Intracellular recording in avian brain of a nicotinic response that is insensitive to K-bungarotoxin

Neuron, Vol. 5, 307-315,September,1990,Copyright © 1990by Cell Press

Intracellular Recording in Avian Brain of a Nicotinic Response That Is Insensitive to K-Bungarotoxin Eva M. Sorenson and Vincent A. Chiappinelli Department of Pharmacology St. Louis University School of Medicine St. Louis, Missouri 63104

Summary We examined nicotinic acetylcholine receptors in the avian brain using a combination of autoradiographic and intracellular electrophysiological techniques. We found that the lateral spiriform nucleus (SPL) in the mesencephalon has a very high density of 3H-nicotine binding sites but no detectable ~2~l-K-bungarotoxin (1251-K-BuTx) or 12Sl-(x-bungarotoxin (12Sl-(x-BuTx) binding sites. Intracellular recordings in brain slices revealed that SPL neurons depolarize in response to nicotine and carbachol (in the presence of atropine). These depolarizations were blocked by the classic nicotinic antagonists d-tubocurarine and dihydro-[3-erythroidine. As predicted for nicotinic receptors with a high affinity for nicotine, neither K-BuTx nor ~-BuTx blocked these nicotinic responses. Thus, although the existence of high-affinity 3H-nicotine binding sites has been known for some time, we now report the in situ detection of a functional nicotinic receptor that has a high affinity for nicotine and is K-BuTx-insensitive. Introduction The nicotinic acetytcholine receptors found in electric organ and skeletal muscle are currently the best characterized of all ligand-gated ion channels, as a result of the early availability of both large numbers of receptors and the snake venom toxin (~-bungarotoxin (~-BuTx), a potent antagonist of these receptors. The characterization of neuronal nicotinic receptors has until recently proceeded at a much slower pace in part because even though ~-BuTx demonstrates saturable, high-affinity binding in autonomic ganglia and brain, electrophysiological studies indicate that ~-BuTx does not block nicotinic neurotransmission in these preparations (Brown and Fumagalli, 1977; Carbonetto et al., 1978). A minor component from the venom of Bungarus multicinctus, K-BuTx (also referred to as toxin F, bungarotoxin 3.1, and neuronal bungarotoxin), does competitively block nicotinic neuro~ transmission in autonomic ganglia (Ravdin and Berg, 1979; Chiappinelli, 1983; Loring et al., 1984). Binding and localization studies in autonomic ganglia demonstrate that K-BuTx recognizes a unique nicotinic site distinct from the ~-BuTx binding site (Chiappinelli, 1983; Halvorsen and Berg, 1986; Loring and Zigmond, 1987). This unique K-BuTx site is associated with the functional nicotinic receptor found in autonomic ganglia and is recognized by all four known snake venom K-neurotoxins (Chiappinelli et al., 1987, 1990).

Multiple classes of neuronal nicotinic receptors in the CNS have been predicted from binding studies. Both high- and low-affinity nicotine sites are found in the chicken brain (Schneider et al., 1985). The high-affinity sites, which are not found in autonomic ganglia, can be divided into two subclasses by immunoaffinity chromatography u sing two different monoclonal antibodies (Whiting et al., 1987). One antibody, MAb 270, recognizes both high-affinity nicotine sites in the chicken brain. The second antibody, MAb 35, recognizes only one of these sites. Neither of the highaffinity sites is recognized by ~-BuTx or K-BuTx (Whiting and Lindstrom, 1986; Wolf et al., 1988). (l-BuTx does bind (Kd = 1 nM) to low-affinity nicotine sites in the brain. K-BuTx subdivides the ~-BuTx sites into two classes, one with a high affinity for K-BuTx (Kd = 1 nM) and another with a low affinity for K-BuTx (Wolf et al., 1988). The significance of ~-BuTx sites in the CNS is unknown, since ~-BuTx generally fails to block nicotinic responses in brain (reviewed in Chiappinelli, 1985). Cloning experiments have also predicted multiple neuronal nicotinic receptors in the CNS. In both rat and chicken brain, at least three putative nicotinic subunits and several 13subunits (also termed non-~ subunits in the chick) have been identified (Boulter et al., 1987; Nef et al., 1988; Schoepfer et al., 1988; Wada et al., 1988; Deneris et al., 1989; Duvoisin et al., 1989). When mRNA or cDNA coding for these subunits was injected into frog oocytes, the ~2, o3, or (~4 subunits ((~1)2131y161 = muscle receptor) formed functional ligand-gated ion channels when combined with either [32 or [34 subunits (Boulter et al., 1987; Ballivet et al., 1988; Wada et al., 1988; Papke et al., 1989; Duvoisin et al., 1989). The only toxin known to differentiate between the neuronal nicotinic receptor subtypes expressed in frog oocytes is K-BuTx. Using rat clones, K-BuTx was shown to block the (~3132 and ~4132 responses at 10 nM and 500 riM, respectively, while having no effect on ~2132and ~3134receptors (Wada et al., 1988; Duvoisin et al., 1989; Luetje et al., 1990). With chicken clones, K-BuTx had no effect on a4132 receptors (Bertrand et al., 1990). Even though these putative nicotinic receptor subunits are expressed in brain, it is not known whether the functional channels expressed in ovo are identical to those expressed in situ. The only previous in situ study of nicotinic responses in the chicken brain was in the retina, where a K-BuTx-sensitive receptor was identified (Loring et al., 1989). We have begun functional characterization of nicotinic responses in chicken brain so that receptors expressed in situ can be compared with nicotinic receptors of known composition expressed in ovo. Prior to our electrophysiological studies, we have used choline acetyltransferase immunohistochemistry and nicotinic receptor ligand autoradiography to locate areas with prominent cholinergic innervation and var-

Neuron 3O8

ious classes of neuronal nicotinic binding sites, respectively. We reasoned that nuclei containing both cholinergic fibers and nicotinic binding sites were likely to contain neurons expressing functional nicotinic receptors. We now report that neurons in the mesencephalic lateral spiriform nucleus (SPL) express functional nicotinic receptors with a high affinity for nicotine. These receptors are unaffected by K-BuTx or ~-BuTx, but are blocked by d-tubocurarine and dihyd ro-~-eryth roidine. Results

Receptor autoradiography in adjacent sections of the chicken brain revealed that a nucleus in the midbrain, the SPL, was unique in demonstrating a very high density of 3H-nicotine sites but no detectable 1251-K-BuTx or 12Sl-a-BuTx binding sites (Figures 1A, 1B, and 1C). Other areas of the brain, including the ventral lateral geniculate and layer 7 of the optic tectum (Figures 1A, 1B, and lC), also showed high densities of 3H-nicotine sites, but both iodinated snake toxins bound to these structures as well. Choline acetyltransferase (CHAT) immunohistochemistry demonstrated a cholinergic fiber tract immediately lateral to the SPL (Figure 1D; Sorenson et al., 1989). ChAT-positive fibers leave this tract and innervate neurons within the SPL. As no cholinergic cell bodies were found in the SPL, the ChAT-positive fibers localized in the nucleus were apparently all cholinergic afferents. The colocalization of cholinergic fibers and 3H-nicotine sites in the SPL suggested that SPL neurons expressed functional nicotinic receptors that had a high affinity for nicotine. As the ventral lateral geniculate and layer 7 of the optic tectum contained high densities of cholinergic fibers and both high-affinity nicotine and toxin binding sites, it is likely that there are functional nicotinic receptors in these areas as well. This possibility will be examined in future electrophysiological studies. To test whether SPL neurons expressed functional high-affinity nicotine receptors, intracellular records were obtained from 103 SPL neurons in chick brain slices. The membrane characteristics of SPL neurons are given in Table 1. The input resistance of SPL neurons suggests that they are of medium to large size. SPL perikarya have been reported to measure between 20 and 40 I~m in diameter (Reiner et al., 1982; Morris et al., 1990), which would be in keeping with the values we obtained for cell input resistances. Sixty percent of the neurons exhibited spontaneous action potentials, and most cell recordings displayed spontaneous synaptic potentials. Figure 2A is a record from a spontaneously active SPL neuron. Several subthreshold spontaneous synaptic potentials can be seen, and at one point in the record these summate, resulting in an action potential. The action potentials of SPL neurons had an amplitude of 67 + 2 mV (mean + SEM, n = 22) from resting membrane potential, with a duration of 1.1:1:0.1 ms (mean + SEM, n = 22) at halfamplitude. The different rise rates of subthreshold

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K-BuTx-lnsensitiveNicotinic Responsein Brain 309

Table 1. 'Membrane Properties of Neurons in the SPL Membrane Potential (mV)

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-62 + 3 (n = 10)

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27 + 3 (n = 22)

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Values are mean + SEM.

potentials observed in individual cells suggest that some SPL neurons are multiply innervated (Figure 2C). Some of the neurons exhibit an inward rectification similar to a Q-current when injecting increasing steps of hyperpolarizing pulse current into the neuron (Figure 2D). All SPL neurons displayed excitatory responses when nicotine or carbachol (in the presence of 1 tiM atropine) was applied to the brain slice during intracellular recording (Figure 3). A response to a pressure ejection of carbachol is shown in Figure 3A. The typical response was a depolarization accompanied by evoked action potentials and an increase in the baseline noise level. The mean depolarization was 12 + I mV (n = 20) from a resting membrane potential of -70 mV in response to a pressu re ejection of 5 ~I of 10 mM carbachol into the bath 6 mm upstream from the brain slice. The apparent decrease in input resistance evident during depolarization was less pronounced when the cells were clamped back to the initial membrane potential (Figure 4A). Nicotine often produced more robust responses than carbachoi. A more robust response included any or all of the following characteristics: a longer lasting response, a larger depolarization, or an increase in the number of action potentials elicited. Nicotine also readily desensitized the neurons, producing reduced responses to either carbachol or nicotine for up to I hr after a single pressure ejection (Figure 3A), whereas desensitization occurred only at higher doses of carbachol. In addition, higher concentrations of antagonists were required to block the actions of nicotine than to block carbachol re-

Figure1. AutoradiographicComparisonof 3H-Nicotine,1251-K-BuTx, and 12Sl-~-BuTxBinding in the SPLof the Chicken Mesencephalon (A, B, and C) Autoradiographswere made of adjacenttransverse sections exposed to either 3H-nicotine (A), 12Sl-E-BuTx(B), or 12Sl-~-BuTx(C). In the midbrain, the SPL contained a very high density of 3H-nicotine binding sites(A), but no detectableiodinated toxin binding sites (B and C). All three ligands were colocalized in the ventral lateralgeniculate(GLv)and in layer7 of the optic tectum (TeO). (D) ChAT immunohistochemistry,in a transversesection from a different chicken brain, showsa cholinergic fiber tract immediately lateralto the SPL Cholinergic fibers are seen leavingthe tract and projecting into the SPL.No cholinergic cell bodies are found within the SPL.The ventral lateralgeniculateand layer7 of the optic tectum also contain high densities of cholinergic fibers. Bars, I mm.

sponses (Figure 4B). These observations provided evidence that nicotine was a more potent agonist at these receptors than was carbachol. SPL neurons responded to perfused concentrations of nicotine as low as 10 nM (Figure 3B). The initial depolarization, increased spiking activity, and increased noise level in response to nicotine perfusion were followed after several minutes by a return to resting potential, although in the example shown the input resistance of the cell remained decreased by 45% for another 4.5 min (Figure 3B, middle sweep). The return to resting membrane potential during continuous nicotine perfusion was probably due to desensitization of a portion of the nicotinic receptors. After 15 min of 10 nM nicotine perfusion, both membrar~e potential and input resistance had generally returned to control values, but the response to carbachol was attenuated (Figure 3B, bottom sweep). This was consistent with a partial desensitization. Perfusion of nicotine at higher concentrations produced larger depolarizations with higher levels of spiking and increased baseline noise. Perfusion of neurons Wffh 1 llM nicotine was followed by a complete desensitization of the response to carbachol. Nicotine and carbachol acted directly on postsynaptic receptors, since responses to these agonists were unchanged in the presence of 100 llM CdCI2, a concentration that blocked spontaneous activity (Figure 3C). There was no statistical difference in the size of the depolarization in response to carbachol before and during a perfusion with 100 llM CdCl2, as determined by Student's paired t-test (n = 3). Classic nicotinic antagonists blocked the effects of both agonists in a dose-dependent manner. In cell 1 of Figure 4A, the response to carbachol is slightly attenuated by perfusion with 1 p.M d-tubocurarine. In cell 2, the response to carbachol is largely blocked in 10 llM d-tubocurarine. Eighty-two percent (n = 6) of the depolarization in response to carbachol was blocked in the presence of 10 llM d-tubocurarine. All responses to nicotine and carbachol were completely blocked by perfusion with 30 ~M d-tubocurarine. Dihydro-[3-erythroidine (DHBE) also blocked nicotinic responses, but at somewhat higher concentrations. In Figure 4B, the response to 1 Ill of 10 mM carbachol is blocked by 30 llM DHBE. The response of this neuron to the same dose of nicotine was only partially attenuated by 30 llM DHBE, and 100 p.M DHBE was required to block the nicotine response completely. Recovery of agonist responses required 15-45 rain of washing out the antagonists (Figure 4). Perfusion with a nicotinic antagonist frequently reduced the spontaneous activity of SPL neurons (e.g., Figure 2A and Figure 4A, cell 2). However, concentrations of antagonists that completely blocked responses to exogenous nicotinic agonists did not always block all spontaneous activity (e.g., Figure 4B). Since radiolabeled ~-BuTx and K-BuTx did not bind to the SPL, we were interested in knowing whether these snake toxins would have any effects on nicotinic

Neuron 310

Figure2. Intracellular Recordings from SPL Neurons (A) An intracellular record (upper sweep) from a spontaneously active SPL neuron displays several subthreshold synaptic po10 IJM tubocurarirle tentials (arrowheads). At one point these potentials summate, generating an action potential that is truncated in this record. This cell had a resting membrane potential B C D of -70 inV. The bar indicates the voltage response to a 0.1 nA hyperpolarizing current pulse. In the presence of 10 p.M d-tubocurarine (lower sweep) the amplitudes of JI ..... spontaneous responses in the same cell are reduced. (B) Spontaneous action potential from the neuron in (A) at a higher magnification. The action potentials in this neuron had an average amplitude of 62 mV, with a duration of 1.2 ms at half-amplitude. (C) Two subthreshold synaptic potentials from the neuron in (A) have different rise times. (D) Some SPL neurons displayed inward rectification similar to a Q-cu rrent, as seen in the relaxations of the voltage traces (upper sweeps) in response to 0.2 nA and 0.3 nA steps of hyperpolarizing current pulses (lower sweeps).

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responses r e c o r d e d f r o m t h e s e n e u r o n s . The response t o c a r b a c h o l o f an SPL n e u r o n after p r e i n c u b a t i o n of t h e slice for 2 h r w i t h 0.5 ~ M K-BuTx is s h o w n in Figure 5A. T h e d e p o l a r i z a t i o n ( f r o m - 7 0 mV) in r e s p o n s e to c a r b a c h o l (5 ~1 of 10 m M ) after K-BuTx p r e t r e a t m e n t was 8.4 + 1.7 mV ( m e a n + SEM, n = 10 f r o m 3 chicks). This was n o t statistically d i f f e r e n t f r o m t h e m e a n dep o l a r i z a t i o n o f SPL n e u r o n s o b s e r v e d w i t h o u t K-BuTx

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p r e t r e a t m e n t (11.6 + 1.1 mV, m e a n + SEM, n = 20 f r o m 13 chicks), as d e t e r m i n e d b y Student's u n p a i r e d t-test (T = 1.61; P > 0.05). F o l l o w i n g e x p o s u r e to K-BuTx, t h e n i c o t i n i c r e s p o n s e s w e r e still s e n s i t i v e t o b l o c k a d e by d - t u b o c u r a r i n e (Figure 5A). We also saw n o effects o f c~-BuTx p e r f u s i o n (1 I~M for 2 hr) o n n i c o t i n i c r e s p o n s e s r e c o r d e d in SPL n e u r o n s (n = 8 f r o m 3 chicks; Figure 5B).

Figure 3. Direct Actions of Nicotinic Agonists on SPL Neurons (A) The traces show an SPL neuron depolarizing in response to pressu re ejection (at arrow) of 5 I~1of 10 mM carbachol (carb) carb lllll ~ lOOpM CdO, or nicotine (nic) into the recording chamber approximately 6 mm upstream from . ~ l i I' , . • . ...... the recording microelectrode. The response to nicotine produced more action potentials than the response to carbachol. The carb Desensitization tO~ + agonists were applied to this neuron at 15 rain intervals. The response to the second 30 -7Ot-I~i~llipl"i II tlllllllllll+lllllllllllll~llllll]llllllll application of carbachol was largely blocked as a result of the long-term desensitization caused by the previous application of nicotine. Nicotine desensitized SPL neurons much more readily than carbachol. The scale is the same as in (C). (B) Fifteen minutes after a control response +,, j L .... to a pressure ejection of carbachol (3 I11 of 10 raM; top record), this SPL neuron was perfused with 10 nM nicotine (bar}. The response to 10 nM nicotine was seen as an increase in baseline noise, a gradual decarb IHl[llllllll'~ ++l polarization, and evoked action potentials. The response gradually "faded" during the continuous perfusion, the membrane potential returning to baseline about 3 rain after the beginning of the response (middie record). The response to a second application of carbachol, pressure ejected into the recording chamber 15 min after the beginning of nicotine perfusion, was partially reduced as a result of the nicotine perfusion. Sections of the record between responses are not shown. (C) After a control response to carbachol (3 I~1 of 10 raM; top record), an SPL neuron was perfused with 100 I~M CdCl2 for 20 rain to block the release of neurotransmitters and then exposed to carbachol again (bottom record). Both depolarizations, before and during CdCl2 perfusion, measure 8 inV. Thus, carbachol acted directly on postsynaptic nicotinic receptors. A

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K-BuTx-lnsensitive Nicotinic Response in Brain 311

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Figure 5. Nicotinic Receptors on SPL Neurons Are Insensitive to K-BuTx and c~-BuTx (A) Brain slices were preincubated in 0.5 llM K-BuTx (KBgT) for 2 hr and then placed in the recording chamber, where they were perfused with ACSF containing 1 llM atropine. Only responses obtained within 1 hr of placing the slice in the recording chamber were considered. This neuron had been in the recording chamber for 14 rain before initial application of agonist. The nicotinic response was not blocked by K-BuTx, as seen by the 20 mV depolarization in response to carbachol (5 p,I of 10 raM; top sweep). The response to carbachol was blocked by 30 pM d4ubocurarine (middle sweep) and recovered after 38 m i n of washing out d-tubocurarine. (B) c¢-BuTx (ABgT; 1.0 p,M) was perfused on the slice containing this neuron for 2 hr before examining the effects of nicotinic agonists. The responses to carbachol (2 Ill of 10 mM) and nicotine (1 p,I of 10 raM) were unaffected by the presence of ~-BuTx.

Neuron 312

Discussion

The noncholinergic neurons of the mesencephalic SPL are heavily innervated by ChAT-positive fibers, which are likely to originate in the cholinergic nucleus semilunaris (Reiner et al., 1982; Sorenson et al., 1989; present study). Our autoradiographic studies revealed that the SPL contains the highest density of 3H-nicotine sites observed in the chicken brain, but lacks detectable high-affinity 12SI-K-BuTxor 12Sl-~-BuTx sites. All of our electrophysiological data are consistent with the hypothesis that the 3H-nicotine sites in the SPL are localized on functional postsynaptic nicotinic receptors. Our intracellular recordings in brain slices demonstrate that functional nicotinic receptors are expressed on SPL neurons, since these cells were directly excited by nicotinic agonists. The receptors appear to have a high affinity for nicotine, as SPL neurons respond to concentrations of nicotine as low as 10 nM. As with other subtypes of nicotinic receptors, the SPL receptors are readily desensitized by nicotine, d-Tubocurarine and DHBE, antagonists of nicotinic responses in skeletal muscle and ganglia, block nicotinic responses in the SPL in a dose-dependent manner. The effective concentrations of ligands on the nicotinic responses of SPL neurons are similar to the concentrations required to inhibit the binding of 3H-nicotine to immunoisolated nicotinic sites from chicken brain (Whiting and Lindstrom, 1986). The ICs0 value of 13 nM for nicotine is very close to the lowest concentration (10 nM) producing a response in SPL neurons. Carbachol had a lower affinity than nicotine for the immunoisolated sites, as seen from an ICs0 value of 370 nM. Likewise, carbachol appeared to be a less potent nicotinic agonist in SPL neurons, since higher doses of carbachol were required to produce desensitization of the receptors and lower concentrations of antagonists were required to block responses to carbachol. Carbachol also had a lower affinity than nicotine for the putative chicken brain nicotinic receptors composed of ~4 and 132 subunits when these were expressed in frog oocytes (Bertrand et al., 1990). d-Tubocurarine completely blocked nicotinic responses in SPL neurons at 30 ~M, whereas the ICs0 value for d-tubocurarine reported from binding studies was 28 I~M (Whiting and Lindstrom, 1986). In frog oocytes, 2.5 ~M d-tubocurarine blocked 50% of the peak current induced by 1 gM acetylcholine in ~4[~2 receptors from chicken (Bertrand et al., 1990). d-Tubocurarine has also been reported to block central nicotinic receptors in the rat ventral tegmental area, in acutely dissociated rat medial habenula neurons, and in rat retinal ganglion cells in culture (Calabresi et al., 1989; Mulle and Changeux, 1990; Lipton et al., 1987). DHBE blocked nicotinic responses in rat locus ceruleus and medial habenula neurons (Egan and North, 1986; Mulle and Changeux, 1990). These two antagonists have recognized all excitatory neuronal nicotinic re-

ceptors on which they have been tested at similar concentration ranges. It would be valuable to have selective pharmacological agents for all subtypes of neuronal nicotinic receptors, especially since more than one subtype may be expressed in some neurons. K-BuTx has been shown to differentiate between muscle and ganglionic nicotinic receptors, and between putative neuronal nicotinic receptor subtypes when these were expressed in frog oocytes (Chiappinelli, 1983; Luetje et al., 1990). K-BuTx blocked the rat ~3~2 and ~4132 responses in frog oocytes at 10 nM and 500 nM, respectively (Luetje et al., 1990). In contrast, the rat ~2132and chicken ~4[~2 receptors were not blocked by 500 nM K-BuTx (Wada et al., 1988; Bertrand et al., 1990). Unlike the ganglionic receptor, which appears to be an ~3-containing receptor (Boyd et al., 1988), the nicotinic receptor in the SPL is not sensitive to K-BuTx at concentrations of up to 500 nM. Thus, on the basis of the oocyte studies described above, the receptors expressed in the SPL would be predicted to be composed of receptors containing ~2 or ~4 subunits. A recent in situ hybridization study has demonstrated that SPL neurons express the a2, ~4, and [32 nicotinic receptor subunits, but not the ~3 subunit found in the ganglionic receptor (Morris et al., 1990). Any combination of the subunits found in the SPL would be expected to be K-BuTx-insensitive in the chicken. At least 90% of the SPL neurons expressed ~2 and [32 subunits, but only 30% expressed the ~4 subunit (Morris et al., 1990). It can be reasoned then that most SPL neurons express nicotinic receptors that are composed of the ~2 and [32 subunits. Thirty percent of the neurons may express another subtype of the nicotinic receptor, for example, ~4132or g2~4~2. It is also possible that two or more receptor subtypes are expressed within the neurons of this SPL subpopulation. Since 70% of SPL neurons contain message for only ct2 and [32, we believe that most of our intracellular responses are typical of an a2132 receptor expressed in situ. We did not detect any major differences in our nicotinic responses between cells and can therefore only speculate on the significance of the expression of both the ~2 and the ~4 message in some SPL neurons. The ~2132and ~4~2 receptor subtypes, if they are both functional, may have similar properties. Studies at the level of single channels may be necessary to detect differences between these receptor subtypes. Specific probes for the putative nicotinic receptors expressed in oocytes may need to be developed in order to elucidate the role of various neuronal nicotinic receptor subtypes found in situ. An alternative theory is that one of the ~ subunit messages detected in the SPL codes for a presynaptic receptor found primarily on the axon terminals of SPL neurons. There is immunohistochemical evidence for such a presynaptic nicotinic receptor on SPL terminals, since MAb 35 recognizes not only SPL cell bodies, but

K-BuTx-lnsensitiveNicotinic Responsein Brain 313

also detects their terminals in the optic rectum (Swanson et al., 1983; Reiner et al., 1982). Lesioning of the SPL reduces MAb 35 binding in the optic tectum (Swanson et al., 1983). Such presynaptic receptors w o u l d not be autoreceptors, since SPL neurons are not cholinergic (Sorenson et al., 1989). However, there are cholinergic fibers in the appropriate layers of the optic tectum that may form axon-axon connections with SPL terminals (Sorenson et al., 1989). The o n l y other report of a K-BuTx-insensitive receptor expressed in situ has been in acutely dissociated neurons from the rat medial habenula (Mul[e and Changeux, 1990). Nicotinic responses persisted after exposure of these dissociated cells to 500 n M K-BuTx for 10 min. Although this is a short exposure time for a blockade of receptors by the toxin, the study was limited by the loss of nicotinic responses in control experiments after this period of time. Since medial habenula neurons do not express the ~2 subunit (Duvoisin et al., 1989; Wada et al., 1989), the K-BuTxinsensitive receptors in these neurons are likely to be of a subunit composition different from those we have described in the SPL. K-BuTx blocks nicotinic responses in the rat ventral tegmental area (Calabresi et al., 1989). The lot of K-BuTx and the m e t h o d o l o g y employed by Calabresi et al. (1989)were identical to those used in the present study of SPL neurons, thus confirming that these procedures can detect sensitivity of nicotinic receptors in brain slices to K-BuTx. A l t h o u g h our protocol might fail to detect a rapidly reversing effect of the toxin on the responses of SPL neurons, the blockade of neuronal nicotinic responses by K-BuTx typically lasts for several hours in K-BuTx-sensitive neurons of avian and rat CNS and autonomic ganglia (Chiappinelli, 1983; Lipton et al., 1987; Calabresi et al., 1989). In situ hybridization has demonstrated (~3, 0.4, and 132 subunit expression in the ventral tegmentum (Wada et al., 1989). Receptors consisting of these subunits w o u l d be expected to be blocked by K-BuTx (Luetje et al., 1990). Retinal ganglion cells from the chicken and rat also exhibit a nicotinic response that is sensitive to K-BuTx (Lipton et al., 1987; Loring et al., 1989). Retinal ganglion cells in the rat have been shown to express the 0.3, ~4, and l~2 subunits (Wada et al., 1989), and thus nicotinic responses in these cells w o u l d be predicted to be sensitive to K-BuTx. Clearly, K-BuTx can distinguish between some of the nicotinic receptor subtypes expressed in the CNS of birds and mammals. In conclusion, the K-BuTx-insensitive nicotinic receptors we have identified on SPL neurons are likely to be composed of subunits different from those constituting the K-BuTx-insensitive nicotinic receptor described in the medial habenula or the K-BuTx-sensitive nicotinic receptors found in the ventral tegmentum, since ~2 subunits are expressed in the SPL but not in the medial habenula or the ventral tegmentum. Whether more than one class of functional nicotinic channels is expressed in the SPL will not be known until

single-channel studies are performed on dissociated SPL neurons, or until more specific nicotinic antagonists are developed. These neurons w o u l d appear to be a valuable source of K-BuTx-insensitive nicotinic receptors for future studies, since the majority of SPL neurons may express only one class of neuronal nicotinic receptor.

Experimental Procedures Nicotinic Receptor Autoradiography Four adult female White Leghorn chickens were decapitated after pentobarbital anesthesia.Their brains were removed, frozen in O.C.T.compound (Tissue-Tek),and cut into 20 pm thick transverse sections that were thaw-mounted onto subbed microscope slides. The sections were stored at -20°C to -70°C until use. Adjacent or nearly adjacent sections from the same brain were incubated with 3H-nicotine, 12Sl-K-BuTx,or 1251-(~-BuTx.Sections incubated with iodinated toxins were preincubated in 50 mM Tris-HCl buffer containing I mglrnl bovine serum albumin (Sigma) for 30 min. They were then incubated in'buffer containing 0.5 nM 1251-K-BuTx(202-649 Ci/mmol) or 1251-a-BuTx(429-842 Ci/mmol) for 1 hr. K-BuTxand ct-BuTxwere purified and iodinated as previously reported (Chiappinelli, 1983;Wolf et al., 1988).After three 10 rain washes in Tris buffer, the slides were quickly rinsed in distilled water. To assess nonspecific binding, native ~-BuTx (1 t~M) was added to the preincubation and incubation buffers used for half of the sections. L-Nicotine bitartrate (1 raM, Sigma) and K-BuTx (1 pM) were also used as cold ligands. For 3H-nicotine binding, the sections were preincubated for 5 rain in 50 mM Tris-HCI buffer containing 8 mM CaCl2. They were then incubated for 30 rnin in buffer containing 2 nM 3H-L-nicotine (N-methyl-3H, 70-80 Ci/mmol; New England Nuclear). Nonspecific binding was assessedby adding 10 I~M L-nicotine bitartrate to the incubation buffer. Finally, the slides were washed three times for 5 s and quickly rinsed in distilled water. For sections bound with nicotine, all incubations and washes were done at 0-4°C. After washing, the slides were dried under a stream of cool, desiccated air and desiccated overnight. Sections, along with radioactive standards (American Radiolabeled Chemicals, Inc.), were placed tightly against tritium-sensitive film (Amersham) in X-ray cassettes for 7-14 days or 12 weeks for 12st-toxins and 3H-nicotine, respectively. Film was developed in Kodak developer D-19for 2 min and then fixed in Kodak RapidFix for 4 min. Enlarged photographs of the autoradiograrns were compared with sections stained with cresyl violet. The atlas of Karten and Hodos (1967) was used to identify areas of the avian brain. ChAT Immunohistochemistry The methodology used to label ChAT-containingfibers and cell bodies in slices of chicken brain has been previously described by Sorenson et al. (1989). Electrophysiology One day hatched White Leghorn chicks (n = 76) were decapitated, and their brains were removed and glued onto a mounting block. The mounted brains were then placed in cold ACSF(126 mM NaCI, 2.5 mM KCI, 2.5 mM CaCI2, 1.3 mM MgCI2, 1.2 mM Na2HPO4,25 mM NaHCO3,11 mM glucose)continuously bubbled with 95% 02 and 5% CO2 to bring it to pH 7.4.Transversebrain sections, 400 p.m thick, were cut on a vibratome, and sections containing the SPLwere placed in a Nicoll-type recording chamber or in a holding chamber at room temperature until needed (Nicoll and Alger, 1981). In the recording chamber, the sections were submerged betweentwo nets in 1 ml of ACSFand were continuously perfused at a rate of 2 ml/min at 29°C-30°C. lntracellular recordings were made with microelectrodes pulled from filamented capillary glass(1.0mm OD, 0.5 mm ID; Frederick Haer Co.) on a Brown-Flaming P-80microelectrode puller. They were filled with 3 M KCI and had resistancesof 60-120 M~, Signals

Neuron 314

were amplified with an Axoclamp-2A amplifier (Axon Instruments, Inc.) and continuously displayed on an oscilloscope. Data were recorded on a Gould 2200S chart recorder and a Vetter video cassette instrumentation recorder. Records were analyzed on a Nicolet 3091 digital oscilloscope. Membrane resistance was monitored by passing brief hyperpolarizing current pulses through the microelectrode. The bridge balance was monitored and adjusted during the delivery of current pulses. A DC current was passed to bring the cells to a common membrane potential, usually -65 or -70 mV, before application of drugs so that the resulting voltage changes could be compared between neurons. Some neurons were also hyperpolarized with DC current if they exhibited a large number of spontaneous action potentials so that the responses to drugs could be more easily monitored. Drugs were applied by two methods, perfusion at known concentrations and manual ejection of drug in a small volume of solution from a pressure pipette into the recording chamber 6 mm "upstream" from the recording microelectrode. Atropine (1 I~M) was always included in the perfusion ACSF. DHBE (Merck Sharp & Dohme Research Laboratories), d-tubocurarine (Sigma), and CdCl2 (Sigma) were added to separate flasks of ACSF in known concentrations, and perfusion solutions were Switched by manually controlled valves. Antagonists and CdCl2 were perfused for at least 15 rain before testing the effects of agonists on SPL neurons. The nicotinic agonists, L-nicotine bitartrate (Sigma) and carbachol (Sigma) were usually applied in bolus doses (1-5 l~l of 10 mM) by pressure ejection to help prevent desensitization. Carbachol was found to be much less likely to cause desensitization and so was used as the nicotinic agonist in most experiments. Known concentrations of nicotine and carbachol were also perfused over the brain slice so that the effects of known concentrations of agonists could be observed, c~-BuTx (1 I~M) was perfused over the brain slices for at least 1 hr before testing nicotinic responses in SPL neurons. Due to the expense and limited availability of K-BuTx, the brain slices could not be continuously perfused with the toxin. Instead, slices were preincubated in 0.5 I~M K-BuTx for at least 2 hr before being placed in the recording chamber, where they were perfused with ACSF containing 1 p,M atropine. This protocol is identical to that used by Calabresi et al. (1989) to examine the K-BuTx sensitivity of rat ventral tegmental neurons in brain slices. Neurons were impaled and recorded from for only 1 hr after placing a K-BuTxpreincubated slice in the recording chamber. Since K-BuTx exists in solution entirely as a dimer (Chiappinelli and Lee, 1985), the formula weight of the dimer (14,600daltons) was used in calculating concentrations of the toxin.

Acknowledgments We would like to thank Ms. Linda Russell for preparation of the manuscript. DHBE was a generous gift from Merck Sharp & Dohme Research Laboratories. This work was supported by National Institutes of Health grant NS17574 to V. A. C. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received April 25, 1990; revised June 15, 1990.

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