar ganglia (Ll-L5) were traced with a cursor. The optical density cell basis, as well as spatially segregated from one another on (OD) of the circumscribed area directly correlates with the density of The Journal of Neuroscience, January 1993, 13(i) 15 reaction product and hence the level of CO activity in the individual cell (Mawe et al., 1990). , Sympathetic preganglionic nucleus Preparation of acutely dispersed neurons for electrophysioIogica1 re- cording. All electrophysiological recording [with the exception of neu- ronal bungarotoxin (NBT) experiments; see below] was done on acutely dispersed sympathetic neurons. Briefly, lumbar sympathetic chains from ED 11 and ED 17 chickens were dissected directly into Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with horse serum (lo%), penicillin (50 U/ml), streptomycin (50 &ml), glutamine (2 mM), nerve growth factor (NGF, 2.5 S, 0.01 clg/ml; kindly provided by P. Osboume and G. Johnson, Washington University, St. Louis, MO), and ED 11 chick embryo extract (596, v/v). After removing as much of the connective tissue sheath as possible, the ganglia were dispersed to single cells by repeated passage through a fire-polished Pasteur pipette. The cells were then plated at one chain per 35 mm dish on a 0.1% poly-(L- omithine) substrate. Neurons dispersed in this manner were well iso- lated, phase bright, and spherical with no or few processes. Cells were used for physiological recording l-2 hr after plating. Macroscopic current recording. For macroscopic current recording, we used the whole-cell tight-seal configuration of the patch-clamp tech- Sympathetic ganglion nique (Hamill et al., 1981). In each experiment, the plating medium was removed and the neurons were gently rinsed with three changes of L- 15 tissue culture medium (GIBCO) supplemented with 4.5 mM cal- cium. The culture dish was placed on the stage of a Zeiss inverted microscope equipped with phase-contrast optics. Cells were viewed at 400 x with a 40 x objective. All experiments were done at room tem- perature (22-24°C). Patch pipettes were pulled from Omega Dot tubing (Glass Co. of America, Bargaintown, NJ) on a Kopf vertical puller and coated with Sigmacote (Sigma) to reduce capacitance. The recording pipette was connected to a Dagan 8900 patch-clamp amplifier. Pipette resistances were 8-12 MB. The recording pipette was filled with an intracellular solution con- sisting of 140 mM KCl, 2 mM MgCl,, 11 mM EGTA, 1 mM CaCl,, and 5 mM Mg-ATP buffered to pH 7.2 with 10 mM HEPES. The pressure ejection pipette used to apply ACh contained ACh (500 PM) dissolved in an extracellular solution consisting of 150 mM NaCl, 3 mM KCl, 1 mM MgCl,, 1 mM CaCl,, and 10 mM HEPES titrated to pH 7.2 with 1 M NaOH. For each cell, rest potential was measured as soon as the whole-cell configuration was achieved. Only cells with rest potentials more negative than -40 mV were used; these cells were then voltage clamped at -60 mV. ACh at 500 PM was pressure-applied (approximately 8 psi) for 10 set from a pipette placed approximately 50 pm from the soma. Previous Figure 1. DiI injected into the dorsal spinal cord labels the region of experiments have demonstrated that agents applied in this manner are the sympathetic preganglionic nucleus. A, Schematic diagram of a cross diluted by < 10% (Choi and Fischbach, 198 1). The peak amplitudes of section through the spinal cord and sympathetic chain illustrating mi- ACh-evoked currents were determined from the average of 5-10 points croelectrode injection of DiI (shaded area) into the preganglionic nu- at the peak of the digitized currents recorded with 4 kHz filtering. cleus. B, Fluorescence micrograph of an ED 11 spinal cord that was Single-channel recording. Single-channel recording was done using embedded and vibratome sectioned immediately following DiI injec- conventional cell-attached and inside-out patch-clamp techniques tion. The section is oriented as in the schematic diagram above. The (Hamill et al., 198 1). Detailed methods for cell-attached patch recording region of the preganglionic nucleus has been completely labeled. Scale and single-channel analysis have been described previously (Moss et bar, 200 pm. al., 1989). For inside-out patch recording, the plating medium was replaced with an intracellular solution consisting of 140 mM CsCl, 1 mru CaCl,, 5 mM Mg-ATP, 11 mM EGTA, and 10 mM HEPES titrated vidual ceils, and plated under conditions that suppress proliferation of to pH 7.3 with KOH. The recording pipette contained ACh dissolved non-neuronal cells as previously described in detail (Role, 1984). Cul- in an extracellular solution (see above). After the membrane patch was tures were used for physiological recording 4-10 d after plating. excised, channel openings were routinely examined for distortion in- Each NBT experiment was performed using the cell-attached patch dicative of vesicle formation. In such cases, it was sometimes possible configuration according to the following protocol. The initial level of to disrupt the vesicle by passing the pipette tip through the solution- AChR channel activity was determined from three to five patches on air interlace. neurons bathed in L-15 tissue culture medium. The cells were then Identification of channel amplitude classes. Channel openings in cell- incubated in NBT ( 15- 100 nM in L- 15; provided by Dr. Ralph Loring, attached patches were assigned to the S, M, or L (or S,,, M,,, L,,, or Northeastern University, Boston, MA) for 30-45 min at room temper- XL) channel classes based on their current amplitude at a patch potential ature. Channel activity was then determined from three to five patches of -50 mV relative to rest potential. As shown in Figures 7C and SC, on neurons incubated with NBT and with NBT included in the ACh the channel classes are well separated at this hyperpolarized potential. solution. Recovery was determined from three to five additional patches For each embryonic age, amplitude histograms from patches containing after the neurons were rinsed three times with L-l 5 (30-60 min). all channel classes were used to determine the mean amplitude and For each patch, we recorded for 5 min at a patch potential of -50 range for each class. In patches containing a subset ofthe channel classes, mV relative to rest potential (ACh = 2.5 PM). We then scored every an opening was assigned to a given class if its current amplitude fell patch recording for the presence of each channel class. A patch was within one standard deviation for that class. Events that could not be considered an “active patch” for a given class if one or more openings classified according to these criteria were considered ambiguous and belonging to that amplitude class were observed. were ignored. Distribution of surface AChRs. To map the cell surface distribution AChR block by NBT. Experiments examining AChR block by NBT of the S, M, L, and XL channel classes, three to five cell-attached patches were done using neurons that had been maintained in vitro. Lumbar were obtained on individual neurons. The patch pipette was positioned chain ganglia were dissected from ED 11 chickens, dispersed to indi- at approximately 3-5 pm intervals around the perimeter of the soma 16 Moss and Role Regulation of Neuronal AChRs during Innervation l cell capacitance using the capacitance compensation circuit of the patch- clamp amplifier (List EPC-7). In the cell-attached patch configuration, a 5 msec, 10 mV hyperpolarizing pulse was applied and the fast capa- citative transients (due primarily to pipette capacitance) were canceled with the capacitance circuit. The patch was then disrupted and the cell was voltage clamped at rest potential (only cells with rest potentials more negative than -40 mV were used). Series resistance ranged from 5 to 10 MQ. The increase in capacitance after breaking the patch, due to the additional cell capacitance, was estimated by noting the level of capacitance required for subsequent cancellation. Cell surface area was then calculated by dividing cell capacitance by specific membrane ca- pacitance (= 1 pF/cm*). We found that, for each embryonic age, the relationship between the surface area determinations based on the two approaches was linear; a plot of surface area from cell diameter versus surface area from cell capacitance yielded a straight line with a slope of approximately 0.5 (ED 11: r = 0.83, n = 24; ED 17: r = 0.77, n = 20). Results The number of preganglionicjibers and bouton-like structures in lumbar chain ganglia increases between ED 1 I and ED I7 To determine the time of arrival of preganglionic fibers to lum- bar sympathetic ganglia, we labeled the region of the pregan- glionic cell bodies with DiI and examined the ganglia for or- thograde labelingo f incoming fibers( seeM aterials and Methods, Fig. 1). Examination of ganglia and the interganglionic connec- tives from four ED 11 embryos (eight sympathetic chains) re- vealed very few labeledn eurites. In serials ectionso f four chains (56 sections)f rom two DiI-injected embryos, labeledf ibers were found in only seven sectionsf rom one embryo (Fig. 2). Most DiI-labeled neurites were within fiber tracts (Fig. 2&C’). Only one DiI-labeled processw as observed among cell bodieso f the ganglionic neurons( Fig. U), which were visualized by staining adjacent sections with cresyl violet (data not shown). These resultsi ndicate that there is little preganglionici nput to lumbar sympathetic ganglion neurons by ED 11. In contrast, injection of DiI into the sympathetic pregangli- onic nucleuso f ED 17 embryos labeledm any fibersa nd bouton- like structures within the lumbar sympathetic chain. Exami- nation of serial sectionso f three chains (73 sections)f rom ED 17 embryos revealed both labeledf ibers and bouton-like struc- tures in all sections( Fig. 3). In contrast to ED 11, many labeled processesw ere found among ganglion cell bodies (Fig. 3A,B), as well as within fiber tracts (Fig. 3C). Most DiI-labeled fibers were studded with a serieso f discrete, bouton-like swellingsa nd Figure 2. Few DiI-labeled preganglionic fibers are observed in ED 11 often appearedt o envelope cell bodieso f neuronsi n more rostra1 lumbar s.y mpathetic ganglia: fluorescence micrographs of vibratome- eanelia( Fin. 3D-F). In somec asese. xamination of suchs ections sectioned lumbar sympathetic ganglia from an elm bryo fixed and DiI a” t dYif fer\e n-t planes’ of focus revealed labeled axons leading to injected on ED 11 showing the most intensely lat Eled fibers observed. theseb outon-like swellings( data not shown). It should be noted A, Single labeled fiber among ganglion cell bodies that were visualized by sta-in_ine_ ____ a-d-_ia,c -e nt __.._s_e c.ti.o.n.s- - with cresvl violet. Suc h staining indicated that these results indicate a relative increase in the level of aDDl .oximatelv 20-25 cell bodies in the-ield shown here (data not shown). innervation between ED 11 and ED 17. rather than the absolute B and C, Labeled fibers within ganglionic connectives. Scale bar, 2b degreeo f innervation at either embrybnic age. Becauseo f the pm. thicknesso f the sections( approximately two to three cellst hick), only the most brightly labeled cells are readily visible. (a 10-20 pm in diameter). If the quality of the recording was compro- Synaptic activity, measured by CO histochemistry, increases mised (i.e., cell swelling, blebbing, or apparent membrane breakdown), mapping was discontinued. We recorded from each patch for 3 min at with the arrival of preganglionic inputs a p%h-potential of -50 mV relative to rest potential (2.5 PM ACh). Since preganglionic fibers and bouton-like structures can be vi- We then scored all patch recordings for the presence of each channel sualized within lumbar sympathetic ganglia by ED 17, we ex- class and determined the overall opening frequency (number of open- ings/sec, all classes combined). amined whether there was a concurrent increase in synaptic Neuronal membrane surface area. In most experiments, surface area input to ganglionic neurons.E lectrophysiologicalr ecordingf rom was calculated using the formula for the surface area of a sphere, A = embryonic ganglia, however, is both technically difficult and ud* (A = surface area and d = diameter). Cell diameter was measured restricted to neurons close to the surface of the ganglia( Dryer with a calibrated reticle at 500 x . To check the accuracy of this method, in some cases we also calculated surface area from measurements of and Chiappinelli, 1985). To determine the innervation status cell capacitance. The spherical shape of the cells enabled us to estimate of a large population of neurons, we assayed CO activity of The Journal of Neuroscience, January 1993. 73(l) 17 Figure 3. The number of DiI-labeled fibers and bouton-like structures is increased substantially by ED 17: fluorescence micrographs of a vibratome- sectioned lumbar sympathetic chain from an embryo fixed and injected with DiI on ED 17. A and B, Labeled fibers among ganglion cell bodies. Cresyl violet staining of adjacent sections indicated approximately 20-25 neuronal cell somas in each panel (data not shown). Note that most of the fibers have several bouton-like swellings. C, Labeled fibers within a fiber tract. SF, Neuron cell bodies surrounded by labeled bouton-like structures. In the cell shown in F, the nucleus appears in outline reflecting the lack of fluorescent structures over this region of the soma. Scale bar: 15 pm for A-C,; 10 pm for D-F. neuronsa t ED 11 and ED 18. The level of activity ofthis enzyme (Fig. 4B). The overall mean OD increasedx 54%, from 37 f 2 has previously been shown to correspond directly to the inner- (n = 50) at ED 11 to 57 + 1 (n = 50) at ED 18. vation status of sympathetic neurons (Mawe et al., 1990). Cry- ostat sectionso f an ED 11 lumbar sympathetic chain, processed ACh sensitivity increases during innervation in vivo for histochemicald emonstration of CO activity (enzyme activ- Previous experiments have demonstrated that innervation of ity is proportional to density of reaction product), revealed that chicken lumbar sympathetic neurons by explants of pregangli- the majority of cells were lightly stained, some barely above onic neuronsi n vitro is accompanied by increasedp ostsynaptic background levels (Fig. 4A). An occasional cell at this stage, ACh sensitivity (Role, 1988; Gardette et al., 1991 ). Comparison however, was more darkly stained( Fig. 4A, arrowheads).F igure of ACh-induced macroscopic currents of acutely dispersedE D 4B showsa histogram of the OD values from individual neurons 11 and ED 17 sympathetic neurons indicates that ACh sensi- in the samet issue. tivity also increases during this time of innervation in vivo (Fig. ED 18 lumbar sympathetic ganglia were processedf or CO 5A; ACh = 500 I.LM;m embrane potential = -60 mV). In ED activity in parallel with the ED 11 tissue. The enzyme activity 11 neurons, ACh evoked small, often barely detectable, re- of the neurons at ED 18 was considerably greater than at ED sponses.I n contrast, ACh induced robust responsesin ED 17 11 (Fig. 4A), and examination of a histogram of the OD deter- neurons. Figure 5B is a histogram displaying all peak current minations revealed little overlap between the two distributions values obtained for each age;t he mean peak macroscopicc urrent 18 Moss and Role l Regulation of Neuronal AChRs during Innervation --J B 15 1 B I I II 0 ED11 15 n ED18 I fl ED11 10 2 v n ED17 % n” * 5 0 8 x Optical density Figure 4. The level of CO activity, an indicator of electrical activity, Peakcurrent@A) increases between ED 11 and ED 18, indicating an increase in synaptic input to sympathetic neurons during this period in vivo. A, Cryostat sections from ED 11 and ED 18 lumbar sympathetic chains processed Figure 5. ACh sensitivity of sympathetic neurons increases during simultaneously for CO histochemistry. The density of reaction product innervation in vivo. A, Representative ACh-induced macroscopic cur- is proportional to the level of enzyme activity. Arrowheads indicate rents from acutely dispersed ED 11 (top) and ED 17 (bottom) lumbar unusually darkly staining cells observed in ED 11 ganglia. Scale bar, 15 sympathetic neurons. ACh = 500 PM. Membrane potential = -60 mV. pm. B, Histogram comparing ED 11 and ED 18 OD values from the Calibration: 25 pA, 2 sec. B, Histogram displaying all peak ACh-evoked tissue shown above. Each value is the mean OD per neuron; cells were current values obtained for each embryonic age. The mean peak current randomly selected from all lumbar ganglia (Ll-L5). Mean OD increased (arrowheads) increased from 31 + 7 pA (+SEM; n = 22) at ED 11 to *54% from 37 f 2 at ED 11 to 57 -+ 1 at ED 18. 137 + 10 pA (n = 22) at ED 17. Note that 23% (5 of 22) of the ED 11 cells had no detectable ACh response. increased 4.4-fold, from 31 + 7 pA (n = 22) at ED 11 to 137 + 10 pA (n = 22) at ED 17. Twenty-three percent (5 of 22) of the ED 11 cells had no detectable ACh response, whereas all ED 17 neurons were positive. The Journal of Neuroscience. January 1993. 13(l) 19 A The observed increase in sensitivity is not due to increased cell surfacea rea (with AChR density remaining constant). The 25 , mean surfacea rea of ED 11 and ED 17 neuronsw as calculated q ED11 from measurementso f cell diameter (surface area determined from cell diameter is directly correlated to surface area deter- ED 17 mined from cell capacitance;s eeM aterials and Methods). Com- parison of population histogramso f surfacea reav alues for ED 11a nd ED 17 acutely dispersedn euronsr evealed a 55% increase (from473+45~m2atED11,n=50,to731 +94pm*atED 17, n = 50; Fig. 6A) that cannot account for the =350% increase in sensitivity. ACh sensitivity also does not correlate with sur- face area on a cell-by-cell basisa t either embryonic age. Plots of peak ACh-evoked current versuss urfacea rea for ED 11 and ED 17 neuronsr eveal no correlation (Fig. 6B; ED 11: r = -0.28, n = 10; ED 17: r = -0.29, n = 10). Biophysical and pharmacological properties of AChRs expressedb y neuronsf rom ED 1I animals The increasedA Ch sensitivity of neuronsf ollowing innervation in vivo might be due to changesin the number and/or biophysical properties of the AChRs. We evaluated a possiblec ontribution of changesi n AChR channel properties by comparing ACh- activated single-channelc urrents in neurons acutely dispersed on ED 11 with currents in neuronsr emoved on ED 17. Cell-attached patch recordings from neurons acutely dis- persedf rom ED 11 animals revealed three amplitudeso f ACh- ED 11 activated channels. Nonconsecutive segmentsf rom a cell-at- tached patch recording illustrate each of the three classes(F ig. 7A; 2.5 PM ACh; patch membranep otential = - 50 mV relative to rest potential). Figure 7C is an amplitude histogram in which the values of all points in the sampledp ortion of the record are plotted to provide an unbiased estimate of the single-channel amplitudes. The three peaks indicate three amplitude classes. Superimposed on the histogram is the sum of three Gaussian curves with means + SD of 1.9 rt 0.3 pA, 3.5 + 0.3 pA, and 4.5 rfr 0.2 pA. We refer to these three classesa s S, M, and L (for small, medium, and large) sinces teady-statec urrent-voltage curves in the cell-attached configuration are identical to those of AChRs in ED 10 lumbar sympathetic neurons maintained in vitro (Moss et al., 1989;T able 1). Thus, the multiple subtypes and their characteristic conductancesa re apparently faithfully Surface area (clm2) retained by neurons in vitro (seeD iscussion). More detailed study of the properties of thesec hannelsi n the inside-out patch-clamp configuration indicates that the three classesd iffer both in slope conductance and in the extent of ED 17 rectification at more negative holding potentials (Fig. 7B). Vary- ing membrane potential from - 50 mV to -70 mV resulted in ohmic (i.e., linear) changesi n current amplitude for each class. Linear regressiono f data from inside-out patchesy ielded slope conductance values of 20 pS, 26 pS, and 40 pS. t Figure 6. ACh sensitivityd oesn ot correlatew ith cells ize.A , Observed increasein ACh sensitivityi s not duet o an increasien cells urfacea rea. Histogramo f surfacea read eterminationfso r both ED 11 and ED 17 acutely dispersedlu mbar sympatheticn euronsS. urfacea reaw ase al- culatedf rom celld iameterC. omparedto the observed~ 350%i ncrease in peakA Ch-evokedc urrent,t he means urfacea reain creaseodn ly 55%, from 473 + 45 pm* (n = 50) at ED 11 to 731 + 94 Mm2( n = 50) at ED 17. B, ACh sensitivity doesn ot correlatew ith cell surfacea reaa t 500 1000 1500 either embryonica ge:p eak ACh-inducedc urrent plotted versusc ell Surface area( pm*) surfacea reaf or both ED 11a nd ED 17a cutelyd ispersedsy mpathetic neuronsE. D 11: r = -0.28, n = 10; ED 17: r = -0.29, n = 10. 20 Moss and Role Regulation of Neuronal AChRs during Innervation l Figure 7. ACh activates three distinct channel amplitude classes in acutely dispersed ED 11 neurons. A, Nonconsecutive traces of a cell-attached patch recording from an acutely dispersed ED 11 neuron showing the S, M, and L channel classes. The traces were digitally low-pass filtered at 1.5 kHz. ACh concentration = 2.5 PM. Patch potential = -50 mV relative to rest potential. Calibration: 7.5 pA, 5 msec. B, Single-channel current amplitude versus membrane potential. These data were obtained in the inside-out patch-clamp configuration; each current-voltage curve was obtained from a separate patch. In this example, the S, M, and L classes had slope conductances of 20 pS, 26 pS, and 40 pS, respectively. The S class rectifies significantly at hyperpolarized potentials; the conductance between -50 mV and -70 mV is 20 pS, whereas between -50 mV and - 100 mV, the conductance is only 12 pS. C, Amplitude histogram showing that the three classes give rise to three peaks in the distribution. Superimposed on the histogram is the sum of three Gaussian curves with means 2 SD of 1.9 + 0.3 pA, 3.5 + 0.3 pA, and 4.5 + 0.2 pA. Table 1. Comparison of AChR channel properties in acutely dispersed neurons versus neurons maintained in vitro Conductance (pS) Relative frequency of occurrence Neurons Neurons Acutely dispersed maintained Acutely dispersed maintained neurons in vittw neurons in vitr@ Class ED 11 ED 17 ED 10 ED 17 ED 11 ED 17 ED11 ED17 S 17 + 1 22 f 1 14+ 1 23 z?z 1 100% 80% 63% 30% M 28 rf: 2 35 + 1 27 I!I 1 38 -+ 2 27% 27% 83% 57% L 46 f 3 47 f 1 49 + 2 50 + 1 23% 57% 50% 67% XL NP 72 + 2 NP 69 + 6 NP 3% NP 8% AChR conductance and frequencyo foccurrence of each channel classw ere determined for both acutely dispersed neurons and neurons from animals of the same developmental stage but then maintained in vitro for 6-8 d prior to recording. Conductance values from acutely dispersed neurons were either determined from cell-attached patch recordings at 30, 50, and 70 mV applied potential or calculated from channel amplitude determinations at 50 mV, assuming a cellular rest potential of -50 mV, with identical results. NP, Not present. * Data from Moss et al. (1989). The Journal of Neuroscience, January 1993. 13(l) 21 Recordings from inside-out patches at more hyperpolarized A potentials revealed considerable rectification of the S class (i.e., the current-voltage relationship is nonlinear). The conductance 100 nM NBT of the S class over the linear range of the curve (between -50 El Cont mV and -70 mV) is approximately 20 pS, whereas between H NBT - 50 mV and - 100 mV the conductance is = 12 pS. The cur- rent-voltage relationship for both the M and L channel classes 80 remains ohmic over this range (data not shown). The S, M, and L channel classes also differ in their relative contribution to overall channel activity. The frequency of oc- currence of each class, determined from 30 “active” cell-at- tached patches (i.e., patches containing channel activity, 3 min recordings, 2.5 PM ACh, patch potential -50 mV relative to rest) revealed that S is the most common class, occurring in 100% of the patches. The M and L classes contributed about equally to overall channel activity, occurring in 27% and 23% of patches, respectively. Note that this analysis reveals the rel- ative frequency of appearance of the classes, rather than differ- ences in the frequency of channel opening; opening frequencies of the three classes are quite similar (median opening frequency for each class = 0.03 openings/set; 2.5 I.~MA Ch). The S, M, and L classes dl$er in sensitivity to NBT B ED 11 neurons, whether examined just following dispersal or after being maintained 1 week in vitro, express the same three 15-25 nM NBT AChR amplitude classes. Since the S, M, and L classes are observed in isolation (Moss et al., 1989), the three classes rep- 1001 R Cont resent distinct AChR subtypes. To probe possible structural H NBT differences between the subtypes, we examined their relative sensitivity to NBT, an antagonist specific for some neuronal 80 nicotinic AChRs (Ravdin and Berg, 1979; Chiappinelli, 1983; Loring et al., 1984). To test whether each channel class is sensitive to NBT, we examined channel block by 100 nM NBT, a concentration that effectively blocks NBT-sensitive AChRs in other cultured neu- rons (Sah et al., 1987). To determine the control level of channel activity, cell-attached patches were first scored for the presence of each channel class. After a 45 min incubation in toxin, a second set of patches was scored in the same manner. Figure 8A shows the combined results of two experiments (a total of 10 control patches, 10 experimental patches) showing that all classes were substantially inhibited, The S class decreased to 33% of the control level of activity, and the M and L conductance classes were both completely blocked. It should be noted that Figure 8. S, M, and L classes differ in sensitivityt o NBT. A, Each these experiments were performed on lumbar sympathetic neu- channel class is substantiallyb lockedb y 100n o NBT. The S class rons maintained in vitro. Thus, the control levels of channel decreasetdo =33% of the control level of activity. Both the M andL classews erec ompletelyb locked( two experiments1, 0p atchesp erc on- activity for each class differ from that in acutely dispersed neu- dition). B, Block by 15-25 nM NBT revealed that the S, M, and L classes rons. Inhibition of AChR channel activity by NBT was only differ in sensitivitv to NBT. The S class is the least sensitive to toxin partially reversible. After a 60 min incubation in toxin-free block, decreasing only slightly to =93% of control. In contrast, the M media, S, M, and L channel activity increased significantly but channel was significantly inhibited by toxin, decreasing to =63% of not to control levels (data not shown). This partial recovery over control. The L class is the most sensitive to NBT, decreasing to ~471 of control (five experiments, 23 patches per condition). It should be 1 hr is consistent with previous studies of recovery of ACh- noted that these experiments were performed on lumbar sympathetic activated currents following NBT treatment in other neurons neurons maintained in vitro. Thus, the control levels of channel activity (Lipton et al., 1987; Sah et al., 1987). for each class differ from that in acutely dispersed neurons. To test for differences in NBT sensitivity between S-, M-, and L-type channels, we examined the effects of lower concentrations of toxin (15 nM and 25 nM). Figure 8B shows the combined nificantly inhibited by NBT, decreasingt o 64% of control. Fi- results of five experiments (a total of 23 control patches, 23 nally, the L channelc lassi s the most sensitivet o the toxin, with experimental patches) indicating that the AChR subtypes may channel activity reducedt o 47% of control. Analysis of opening be distinguished by their differential sensitivity to NBT. The S frequency data for each classu nder thesec onditions confirms class is the least sensitive to toxin block, decreasing only slightly the differential sensitivity of the subtypes to NBT block (data to 93% of control. In contrast, the M class openings were sig- not shown). 22 Moss and Role Regulation of Neuronal AChRs during Innervation l A s17 Ml7 B M17 V NW -100 -80 -60 40 -20 I I I I I I (PA) S 17’ Ml7 T L17 Figure 9. AChR channel properties change in a manner consistent with the observed increase in ACh sensitivity. A, A fourth, larger ACh-activated amplitude class, denoted XL, is present: nonconsecutive traces from four cell-attached patch recordings from an acutely dispersed ED 17 neuron illustrating the S,,, M,,, L,,, and XL channel classes. The traces were digitally low-pass filtered at 1.5 kHz. ACh concentration = 2.5 PM. Patch potential = -50 mV relative to rest potential. Calibration: 7.5 pA, 5 msec. B, The slope conductances of both the S and M (but not L) classes increase by =3MO%, with the shift in the S class possibly due to a decrease in the extent of rectification. Single-channel current amplitude is plotted against membrane potential. The current-voltage curve for S,, was obtained from a separate patch. C, Amplitude histograms from two different cell-attached patches showing that the XL class gives rise to a distinct peak in the amplitude distribution. The rap histogramsh ows the S,,, M,,, and L,, channel classes; superimposed on the histogram is the sum of three Gaussians with means (&SD) of 1.9 + 0.2 pA, 3.4 + 0.3 PA, and 5.0 f 0.2 PA. The bottomh istogramsh ows the M,, and XL classes; superimposed on the histogram is the sum of two Gaussian curves with means (*SD) of 3.7 + 0.3 pA and 7.6 + 0.4 pA. (*SD) of 3.7 + 0.3 pA and 7.6 + 0.4 PA. The top histogram AChRs undergo developmental changes in channel properties showst he S,,, M,,, and L,, channel classess; uperimposedo n consistent with an increase in ACh sensitivity the histogram is the sum of three Gaussiansw ith means( &SD) Cell-attached patch recordings from acutely dispersedE D 17 of 1.9 + 0.2 pA, 3.4 f 0.3 pA, and 5.0 + 0.2 pA. The low neurons revealed a fourth, larger-amplitude ACh-activated frequency of occurrenceo f XL channel openings( ~3% of cell- channelc lass( denotedX L). Nonconsecutive segmentsfr om cell- attached patches) prohibited determination of slope conduc- attached patch recordingsi llustrate all four channelc lassesn, ow tance; however, its amplitude at a membranep otential of - 100 denoted S,,, M,,, L,,, and XL, seena t this embryonic age( Fig. mV (membrane potential = -50 mV below an assumedr est 9A; ACh = 2.5 PM; patch potential = -50 mV relative to rest potential of - 50 mV) indicates a conductanceo f %7 2 pS, con- potential). Figure 9C includes two amplitude histogramsf rom sistent with results obtained in ED 17 neurons maintained in two different cell-attached patches showing that the XL class vitro (Moss et al., 1989; Table 1). Some patchesf rom neurons gives rise to a distinct peak in the amplitude distribution. The dispersedo n ED 17c ontained only XL openings,i ndicating that bottom histograms howst he M,, and XL classess; uperimposed XL representsa new AChR subtype, rather than the simulta- on the histogrami s the sum of two Gaussianc urves with means neouso pening of smaller conductance channels.