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Purinergic synapses formed between rat sensory neurons in primary culture
Neuroscience 126 (2004) 195–201
PURINERGIC SYNAPSES FORMED BETWEEN RAT SENSORY NEURONS IN PRIMARY CULTURE M. M. ZAREI,a* B. TOROb AND E. W. MCCLESKEYc
In addition to in vivo studies, there has been one previous report of synapses formed between embryonic sensory neurons from nodose ganglia in dissociated tissue culture (Cooper, 1984). In light of such data obtained in vivo, and in vitro, we asked whether DRG neurons could communicate with each other in tissue culture. Finding that they can, we asked whether this occurs through explicit, Ca2⫹-dependent synapses, and we sought the identity of the neurotransmitter. Experiments were performed in dissociated primary culture and DRG explant culture using whole cell patch clamp and an immunocytochemical measure of active synapses.
a
Center for Biomedical Studies, The University of Texas at Brownsville and Texas Southmost College, Brownsville, TX 78520, USA b
Arthritis and Osteoporosis Center, Brownsville, TX 78526, USA
c
Vollum Institute, Oregon Health and Science University, Portland, OR 97239, USA
Abstract—Though there is some evidence to the contrary, dogma claims that primary sensory neurons in the dorsal root ganglion do not interact, that the ganglion serves as a through-station in which no signal processing occurs. Here we use patch clamp and immunocytochemistry to show that sensory neurons in primary culture can form chemical synapses on each other. The resulting neurotransmitter release is calcium dependent and uses synaptotagmin-containing vesicles. On many cells studied, the postsynaptic receptor for the neurotransmitter is a P2X receptor, an ion channel activated by extracellular ATP. This shows that sensory neurons have the machinery to form purinergic synapses on each other and that they do so when placed in short-term tissue culture. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.
EXPERIMENTAL PROCEDURES Tissue culture Adult Sprague–Dawley rats (Charles River) were initially anesthetized with halothane (Halocarbon Ind.) and then beheaded according to institutionally approved protocol. All experiments conformed to NIH and international guidelines on the ethical use of animals. Minimum number of animals was used to achieve statistical significance and high dose of anesthetic was used to minimize animal suffering. Trigeminal or DRG were dissected from 200 to 300 g Sprague–Dawley rats, dissociated with enzymes (papain, dispase and collagenase) and plated on glass coverslips coated with poly-D-lysine/laminin as previously described (Eckert et al., 1997). Cells were grown for 1–3 days at 37 °C in 5% CO2 in F12 media plus 10% heat-inactivated fetal calf serum, 50 U/ml penicillin, 50 g/ml streptomycin and 50 ng/ml nerve growth factor. Explants were prepared from DRG dissected from newborn rats and grown with the same media as above. The DRG were stripped of their outer connective tissue and grown for 24 h with a volume of media just sufficient to cover them; this kept the ganglion from floating off the coverslip before it attached to the substrate. Dissociated DRG or trigeminal neurons were added to the culture 24 h after the explants were started and experiments were typically done 48 –72 h after plating when the field of axons growing from the explant had passed many dissociated neurons.
Key words: dorsal root ganglion, P2X receptors, sensory processing.
Scattered evidence challenges the dogma that sensory neurons do not communicate within sensory ganglia. Peptide-containing presynaptic terminals clearly form between sensory neurons after peripheral nerve damage (McLachlan and Hu, 1998), confirming the occasional observations of sensory–sensory synapses reported to occur rarely in ganglia or nerve in the absence of damage (Miller et al., 1970; Herrick, 1924; Kruger and Halata, 1996). Calcium-dependent exocytosis of sensory peptides— substance P and calcitonin gene-related peptide (CGRP)— occurs within sensory ganglion (Huang and Neher, 1996; Ulrich-Lai et al., 2001), as does co-release of ATP and substance P (Matsuka et al., 2001). Particularly intriguing physiological studies by Devor and colleagues (Amir and Devor, 1997; Amir and Devor, 1996; Liu et al., 1999) report that the membrane potential of individual dorsal root ganglion (DRG) neurons depolarizes when other neurons in the ganglion fire action potentials at a high rate. This behavior may contribute to pathological pain states (Devor et al., 2002).
Electrophysiology Whole-cell currents were measured using standard patch-clamp techniques with 2–5 M⍀ pipettes and an Axopatch 200 amplifier (Axon Inst.). Currents were filtered at 5 kHz and sampled at 0.1 ms intervals. Acquisition and analysis were done with PClamp 6 (Axon Inst) software and Origin (Microcal). Rapid agonist and drug applications were achieved using a linear array of flow pipes with solution changes accomplished by opening or closing latching valves (General Valve). External solution contained (mM): 150 NaCl, 2 CaCl2, 1 MgCl2, 3 KCl, 10 glucose, 10 HEPES. In all experiments except reversal potential measurements, internal (pipette) solution contained (mM): 140 KMeSO3, 10 KCl, 1 Na2ATP, 2 MgCl2, 0.3 Na3GTP, 5 EGTA, 10 HEPES. For reversal potential measurements, internal solution contained (in mM): 140 NaMeSO3, 10 NaCl, 1 Na2ATP, 2 MgCl2, 0.3 Na3GTP, 5 EGTA, 10 HEPES; this solution eliminated steady outward potassium
*Corresponding author. Tel: ⫹1-956-554-5069; fax: ⫹1-956-554-5065. E-mail address: [email protected] (M. M. Zarei). Abbreviations: CNQX, 6-cyano-7-nitroquinoxiline-2,3-dione; DRG, dorsal root ganglion; P2X receptor, an ion channel that opens in response to extracellular ATP; PBS, phosphate buffered saline.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.03.019
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Fig. 1. Consistent amplitude of spontaneous currents in cultured sensory neurons. Insets show spontaneous currents from two different cells, one held at ⫺60 mV (A) and the other at ⫾40 mV (C). Asterisks in (A) indicate currents shown at expanded scale in (B). Histograms of the amplitudes of all current events (A, C) have a major peak at a certain minimal amplitude with occasional larger events. The cell in C had many currents larger than the minimum, so we attempted to fit them with the indicated Gaussian distribution. The data seem insufficient to conclude that the larger amplitudes are multiples of the smallest. A plot of the average amplitude of the smallest currents at different voltages for the cell in C reveals strong inward rectification (D); that is, outward currents do not occur above the apparent reversal potential near 0 mV. Scale bar in A⫽100 pA, 50 ms; B⫽50 pA, 25 ms; C⫽100 pA, 20 ms.
currents. All solutions had pH 7.35–7.4; 315–325 mmol/kg, and all experiments were conducted at room temperature, 23 °C. To evoke synaptic currents in sensory neurons, we recorded, with whole cell patch clamp, from a dissociated sensory neuron that was in co-culture with an explanted ganglion. To simultaneously stimulate many neurons at once within the explant ganglion, a flat, concentric bipolar extracellular electrode (200 m outer pole, 25 m inner pole; Stoelting Co. 50691) was pushed into its center and 0.1 ms current pulses were applied once per second. Stimulating strength was adjusted upward while responses were measured in the individual sensory neuron.
Immunofluorescence of active presynaptic terminals To visualize active synaptic terminals, we used an antibody raised against the lumenal domain of a protein that spans synaptic vesicles, synaptotagmin I, in cells whose surface membrane was intact, as previously described (Perin et al., 1991; Matteoli et al.,
1992; Zarei et al., 1999). Neurons were incubated with 10 nM of the toxin, taipoxin, that was added in complete media and incubated at 37 °C, 5% CO2 for 40 min. Taipoxin retards the recycling of synaptic vesicles, and consequently, the lumenal domain of the synaptic vesicles containing synaptotagmin gets exposed on the surface of the neuron. The cells were washed with phosphate buffered saline (PBS; in mM: 10 Na2HPO4, 2.3 NaH2PO4, 138 NaCl, and 2.7 KCl, pH 7.4), fixed with a 4% paraformaldehyde solution for 20 min, and washed again with PBS (note that no detergent is used to permeabilize the cells). Fixed neurons were incubated overnight with rabbit anti-synaptotagmin I IgG (anti-Syn I), washed for 30 min, incubated with fluorescein-labeled goat anti-rabbit antibody (Vector Laboratory) for 20 min, washed for 20 min with PBS, mounted (Molecular Probe Prolong), and phase and fluorescent images were acquired using a confocal microscope. Anti-Syn I was developed against the NH2 terminus of synaptotagmin I, which is located in the lumenal domain of synaptic vesicles (Perin et al., 1991; Matteoli et al., 1992).
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Fig. 2. Spontaneous currents are purinergic. (A) Top five traces show representative spontaneous currents in control conditions and bottom five show representative currents from the same cell in the presence of 100 M suramin. Amplitude histograms show an obvious decrease in current amplitude in suramin (black bars). (B) Results from another cell showing amplitudes of spontaneous currents plotted sequentially. Bar shows application of suramin; arrows show when the three sets of records shown above occurred. (C) Summary from seven cells showing the average fractional change in amplitude and frequency of spontaneous currents compared with records before suramin application. Suramin decreases amplitude without a consistent effect on frequency. Nothing occurred with a cocktail of blockers of glutamate, nicotinic, and acid-sensing channels (CNQX, 10 M; d-tubocurarine, 100 M; and amiloride, 100 M; n⫽4). (D) Perfusion of 0.5 mM ATP eliminated the spontaneous currents (n⫽3). Histograms give the number of spontaneous current events in 1 s bins vs. experimental time for a representative cell. Upper bar shows the time of ATP application; lower bars show the times for the stretches of raw data shown above (before and during ATP) and at inset (after recovery). ATP application causes a standing current that desensitizes and eliminates spontaneous current events. Scale bar in A⫽50 pA, 25 ms; B⫽100 pA, 25 ms; D⫽200 pA, 1.2 s.
RESULTS Spontaneous currents in cultured sensory neurons Fig. 1A, B shows spontaneous currents recorded with a whole cell patch clamp from a sensory neuron. These neurons were dissociated from sensory ganglia and cultured with nerve growth factor for 2–5 days; of 55 neurons tested, 20 exhibited such spontaneous currents. Most currents on a given cell had particular amplitude (about 100 pA in Fig. 1A, 40 pA in Fig. 1C). Almost all other amplitudes seen are larger than the most common one (Fig. 1C). Because only sensory neurons are present in these cultures, this result suggests that sensory neurons can form synapses on each other.
Evidence for purinergic synapses Consistent with excitatory synaptic currents, these spontaneous currents approach zero amplitude when the holding voltage approaches 0 mV. But unlike most synaptic currents they fail to conduct outward current (Fig. 1C, D). Such inward rectification is a property of P2X receptors, ion channels that are activated by extracellular ATP (North, 2002). Furthermore, application of suramin, a blocker of P2X receptors, diminishes the amplitude (Fig. 2A) of the spontaneous currents without any consistent effect on frequency (Fig. 2B, C). This is the effect expected of a postsynaptic blocker of an ion channel; thus, P2X receptors are localized at post-synaptic junctions. A mixture of other ion
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Fig. 3. Spontaneous currents require external calcium. Spontaneous currents from a particular cell (A) occur infrequently when extracellular Ca2⫹ is 0.5 mM compared with 2 mM. Amplitude histogram of all current events (B) shows that the primary amplitude appears unchanged in 0.5 mM Ca2⫹ (dark bars) though the number of events is greatly diminished; inset shows fits to data only. Time course of the number of events per (3.6 s) bin (C) with the time of the change to 0.5 mM Ca2⫹ indicated. Summary from four cells (D) shows that dropping Ca2⫹ concentration from 2 mM to 0.5 mM does not alter the primary amplitude of spontaneous currents, but does decrease the frequency at which they occur. Scale bar in A⫽200 pA, 50 ms.
channel blockers (6-cyano-7-nitroquinoxiline-2,3-dione [CNQX], 10 M, for glutamate channels; d-tubocurarine, 100 M, for nicotinic acetylcholine receptors; and amiloride, 100 M, for acid-sensing channels) had no effect (Fig. 2C, blockers), leaving suramin as the only effective blocker we used. Finally, if ATP is indeed the neurotransmitter, exogenous application of ATP should eliminate the spontaneous currents by desensitizing P2X receptors. Indeed, high concentration of ATP significantly reduces spontaneous events in a reversible manner (Fig. 2D, solid bar). Since Ca2⫹ triggers release of neurotransmitter vesicles, we asked whether the spontaneous currents were sensitive to extracellular Ca2⫹ concentration. Dropping extracellular Ca2⫹ from 2 mM to 0.5 mM greatly decreased the frequency of spontaneous currents in the cell in Fig. 3A. There was no shift in the predominant amplitude of the
currents (amplitude histograms, Fig. 3B). Fig. 3C shows the frequency of spontaneous currents over time and Fig. 3D collects results from four cells on the effect of Ca2⫹ on average amplitude and frequency. Clearly, Ca2⫹ decreases the frequency of spontaneous currents without affecting their amplitude, opposite to the action of suramin (Fig. 2C). This indicates that Ca2⫹ controls presynaptic release whereas suramin blocks the postsynaptic receptor. To further test this, we attempted to evoke synaptic currents between sensory neurons. Evoked synaptic current between sensory neurons Can stimulation of one sensory neuron evoke synaptic currents in another? Connections between single pairs of neurons are rare even when sensory neurons are cultured with spinal neurons (Jahr and Jessell, 1985); thus, we
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Fig. 4. Evoked synaptic currents. Whole ganglia were cultured together with isolated sensory neurons so that the field of axons emanating from the ganglion contacted neurons that could be patch clamped. The photograph (A) shows a patch pipette recording from an individual cell (expanded in inset) that lies within the axons coming from the ganglion contacted by the large stimulating electrode, S. Recordings from the neurons (B) have a sharp shock artifact followed by an inward current that occurs a consistent time after the shock and usually has the same amplitude (nine successive records shown). Suramin fully blocked the inward current in four cells from three preparations (C, left), but had no effect in six other cells (C, middle and right); a cocktail of blockers of other fast ligand gated channels also failed to block this suramin-insensitive current (C, right; suramin, 500 M; CNQX, 10 M; d-tubocurarine, 100 M; amiloride, 100 M). These block P2X, AMPA, nicotinic and acid-sensing ion channels respectively. Scale bar in B⫽100 pA, 50 ms; C⫽100 pA, 10 ms.
enhanced the number of synapses by co-culturing whole DRG with dissociated sensory neurons. In this approach, whole ganglia were cultured for several days together with dissociated sensory neurons until the outgrowing field of axons passed over single isolated neurons (Fig. 4A). Then, electrical shocks were delivered through a flat extracellular electrode pressed against the ganglion while recording from a single cell in the ganglion’s field of axons. We were able to evoke synaptic currents between sensory neurons, and the currents had a regular amplitude over time (Fig. 4B). Suramin totally blocked these evoked currents in four of 10 cells tested (Fig. 4C, left). However, suramin did not affect some cells (Fig. 4C, middle). A mix of blockers (CNQX, 10 M; d-tubocurarine, 100 M; and amiloride, 100 M) also failed to block these suramin-insensitive currents (Fig. 4C, right), indicating that fast glutamate, nicotinic, or acid-sensing ion channels were not involved. Future studies are needed to determine the nature of suramin-insensitive evoked responses. Visualizing active sensory–sensory synapses To independently test for the presence of sensory–sensory synapses, we used taipoxin, which inhibits recycling of synaptic vesicles and thereby leaves their lumenal face exposed on the surface of cells after vesicles have fused
(Zarei et al., 1999). This exposed lumen can be labeled with anti-Syn I, an antibody that is targeted to the lumenal side of a vesicle protein, synaptotagmin. The antibody is applied to cells that have not been permeabilized so it labels only surface proteins and not intracellular vesicles. The procedure labels only those synaptic sites that were active during the taipoxin treatment period. Fig. 5 shows phase (left) and confocal fluorescent (right) images of two sensory neuron cell bodies and many passing axons. Many fluorescent puncta decorate the cells and axons. Note on the left cell that some puncta align, suggesting that they come from a single axon lying across the cell. We take these puncta to indicate the presence of active synaptic release sites. Because this culture contains only sensory neurons, this is independent evidence that sensory– sensory synapses are actively formed in culture.
DISCUSSION We report three observations that show conclusively that sensory neurons can form functional synapses on each other in short term tissue culture: 1) about a third of sensory neurons exhibit spontaneous Ca2⫹-dependent currents of consistent amplitude; 2) synaptic currents can be evoked between sensory ganglia and isolated sensory neurons; 3) synaptotagmin-labeled hotspots of synaptic
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Fig. 5. Immunocytochemical evidence of active synapses. After a 40 min exposure to taipoxin, which inhibits endocytosis of synaptic vesicles that have fused to the surface membrane, neurons were lightly fixed, without being permeabilized, and treated with an antibody to the lumenal face of synaptotagmin I (Syn I), a synaptic vesicle protein. Phase (left) and confocal (right) images of a field containing two sensory neurons and axons after labeling with anti-Syn I. Punctate fluorescence demonstrates areas where synaptic vesicles had fused to surface membranes during the taipoxin treatment. Many of these puncta align (see for example on the left cell), as expected if they were on axons.
activity decorate sensory axons and these appear to cross cell bodies. Many of these synapses are purinergic because spontaneous currents and 40% of evoked currents were sensitive to P2X channel blockade and were eliminated by extracellular ATP, which desensitizes P2X channels. A puzzling aspect of the study is that all spontaneous currents were purinergic whereas only 40% of evoked currents were purinergic. One possible explanation is the difference in culture conditions. Spontaneous currents were measured in dissociated tissue culture whereas evoked currents involved explant cultures. The explants would include intact satellite/glial cells whereas these are lost in short term, dissociated culture. The presence or absence of satellite cells greatly influenced the appearance of nicotinic synaptic currents between nodose sensory neurons in culture in another study (Cooper, 1984). Because we did not observe any nicotinic synapses, the relation between the Cooper study and ours is unclear. However, his study clearly demonstrates that changes in tissue culture conditions can influence synaptic formation between sensory neurons. Our present work demonstrates that sensory neurons are capable of communicating with each other through purinergic synapses in short term tissue culture. Furthermore, both in vivo and vitro studies have indicated that DRG neurons can also communicate through crossexcitation (Amir and Devor, 1996; Devor and Wall, 1990; Utzschneider et al., 1992). In cross-excitation, spike activity in one neuron excites spiking in adjacent neurons of the same DRG (Devor and Wall, 1990). This crossdepolarization appears to be mediated by neurotransmitter(s) that are released non-synaptically during neuronal activities (Amir and Devor, 1996; Shinder and Devor,
1994). Cross-excitation may not play a major role in normal communications between sensory neurons since very few synaptic contacts are observed in the healthy DRG neurons (Pannese, 1981). However, during peripheral nerve injury dramatic changes occur within the DRG that may alter the normal sensory communications. Trauma to a peripheral nerve is followed by structural changes in the DRG and spinal cord. These changes include sprouting of sympathetic axons that forms rings of noradrenergic terminals around the DRG neurons (McLachlan et al., 1993; Ramer and Bisby, 1997) and sprouting of small sensory axons that contain calcitonin gene-relegated peptide and substance P (McLachlan and Hu, 1998). In addition, peripheral nerve injury has also been reported to induce sprouting of A-fibers into laminae I–II of the spinal cord (Woolf et al., 1992, 1995). It has been postulated that these structural changes can alter sensory processing and may lead to neuropathic pain (McLachlan and Hu, 1998). It is quite possible that the purinergic synapses we find so common when sensory neurons are in culture several days are a response to the severing of axons that occurs when ganglia are dissociated. Such tissue damage causes release of nerve growth factor, which induces sprouting of sensory neurons (McLachlan and Hu, 1998; Diamond et al., 1992) and might contribute to the formation of synaptic structures between sensory neurons that follows axon damage (McLachlan and Hu, 1998). Experiments with in vivo preparations will be necessary to find whether the purinergic synapses form between sensory neurons in vivo, whether this is more common after nerve injury, and whether this has physiological or pathological significance. Acknowledgements—The NIH supported this work through a grant to Edwin W. McCleskey.
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REFERENCES Amir R, Devor M (1997) Spike-evoke suppression and burst patterning in dorsal root ganglion neurons of the rat. J Physiol (Lond) 501: 183–196. Amir R, Devor M (1996) Chemically mediated cross-excitation in rat dorsal root ganglia. J Neurosci 16:4733–4741. Cooper E (1984) Synapse formation among developing sensory neurones from rat nodose ganglia grown in tissue culture. J Physiol 351:263–274. Devor M, Amir R, Rappaport ZH (2002) Pathophysiology of trigeminal neuralgia: the ignition hypothesis. Clin J Pain 18:4 –13. Devor M, Wall PD (1990) Cross-excitation in dorsal root ganglia of nerve-injured and intact rats. J Neurophysiol 64:1733–1746. Diamond J, Holmes M, Coughlin M (1992) Endogenous NGF and nerve impulses regulate the collateral sprouting of sensory axons in the skin of the adult rat. J Neurosci 12:1454 –1466. Eckert SP, Taddese A, McCleskey EW (1997) Isolation and culture of rat sensory neurons having distinct sensory modalities. J Neurosci Methods 77:183–190. Herrick CJ (1924) An introduction to neurology. London: W. B. Saunders Company. Huang LY, Neher E (1996) Ca(2⫹)-dependent exocytosis in the somata of dorsal root ganglion neurons. Neuron 17:135–145. Jahr CE, Jessell TM (1985) Synaptic transmission between dorsal root ganglion and dorsal horn neurons in culture: antagonism of monosynaptic excitatory postsynaptic potentials and glutamate excitation by kynurenate. J Neurosci 5:2281–2289. Kruger L, Halata A (1996) Structure of nociceptor ending. In: Neurobiology of nociceptors (Belmonte C, Cervero F, eds), pp 37–71. New York: Oxford University Press. Liu CN, Amir R, Devor M (1999) Effect of age and nerve injury on cross-excitation among sensory neurons in rat dorsal root ganglia. Neurosci Lett 259:95–98. Matsuka Y, Neubert JK, Maidment NT, Spigelman I (2001) Concurrent release of ATP and substance P within guinea pig trigeminal ganglia in vivo. Brain Res 915:248 –255. Matteoli M, Takei K, Perin MS, Sudhof TC, De Camilli P (1992)
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Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. J Cell Biol 117:849 –861. McLachlan EM, Hu P (1998) Axonal sprouts containing calcitonin gene-related peptide and substance P form pericellular baskets around large diameter neurons after sciatic nerve transection in the rat. Neuroscience 84:961–965. McLachlan EM, Janig W, Devor M, Michaelis M (1993) Peripheral nerve injury triggers noradrenergic sprouting within dorsal root ganglia. Nature 363:543–546. Miller R, Varon S, Kruger L, Coates PW, Orkand PM (1970) Formation of synaptic contacts on dissociated chick embryo sensory ganglion cells in vitro. Brain Res 24:356 –358. North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067. Pannese E (1981) The satellite cells of the sensory ganglia. Adv Anat Embryol Cell Biol 65:1–111. Perin MS, Brose N, Jahn R, Sudhof TC (1991) Domain structure of synaptotagmin (p65). J Biol Chem 266:623–629. Ramer MS, Bisby MA (1997) Rapid sprouting of sympathetic axons in dorsal root ganglia of rats with a chronic constriction injury. Pain 70:237–244. Shinder V, Devor M (1994) Structural basis of neuron-to-neuron crossexcitation in dorsal root ganglia. J Neurocytol 23:515–531. Ulrich-Lai YM, Flores CM, Harding-Rose CA, Goodis HE, Hargreaves KM (2001) Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from rat trigeminal ganglion: evidence for intraganglionic neurotransmission. Pain 91:219 –226. Utzschneider D, Kocsis J, Devor M (1992) Mutual excitation among dorsal root ganglion neurons in the rat. Neurosci Lett 146:53–56. Woolf CJ, Shortland P, Coggeshall RE (1992) Peripheral nerve injury triggers central sprouting of myelinated afferents. Nature 355:75– 78. Woolf CJ, Shortland P, Reynolds M, Ridings J, Doubell T, Coggeshall RE (1995) Reorganization of central terminals of myelinated primary afferents in the rat dorsal horn following peripheral axotomy. J Comp Neurol 360:121–134. Zarei MM, Radcliffe KA, Chen D, Patrick JW, Dani JA (1999) Distributions of nicotinic acetylcholine receptor alpha7 and beta2 subunits on cultured hippocampal neurons. Neuroscience 88:755–764.
(Accepted 11 March 2004)
PURINERGIC SYNAPSES FORMED BETWEEN RAT SENSORY NEURONS IN PRIMARY CULTURE M. M. ZAREI,a* B. TOROb AND E. W. MCCLESKEYc
In addition to in vivo studies, there has been one previous report of synapses formed between embryonic sensory neurons from nodose ganglia in dissociated tissue culture (Cooper, 1984). In light of such data obtained in vivo, and in vitro, we asked whether DRG neurons could communicate with each other in tissue culture. Finding that they can, we asked whether this occurs through explicit, Ca2⫹-dependent synapses, and we sought the identity of the neurotransmitter. Experiments were performed in dissociated primary culture and DRG explant culture using whole cell patch clamp and an immunocytochemical measure of active synapses.
a
Center for Biomedical Studies, The University of Texas at Brownsville and Texas Southmost College, Brownsville, TX 78520, USA b
Arthritis and Osteoporosis Center, Brownsville, TX 78526, USA
c
Vollum Institute, Oregon Health and Science University, Portland, OR 97239, USA
Abstract—Though there is some evidence to the contrary, dogma claims that primary sensory neurons in the dorsal root ganglion do not interact, that the ganglion serves as a through-station in which no signal processing occurs. Here we use patch clamp and immunocytochemistry to show that sensory neurons in primary culture can form chemical synapses on each other. The resulting neurotransmitter release is calcium dependent and uses synaptotagmin-containing vesicles. On many cells studied, the postsynaptic receptor for the neurotransmitter is a P2X receptor, an ion channel activated by extracellular ATP. This shows that sensory neurons have the machinery to form purinergic synapses on each other and that they do so when placed in short-term tissue culture. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.
EXPERIMENTAL PROCEDURES Tissue culture Adult Sprague–Dawley rats (Charles River) were initially anesthetized with halothane (Halocarbon Ind.) and then beheaded according to institutionally approved protocol. All experiments conformed to NIH and international guidelines on the ethical use of animals. Minimum number of animals was used to achieve statistical significance and high dose of anesthetic was used to minimize animal suffering. Trigeminal or DRG were dissected from 200 to 300 g Sprague–Dawley rats, dissociated with enzymes (papain, dispase and collagenase) and plated on glass coverslips coated with poly-D-lysine/laminin as previously described (Eckert et al., 1997). Cells were grown for 1–3 days at 37 °C in 5% CO2 in F12 media plus 10% heat-inactivated fetal calf serum, 50 U/ml penicillin, 50 g/ml streptomycin and 50 ng/ml nerve growth factor. Explants were prepared from DRG dissected from newborn rats and grown with the same media as above. The DRG were stripped of their outer connective tissue and grown for 24 h with a volume of media just sufficient to cover them; this kept the ganglion from floating off the coverslip before it attached to the substrate. Dissociated DRG or trigeminal neurons were added to the culture 24 h after the explants were started and experiments were typically done 48 –72 h after plating when the field of axons growing from the explant had passed many dissociated neurons.
Key words: dorsal root ganglion, P2X receptors, sensory processing.
Scattered evidence challenges the dogma that sensory neurons do not communicate within sensory ganglia. Peptide-containing presynaptic terminals clearly form between sensory neurons after peripheral nerve damage (McLachlan and Hu, 1998), confirming the occasional observations of sensory–sensory synapses reported to occur rarely in ganglia or nerve in the absence of damage (Miller et al., 1970; Herrick, 1924; Kruger and Halata, 1996). Calcium-dependent exocytosis of sensory peptides— substance P and calcitonin gene-related peptide (CGRP)— occurs within sensory ganglion (Huang and Neher, 1996; Ulrich-Lai et al., 2001), as does co-release of ATP and substance P (Matsuka et al., 2001). Particularly intriguing physiological studies by Devor and colleagues (Amir and Devor, 1997; Amir and Devor, 1996; Liu et al., 1999) report that the membrane potential of individual dorsal root ganglion (DRG) neurons depolarizes when other neurons in the ganglion fire action potentials at a high rate. This behavior may contribute to pathological pain states (Devor et al., 2002).
Electrophysiology Whole-cell currents were measured using standard patch-clamp techniques with 2–5 M⍀ pipettes and an Axopatch 200 amplifier (Axon Inst.). Currents were filtered at 5 kHz and sampled at 0.1 ms intervals. Acquisition and analysis were done with PClamp 6 (Axon Inst) software and Origin (Microcal). Rapid agonist and drug applications were achieved using a linear array of flow pipes with solution changes accomplished by opening or closing latching valves (General Valve). External solution contained (mM): 150 NaCl, 2 CaCl2, 1 MgCl2, 3 KCl, 10 glucose, 10 HEPES. In all experiments except reversal potential measurements, internal (pipette) solution contained (mM): 140 KMeSO3, 10 KCl, 1 Na2ATP, 2 MgCl2, 0.3 Na3GTP, 5 EGTA, 10 HEPES. For reversal potential measurements, internal solution contained (in mM): 140 NaMeSO3, 10 NaCl, 1 Na2ATP, 2 MgCl2, 0.3 Na3GTP, 5 EGTA, 10 HEPES; this solution eliminated steady outward potassium
*Corresponding author. Tel: ⫹1-956-554-5069; fax: ⫹1-956-554-5065. E-mail address: [email protected] (M. M. Zarei). Abbreviations: CNQX, 6-cyano-7-nitroquinoxiline-2,3-dione; DRG, dorsal root ganglion; P2X receptor, an ion channel that opens in response to extracellular ATP; PBS, phosphate buffered saline.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.03.019
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M. M. Zarei et al. / Neuroscience 126 (2004) 195–201
Fig. 1. Consistent amplitude of spontaneous currents in cultured sensory neurons. Insets show spontaneous currents from two different cells, one held at ⫺60 mV (A) and the other at ⫾40 mV (C). Asterisks in (A) indicate currents shown at expanded scale in (B). Histograms of the amplitudes of all current events (A, C) have a major peak at a certain minimal amplitude with occasional larger events. The cell in C had many currents larger than the minimum, so we attempted to fit them with the indicated Gaussian distribution. The data seem insufficient to conclude that the larger amplitudes are multiples of the smallest. A plot of the average amplitude of the smallest currents at different voltages for the cell in C reveals strong inward rectification (D); that is, outward currents do not occur above the apparent reversal potential near 0 mV. Scale bar in A⫽100 pA, 50 ms; B⫽50 pA, 25 ms; C⫽100 pA, 20 ms.
currents. All solutions had pH 7.35–7.4; 315–325 mmol/kg, and all experiments were conducted at room temperature, 23 °C. To evoke synaptic currents in sensory neurons, we recorded, with whole cell patch clamp, from a dissociated sensory neuron that was in co-culture with an explanted ganglion. To simultaneously stimulate many neurons at once within the explant ganglion, a flat, concentric bipolar extracellular electrode (200 m outer pole, 25 m inner pole; Stoelting Co. 50691) was pushed into its center and 0.1 ms current pulses were applied once per second. Stimulating strength was adjusted upward while responses were measured in the individual sensory neuron.
Immunofluorescence of active presynaptic terminals To visualize active synaptic terminals, we used an antibody raised against the lumenal domain of a protein that spans synaptic vesicles, synaptotagmin I, in cells whose surface membrane was intact, as previously described (Perin et al., 1991; Matteoli et al.,
1992; Zarei et al., 1999). Neurons were incubated with 10 nM of the toxin, taipoxin, that was added in complete media and incubated at 37 °C, 5% CO2 for 40 min. Taipoxin retards the recycling of synaptic vesicles, and consequently, the lumenal domain of the synaptic vesicles containing synaptotagmin gets exposed on the surface of the neuron. The cells were washed with phosphate buffered saline (PBS; in mM: 10 Na2HPO4, 2.3 NaH2PO4, 138 NaCl, and 2.7 KCl, pH 7.4), fixed with a 4% paraformaldehyde solution for 20 min, and washed again with PBS (note that no detergent is used to permeabilize the cells). Fixed neurons were incubated overnight with rabbit anti-synaptotagmin I IgG (anti-Syn I), washed for 30 min, incubated with fluorescein-labeled goat anti-rabbit antibody (Vector Laboratory) for 20 min, washed for 20 min with PBS, mounted (Molecular Probe Prolong), and phase and fluorescent images were acquired using a confocal microscope. Anti-Syn I was developed against the NH2 terminus of synaptotagmin I, which is located in the lumenal domain of synaptic vesicles (Perin et al., 1991; Matteoli et al., 1992).
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Fig. 2. Spontaneous currents are purinergic. (A) Top five traces show representative spontaneous currents in control conditions and bottom five show representative currents from the same cell in the presence of 100 M suramin. Amplitude histograms show an obvious decrease in current amplitude in suramin (black bars). (B) Results from another cell showing amplitudes of spontaneous currents plotted sequentially. Bar shows application of suramin; arrows show when the three sets of records shown above occurred. (C) Summary from seven cells showing the average fractional change in amplitude and frequency of spontaneous currents compared with records before suramin application. Suramin decreases amplitude without a consistent effect on frequency. Nothing occurred with a cocktail of blockers of glutamate, nicotinic, and acid-sensing channels (CNQX, 10 M; d-tubocurarine, 100 M; and amiloride, 100 M; n⫽4). (D) Perfusion of 0.5 mM ATP eliminated the spontaneous currents (n⫽3). Histograms give the number of spontaneous current events in 1 s bins vs. experimental time for a representative cell. Upper bar shows the time of ATP application; lower bars show the times for the stretches of raw data shown above (before and during ATP) and at inset (after recovery). ATP application causes a standing current that desensitizes and eliminates spontaneous current events. Scale bar in A⫽50 pA, 25 ms; B⫽100 pA, 25 ms; D⫽200 pA, 1.2 s.
RESULTS Spontaneous currents in cultured sensory neurons Fig. 1A, B shows spontaneous currents recorded with a whole cell patch clamp from a sensory neuron. These neurons were dissociated from sensory ganglia and cultured with nerve growth factor for 2–5 days; of 55 neurons tested, 20 exhibited such spontaneous currents. Most currents on a given cell had particular amplitude (about 100 pA in Fig. 1A, 40 pA in Fig. 1C). Almost all other amplitudes seen are larger than the most common one (Fig. 1C). Because only sensory neurons are present in these cultures, this result suggests that sensory neurons can form synapses on each other.
Evidence for purinergic synapses Consistent with excitatory synaptic currents, these spontaneous currents approach zero amplitude when the holding voltage approaches 0 mV. But unlike most synaptic currents they fail to conduct outward current (Fig. 1C, D). Such inward rectification is a property of P2X receptors, ion channels that are activated by extracellular ATP (North, 2002). Furthermore, application of suramin, a blocker of P2X receptors, diminishes the amplitude (Fig. 2A) of the spontaneous currents without any consistent effect on frequency (Fig. 2B, C). This is the effect expected of a postsynaptic blocker of an ion channel; thus, P2X receptors are localized at post-synaptic junctions. A mixture of other ion
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Fig. 3. Spontaneous currents require external calcium. Spontaneous currents from a particular cell (A) occur infrequently when extracellular Ca2⫹ is 0.5 mM compared with 2 mM. Amplitude histogram of all current events (B) shows that the primary amplitude appears unchanged in 0.5 mM Ca2⫹ (dark bars) though the number of events is greatly diminished; inset shows fits to data only. Time course of the number of events per (3.6 s) bin (C) with the time of the change to 0.5 mM Ca2⫹ indicated. Summary from four cells (D) shows that dropping Ca2⫹ concentration from 2 mM to 0.5 mM does not alter the primary amplitude of spontaneous currents, but does decrease the frequency at which they occur. Scale bar in A⫽200 pA, 50 ms.
channel blockers (6-cyano-7-nitroquinoxiline-2,3-dione [CNQX], 10 M, for glutamate channels; d-tubocurarine, 100 M, for nicotinic acetylcholine receptors; and amiloride, 100 M, for acid-sensing channels) had no effect (Fig. 2C, blockers), leaving suramin as the only effective blocker we used. Finally, if ATP is indeed the neurotransmitter, exogenous application of ATP should eliminate the spontaneous currents by desensitizing P2X receptors. Indeed, high concentration of ATP significantly reduces spontaneous events in a reversible manner (Fig. 2D, solid bar). Since Ca2⫹ triggers release of neurotransmitter vesicles, we asked whether the spontaneous currents were sensitive to extracellular Ca2⫹ concentration. Dropping extracellular Ca2⫹ from 2 mM to 0.5 mM greatly decreased the frequency of spontaneous currents in the cell in Fig. 3A. There was no shift in the predominant amplitude of the
currents (amplitude histograms, Fig. 3B). Fig. 3C shows the frequency of spontaneous currents over time and Fig. 3D collects results from four cells on the effect of Ca2⫹ on average amplitude and frequency. Clearly, Ca2⫹ decreases the frequency of spontaneous currents without affecting their amplitude, opposite to the action of suramin (Fig. 2C). This indicates that Ca2⫹ controls presynaptic release whereas suramin blocks the postsynaptic receptor. To further test this, we attempted to evoke synaptic currents between sensory neurons. Evoked synaptic current between sensory neurons Can stimulation of one sensory neuron evoke synaptic currents in another? Connections between single pairs of neurons are rare even when sensory neurons are cultured with spinal neurons (Jahr and Jessell, 1985); thus, we
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Fig. 4. Evoked synaptic currents. Whole ganglia were cultured together with isolated sensory neurons so that the field of axons emanating from the ganglion contacted neurons that could be patch clamped. The photograph (A) shows a patch pipette recording from an individual cell (expanded in inset) that lies within the axons coming from the ganglion contacted by the large stimulating electrode, S. Recordings from the neurons (B) have a sharp shock artifact followed by an inward current that occurs a consistent time after the shock and usually has the same amplitude (nine successive records shown). Suramin fully blocked the inward current in four cells from three preparations (C, left), but had no effect in six other cells (C, middle and right); a cocktail of blockers of other fast ligand gated channels also failed to block this suramin-insensitive current (C, right; suramin, 500 M; CNQX, 10 M; d-tubocurarine, 100 M; amiloride, 100 M). These block P2X, AMPA, nicotinic and acid-sensing ion channels respectively. Scale bar in B⫽100 pA, 50 ms; C⫽100 pA, 10 ms.
enhanced the number of synapses by co-culturing whole DRG with dissociated sensory neurons. In this approach, whole ganglia were cultured for several days together with dissociated sensory neurons until the outgrowing field of axons passed over single isolated neurons (Fig. 4A). Then, electrical shocks were delivered through a flat extracellular electrode pressed against the ganglion while recording from a single cell in the ganglion’s field of axons. We were able to evoke synaptic currents between sensory neurons, and the currents had a regular amplitude over time (Fig. 4B). Suramin totally blocked these evoked currents in four of 10 cells tested (Fig. 4C, left). However, suramin did not affect some cells (Fig. 4C, middle). A mix of blockers (CNQX, 10 M; d-tubocurarine, 100 M; and amiloride, 100 M) also failed to block these suramin-insensitive currents (Fig. 4C, right), indicating that fast glutamate, nicotinic, or acid-sensing ion channels were not involved. Future studies are needed to determine the nature of suramin-insensitive evoked responses. Visualizing active sensory–sensory synapses To independently test for the presence of sensory–sensory synapses, we used taipoxin, which inhibits recycling of synaptic vesicles and thereby leaves their lumenal face exposed on the surface of cells after vesicles have fused
(Zarei et al., 1999). This exposed lumen can be labeled with anti-Syn I, an antibody that is targeted to the lumenal side of a vesicle protein, synaptotagmin. The antibody is applied to cells that have not been permeabilized so it labels only surface proteins and not intracellular vesicles. The procedure labels only those synaptic sites that were active during the taipoxin treatment period. Fig. 5 shows phase (left) and confocal fluorescent (right) images of two sensory neuron cell bodies and many passing axons. Many fluorescent puncta decorate the cells and axons. Note on the left cell that some puncta align, suggesting that they come from a single axon lying across the cell. We take these puncta to indicate the presence of active synaptic release sites. Because this culture contains only sensory neurons, this is independent evidence that sensory– sensory synapses are actively formed in culture.
DISCUSSION We report three observations that show conclusively that sensory neurons can form functional synapses on each other in short term tissue culture: 1) about a third of sensory neurons exhibit spontaneous Ca2⫹-dependent currents of consistent amplitude; 2) synaptic currents can be evoked between sensory ganglia and isolated sensory neurons; 3) synaptotagmin-labeled hotspots of synaptic
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Fig. 5. Immunocytochemical evidence of active synapses. After a 40 min exposure to taipoxin, which inhibits endocytosis of synaptic vesicles that have fused to the surface membrane, neurons were lightly fixed, without being permeabilized, and treated with an antibody to the lumenal face of synaptotagmin I (Syn I), a synaptic vesicle protein. Phase (left) and confocal (right) images of a field containing two sensory neurons and axons after labeling with anti-Syn I. Punctate fluorescence demonstrates areas where synaptic vesicles had fused to surface membranes during the taipoxin treatment. Many of these puncta align (see for example on the left cell), as expected if they were on axons.
activity decorate sensory axons and these appear to cross cell bodies. Many of these synapses are purinergic because spontaneous currents and 40% of evoked currents were sensitive to P2X channel blockade and were eliminated by extracellular ATP, which desensitizes P2X channels. A puzzling aspect of the study is that all spontaneous currents were purinergic whereas only 40% of evoked currents were purinergic. One possible explanation is the difference in culture conditions. Spontaneous currents were measured in dissociated tissue culture whereas evoked currents involved explant cultures. The explants would include intact satellite/glial cells whereas these are lost in short term, dissociated culture. The presence or absence of satellite cells greatly influenced the appearance of nicotinic synaptic currents between nodose sensory neurons in culture in another study (Cooper, 1984). Because we did not observe any nicotinic synapses, the relation between the Cooper study and ours is unclear. However, his study clearly demonstrates that changes in tissue culture conditions can influence synaptic formation between sensory neurons. Our present work demonstrates that sensory neurons are capable of communicating with each other through purinergic synapses in short term tissue culture. Furthermore, both in vivo and vitro studies have indicated that DRG neurons can also communicate through crossexcitation (Amir and Devor, 1996; Devor and Wall, 1990; Utzschneider et al., 1992). In cross-excitation, spike activity in one neuron excites spiking in adjacent neurons of the same DRG (Devor and Wall, 1990). This crossdepolarization appears to be mediated by neurotransmitter(s) that are released non-synaptically during neuronal activities (Amir and Devor, 1996; Shinder and Devor,
1994). Cross-excitation may not play a major role in normal communications between sensory neurons since very few synaptic contacts are observed in the healthy DRG neurons (Pannese, 1981). However, during peripheral nerve injury dramatic changes occur within the DRG that may alter the normal sensory communications. Trauma to a peripheral nerve is followed by structural changes in the DRG and spinal cord. These changes include sprouting of sympathetic axons that forms rings of noradrenergic terminals around the DRG neurons (McLachlan et al., 1993; Ramer and Bisby, 1997) and sprouting of small sensory axons that contain calcitonin gene-relegated peptide and substance P (McLachlan and Hu, 1998). In addition, peripheral nerve injury has also been reported to induce sprouting of A-fibers into laminae I–II of the spinal cord (Woolf et al., 1992, 1995). It has been postulated that these structural changes can alter sensory processing and may lead to neuropathic pain (McLachlan and Hu, 1998). It is quite possible that the purinergic synapses we find so common when sensory neurons are in culture several days are a response to the severing of axons that occurs when ganglia are dissociated. Such tissue damage causes release of nerve growth factor, which induces sprouting of sensory neurons (McLachlan and Hu, 1998; Diamond et al., 1992) and might contribute to the formation of synaptic structures between sensory neurons that follows axon damage (McLachlan and Hu, 1998). Experiments with in vivo preparations will be necessary to find whether the purinergic synapses form between sensory neurons in vivo, whether this is more common after nerve injury, and whether this has physiological or pathological significance. Acknowledgements—The NIH supported this work through a grant to Edwin W. McCleskey.
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(Accepted 11 March 2004)