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Nicotine-mediated plasticity in robust nucleus of the archistriatum of the adult zebra finch
Brain Research 1018 (2004) 97 – 105 www.elsevier.com/locate/brainres
Research report
Nicotine-mediated plasticity in robust nucleus of the archistriatum of the adult zebra finch Delanthi Salgado-Commissariat a,*, David B. Rosenfield a,b, Santosh A. Helekar a,c a
Speech and Language Center, Department of Neurology, Baylor College of Medicine, 6501 Fannin Street, NB 422, Houston, TX 77030, USA b Department of Otolaryngology, Baylor College of Medicine, Houston, TX 77030, USA c Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA Accepted 17 May 2004 Available online 24 June 2004
Abstract Activation of neuronal nicotinic acetylcholine receptors (nAChRs) modulates the induction of long-term potentiation (LTP), a possible cellular mechanism for learning. This study was undertaken to determine the effects of activation of nAChRs by nicotine on long-term plasticity in the songbird zebra finch, which is a valuable model to study synaptic plasticity and its implications to behavioral learning. Electrophysiological recordings in the robust nucleus of the archistriatum (RA) in adult zebra finch brain slices reveal that tetanic stimulation alone does not produce LTP. However, LTP is induced by such stimulation in the presence of nicotine. The nicotine-mediated LTP is blocked by dihydro-h-erythroidine (DHhE, 1 AM), an antagonist having a greater effect against nAChRs containing the alpha 4 subunit. In the presence of methyllcaconitine (MLA, 10 nM), an antagonist of nAChRs containing the alpha 7 subunit, a long-term depression (LTD) is unmasked, implicating a bi-directional type of plasticity in the zebra finch RA, which is modulated by differential activation of nAChR subtypes. Intracellular recordings from single neurons show a depression of the afterhyperpolarization (AHP) and an increase in frequency of evoked and spontaneous action potentials in the presence of nicotine. These results suggest that nicotinic cholinergic mechanisms may play a critical role in synaptic plasticity in the zebra finch song system and thereby influence song learning and plasticity. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Neural plasticity Keywords: Nicotine; Plasticity; Zebra finch; Songbird; Song nucleus; RA
1. Introduction Exposure to nicotine can lead to an enhancement of cognitive function [17,20]. There are many studies that have investigated the role of nicotine in long term changes in synaptic function and the majority of these studies have been conducted in the rodent hippocampus [11,12,16,23]. In
Abbreviations: nAChR: nicotinic acetylcholine receptor; ACh: acetylcholine; LTP: long-term potentiation; LTD: long-term depression; RA: robust nucleus of the archistriatum; HVC: higher vocal center; LMAN: lateral portion of the magnocellular nucleus of the anterior neostriatum; VP: ventral paleostriatum; OV: nucleus ovoidalis; AFP: anterior forebrain pathway; VMP: vocal motor pathway * Corresponding author. Tel.: +1-713-798-4671; fax: +1-713-7986417. E-mail address: [email protected] (D. Salgado-Commissariat). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.051
addition, it has been demonstrated that local infusion of nicotinic acetylcholine receptor (nAChR) antagonists impairs memory [13,31] while exogenous nicotine reverses lesion-impaired memory deficits [13]. The involvement of nAChRs in learning and memory has also been suggested through gene knock-out studies in mice [32]. However, no such role has yet been demonstrated in natural learning behaviors such as song learning in zebra finches. The effect of nicotine on long-term synaptic plasticity has not been studied in the zebra finch brain. Evidence for a link between the cholinergic system and the song control system of the zebra finch was suggested by the work of Ryan and Arnold [33] who demonstrated the presence of acetylcholinesterase (AChE) positive neurons in several song control nuclei including the robust nucleus of the archistriatum (RA), an important pre-motor nucleus in the zebra finch song system. This was later confirmed with
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studies by another group [47]. Evidence for the cholinergic participation in the song control circuitry has come from the work of Sakaguchi et al. Their studies reported the presence of acetylcholine (ACh) in the song control nuclei of the zebra finch. More interestingly, however, they showed that the ACh content was markedly increased in birds that were 30 –50 days post hatch which is the critical period for song learning [36]. The ACh content decreased in adult birds, however, it stayed higher than post-hatch day 30 levels. Sakaguchi et al. [37] also showed that the increase in ACh concentration paralleled enzyme activity of choline acetyltransferase (ChAT). This could suggest that the increase in ACh content in the song nuclei observed during the critical period of song learning might be related to plastic changes in the synapses of these nuclei. Coincident with these changes was an increase in the ACh agonist carbacholmediated increase in phosphoinositide turnover in synaptoneurosomes of the RA [35]. Subsequent studies showed that the cholinergic innervation to two song nuclei of the vocal motor pathway, higher vocal center (HVC) and RA, originates from the ventral paleostriatum (VP). This study also showed that there is an anterior – posterior topography of the cholinergic neurons in the VP that project to the HVC and RA [21]. The VP in turn receives auditory input from the nucleus ovoidalis (OV), which is a major relay in the main ascending auditory pathway [22]. In a different study by the same investigators it was shown that the VP is sexually dimorphic [38]. The size and density of the ChAT-immunoreactive somata in areas of the VP that project to the RA and HVC were significantly larger in males than in females. Since the female zebra finch never sings, the sexual dimorphism in the cholinergic neurons may be of significance to the sexual dimorphism in song behavior and plasticity. Autoradiographic evidence for the presence of nAChR [45] suggests that nAChRs are present in several of the song nuclei. A recent study on anesthetized zebra finches suggests that cholinergic basal forebrain may regulate auditory feedback in the birdsong system [41]. Despite the evidence of a link between the cholinergic system and song control system in the zebra finch there is a limited amount of data on the functional effects of activation of nAChRs in the zebra finch brain. The zebra finch brain has a well-defined song circuitry and well-delineated series of song nuclei that contribute to song learning and song production. The avian song system is composed of an anterior forebrain pathway (AFP) that is necessary for song learning and a vocal motor pathway (VMP) that is necessary for song production [4,5,14,30,39,42]. The RA, an important pre-motor nucleus of the VMP, receives inputs from the lateral portion of the magnocellular nucleus of the anterior neostriatum (LMAN) of the AFP. The RA, therefore, is at the site of convergence for two functionally distinct parts of the song system [4,14,29,30]. The activity of RA neurons and the modulation of synaptic plasticity may play a critical role in influencing the final motor output of the song system and song-related neuronal plasticity.
Since work from several laboratories has provided evidence for adult-phase song plasticity in the zebra finch, and because of our interest in studying this form of plasticity and its neuromodulation we wanted to determine whether longterm synaptic plasticity could be induced in the adult zebra finch RA. We wanted to study its modulation by activating nAChRs because the ACh content is known to persist at elevated levels in the adult zebra finch brain [36]. In this report we present the initial results of this study. This study could give us an insight into the importance of the cholinergic pathway in song learning in the zebra finch and explore the possibility of the role of nAChR activation in neurobehavioral mechanisms related to song learning and production.
2. Methods In our experiments we used adult male zebra finches (Taeniopygia guttata) that were 90 – 120 days post hatch. Birds were provided ad lib access to food and water and housed in the animal care facility of Baylor College of Medicine according to the guidelines of the Institutional Animal Care and Use Committee. Prior to the preparation of brain slices, birds were anesthetized by injecting xylazine (50 mg/kg body weight) and ketamine (25 mg/kg body weight) intramuscularly, and then decapitated. The brain was quickly removed and placed in ice cold, oxygenated (95% O2, 5% CO2) Artificial Cerebrospinal Fluid (ACSF) which contains (in mM): NaCl 124; KCl 3.5; CaCl2 2.5; MgCl2 1.3; NaHCO3 22; NaH2PO4 1.25; Glucose 10. The brain was transferred to a petri dish and gently placed on blotting paper while totally submerged in cold ACSF. Following this the brain was sectioned mid-sagittally to separate the two hemispheres, after which each hemisphere was sectioned such that a transverse block containing the RA could be obtained. Each block was then fixed to the stage of a vibroslice (Campden Instruments) using cyanoacrylate glue. The slices (400-Am thick) were immediately transferred to a beaker containing oxygenated ACSF maintained at room temperature. After a recovery period of approximately 2 h, a single slice was submerged in a recording chamber and trapped between two nylon meshes. The slices were superfused with oxygenated ACSF warmed to 32 jC using a solution in-line heater (Warner Instruments). Extracellular recording was achieved with borosilicate glass microelectrodes (Garner Glass Claremont, CA) filled with 2 M NaCl pulled to a final resistance of 0– 5 MV using a Brown and Flaming (P80/PC) electrode puller. The microelectrode was advanced onto the slice and visually placed on the RA using an MP-285 micromanipulator (Sutter Instrument). Intracellular recording of RA neurons was conducted with sharp electrodes filled with 3 M potassium acetate and resistance in the range of 120– 160 MV. A tungsten bipolar stimulating electrode (FHC Bowdoinham, ME) was placed on LMAN fiber tracts entering
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the RA. Intracellular/Extracellular responses were evoked by stimulating these afferent inputs. Signals were amplified using an intracellular/extracellular electrometer IE-210 (Warner Instrument). Signals were digitized by an axon instrument digidata 1321A. The software package pClamp 8 (Axon Instruments) was used for data acquisition and data analysis. The amplitude of the extracellularly recorded evoked response was determined by measuring the voltage difference between the negative and positive peaks. The amplitude of the afterhyperpolarization (AHP) of intracellularly recorded action potential was measured relative to the action potential threshold voltage. The firing frequency of evoked action potentials was determined by calculating the instantaneous firing frequency of the first two action potentials evoked with a depolarizing current stimulation. Under control conditions, a depolarizing current stimulus that evoked at least two action potentials were administered. The firing frequency in the presence of a pharmacological agent was determined by applying the same intensity current stimulus as that used during control. Spontaneous firing rates were determined by calculating the frequency between two or more action potentials during spontaneous membrane potential records. In the LTP experiments, a baseline recording of at least 20 min was conducted before administering a tetanic stimulation. Pharmacological agents, when used, were administered 10 min prior to tetanization, unless otherwise noted in the results section. The data from these experiments are expressed as mean ( F SEM) percentage of the response immediately prior to tetanic stimulation (0 min). Changes in the post-tetanic response was assessed by comparing the percent change in response at 60 min post tetani compared to the response at 0 min. Differences in the post-tetanic response between the various treatment groups were determined by comparing the percent response at 60 min. Statistical analysis between two groups was conducted using the Student’s t-test with the software package KyPlot (Kyence). For multiple comparisons we used one-way analysis of variance (ANOVA) followed by the Student Newman-Keuls post hoc test when necessary (Prism version 4.01, GraphPad Software). A p value < 0.05 was considered statistically significant.
3. Results The in vitro slice physiology experiments reveal that bath application of nicotine has significant effects on long-term plasticity in the zebra finch RA. These experiments were conducted on transverse slices (400-Am thick) of adult male zebra finches. Extracellular potentials were recorded from the RA by stimulating LMAN fibers entering the RA. Studies from the laboratories of Mooney and Perkel [24,26,44] have shown using intracellular and patch clamp recording methods, a glutamate receptor sensitive component to the EPSP recorded from RA with LMAN fiber stimulation. In line with this finding we demonstrate that the
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Fig. 1. Extracellularly recorded potentials from RA of a zebra finch. Responses were evoked by stimulating LMAN fibers entering the RA. After a control recording, the brain slice was superfused with ACSF containing the glutamate receptor antagonists, APV (50 AM) and CNQX (10 AM) for 10 min after which the slice was washed with drug-free ACSF for approximately 10 min. ‘‘a’’ depicts measurement of the amplitude of the extracellular response. * Indicates stimulus artifact that has been blanked out for clarity.
extracellular potentials evoked with LMAN fiber stimulation were depressed in the presence of APV (2-amino-5phosphonopentanoic acid) and CNQX (6-cyano-7-nitroquinoxaline-2, 3-dione). From experiments on seven different zebra finch slices, the mean amplitude (in mV) reduced from 0.53 F 0.06 to 0.2 F 0.03. The tracings from one of these experiments are shown in Fig. 1. Although under our experimental conditions the extracellular response was not completely inhibited with glutamate receptor blockade, the decrease in response was statistically significant compared to control (one-way ANOVA and Newman-Keuls post hoc test [ F(2,20) = 14.25, p < 0.001], n = 7). Next we performed tetanic stimulation of LMAN fibers using a protocol (20 pulses at 100 Hz) that has been shown to produce LTP in other preparations [11,12]. However in our preparation, this stimulation protocol did not elicit a long-term potentiation of the response. The response at 30 min post-tetani and 60 min post-tetani were 99 F 4.54% and 91 F 7.68% of the response at 0 min (n = 7, Fig. 2). The changes in response at these time points were not significantly different from each other [ F(2,20) = 0.92, p = 0.41]. Since nicotine application has been shown to enhance or induce LTP in other preparations, we applied nicotine (10 AM) to the superfusate to determine if this would result in an enhancement of the pretetanic response. In the presence of nicotine, the tetanic stimulation of 20 pulses at 100 Hz induced a long-term potentiation of the response (n = 7, Fig. 3). The results from seven different brain slices, show an average potentiation of 182 F 28.8% 1 h after administering a tetanic stimulation. Statistical analysis of the percent response at 30 and 60 min post-tetani revealed that the enhancement of the response is significantly different [ F(2,20) = 5.323, p < 0.05]. The Newman-Keuls post hoc test revealed that the responses at 30 and 60 min are significantly different from 0 min. Although it appears that nicotine by itself attenuates the amplitude of the extracellular potential, this effect of nicotine was not observed in all experiments with nicotine. Irrespective of this nicotine-induced effect on the amplitude of the extracellular potential evoked prior to a tetanic stimulation observed in some of our experiments, the nicotine-mediated
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Fig. 2. The effects of a tetanic stimulation on the extracellular response in zebra finch RA. In this and following figures the changes in the peak amplitude of the response is plotted as the percent of the pre-tetanic response immediately preceding the tetani (0 min). Baseline recordings were conducted for at least 20 min prior to applying a tetanic stimulus (‘‘T’’) to LMAN fibers entering the RA. In this and the following three figures, the bottom panel show representative traces from a single experiment of evoked extracellular potentials immediately prior to the tetanic stimulation, immediately after the tetanic stimulation and one hour after the tetanic stimulation.
LTP was elicited. To demonstrate this, a subset of these experiments have been plotted separately and added to Fig. 3 (Nic3, n = 3, Fig. 3). In these three experiments, there was no change in the baseline recording in the presence of nicotine. Yet, tetanic stimulation resulted in LTP. Here too a statistical analysis of the percent response at 30 and 60 min post-tetani revealed that the enhancement of the response is significantly different to 0 min [ F(2,8) = 73.08, p < 0.001]. Since extracellular potentials evoked in the RA with LMAN stimulation were not completely inhibited with glutamate receptor antagonists, we tested the effects of APV/CNQX on LTP in the presence of nicotine. As shown in Fig. 3, in these experiments, APV/CNQX when added to the superfusate reduced the percent response from 179.67 F 38.78 (at 20 min) to 98.00 F 5.67 (at 10 min). There was no further reduction in the response when nicotine was included into the superfusate. A tetanic stimulation following this combination of APV/CNQX and nicotine in the superfusate did not lead to LTP, indicating that LTP induced in the presence of nicotine reflects the LTP of glutamate receptor-mediated synaptic response, and possibly also the involvement of these receptors in induction of LTP (Nic + APV/CNQX, n = 3, Fig. 3). Statistical analysis of data in Fig. 3 revealed that the percent response at 60 min post-tetani is significantly different between the four groups
[ F(3,19) = 14.17, p < 0.05]. The Newman-Keuls post hoc test revealed that the nicotine group and the subset of nicotine experiments (Nic3) were significantly different from the control and the Nic + APV/CNQX groups. The LTP that was induced in the presence of nicotine was absent during the co-application of nicotine and dihydro-h-erythroidine (DHhE), a nAChR antagonist that shows greater potency against receptors containing the alpha 4 subunit (n = 6, Fig. 4). Statistical analysis revealed that there was no significant difference between the responses at 0, 30 and 60 min [ F(2,16) = 1.174, p = 0.34]. Although the concentration of 1 AM DHhE that was used in our experiments should predominantly block alpha 4 subunit containing receptors, its action on receptors containing the alpha 3 subunit can not be completely ruled out since it has been shown that DHhE at 1 AM blocks 30% of the alpha 3 nAChR-mediated current in rodent hippocampal neurons [1]. We tested another nAChR antagonist, methyllycaconitine (MLA, 10 nM) known to more effectively block alpha 7 subunit containing receptors [1]. Here too, the nicotinic receptor-dependent LTP was inhibited (n = 6, Fig. 5). Although the decrease in the percent response at 30 min and 60 min (73 F 10.81%)
Fig. 3. The effects of a tetanic stimulation on the extracellular response in zebra finch RA in the presence of nicotine (10 AM) or the combination of nicotine and APV/CNQX. In the data sets ‘‘Nicotine’’,‘‘Nic3’’ and ‘‘Nic + APV/CNQX’’, ‘‘D’’ indicates where nicotine was added to the superfusate in the absence (‘‘Nicotine’’,‘‘Nic3’’) or presence (‘‘Nic + APV/ CNQX’’) of APV/CNQX. ‘‘D1’’ applies only to the data set ‘‘Nic + APV/ CNQX and indicates where APV/CNQX was added to the superfusate. ‘‘Nic3’’ represents a subset of experiments from the ‘‘Nicotine’’ group that have been plotted separately to demonstrate that even in the absence of a nicotine-mediated depression of basal response, LTP is elicited. ‘‘Control’’ is the same data from Fig. 2, re-plotted for comparison. ‘‘T’’ indicates tetanic stimulation. The bottom panel shows a representative trace from the ‘‘Nicotine’’ data set.
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analysis revealed a significant difference between the control group and nicotine group. In addition to this, a nicotinemediated increase in spontaneous activity was also observed (n = 5, Fig. 6D). The frequency of spontaneous action potentials significantly increased from 24.27 F 5.15 to 38.88 F 3.39 Hz ( p < 0.05). The increase in neuronal excitability in response to nicotine suggested by these findings might contribute to the induction of LTP in presence of nicotine.
4. Discussion
Fig. 4. Inhibition of the nicotine-induced long-term potentiation with a nAChR antagonist. In these experiments a combination (‘‘D’’) of nicotine and nAChR antagonist DHhE (1 AM) was added to the superfusate 10 minutes before the tetanus (‘‘T’’).
was not statistically significant from the response at 0 min [ F(2,17) = 3.09, p = 0.08] it appears that in the presence of MLA, a long-term depression (LTD) was unmasked. Also included in Fig. 5 are the effects of the combination of the two antagonists MLA and DHhE with nicotine and MLA alone (Nic + MLA + DHhE, n = 3, MLA, n = 4, Fig. 5). A one-way ANOVA of the percent response at 60 min revealed no significant difference between these three groups [ F(2,12) = 1.26, p = 0.33]. The combination of the above results seems to suggest that in the zebra finch RA, there exists a bi-directional type of plasticity, which is dependent on the differential activation of nAChRs. To determine the effects of nicotine on membrane properties and excitability of single neurons within the RA, intracellular recordings of single neurons within the RA was conducted. These experiments revealed that nicotine increased the number of action potential spikes to a given depolarization current stimulus (Fig. 6A). As shown in Fig. 6C, the firing frequency of the first two action potentials evoked by a depolarizing current significantly increased from 42.05 F 4.1 to 64.9 F 8.2 Hz, p < 0.05, n = 6). Concomitantly, the amplitude of the AHP was significantly reduced to 62.28 F 10.1% of control levels. These data are shown in Fig. 6B (n = 6). The amplitude of the AHP during nicotine and wash has been plotted as percent response of control AHP amplitude. One-way ANOVA revealed a significant difference between these three groups [ F(2,13) = 5.73, p < 0.05]. The Newman-Keuls post hoc
This study demonstrates, for the first time, the effects of nicotine on long-term synaptic plasticity in the adult zebra finch brain slice preparation. We demonstrate that tetanic stimulation by itself does not induce LTP in the nucleus RA in vitro; however, activation of nAChRs by nicotine during tetanization causes its induction. It is important to study long-term synaptic plasticity and its neuromodulation in zebra finch song nuclei because the avian song system undergoes a remarkable amount of developmental and adult-phase song-related plasticity [18,25]. Despite evidence for the involvement of songbird neural circuitry in songrelated plasticity publications reporting long-term synaptic potentiation in song nuclei are scarce. A study by Boettiger and Doupe demonstrates the presence of long-term synaptic plasticity in the song nucleus LMAN of the zebra finch [2]. In their study, they delivered a postsynaptic depolarizing
Fig. 5. The effects of a tetanic stimulation on the extracellular response in zebra finch RA in the presence of another nAChR antagonist, MLA (10 nM). The combination of nicotine and MLA (‘‘Nic + MLA’’), nicotine, MLA and DHhE (‘‘Nic + MLA + DHhE’’), or MLA alone (‘‘MLA’’) was added (‘‘D’’) to the superfusate 10 min before tetanic stimulation (‘‘T’’). The bottom panel shows a representative trace from the ‘‘Nic + MLA’’ data set.
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Fig. 6. Intracellular recordings to determine the effect of nicotine on single neurons in male zebra finch RA. Panel A is an example of an increase in evoked action potentials in the presence of nicotine. Panel B is the summary of data showing a significant decrease in percent AHP amplitude in the presence of nicotine. Panel C shows an increase in the frequency of evoked action potentials in six different neurons. Panel D is an example of an increase in spontaneous action potentials in the presence of nicotine. * Indicates significant difference from control, p < 0.05.
current paired with the stimulation of recurrent axon collateral inputs that interconnect neurons within the LMAN nucleus. This protocol repeated 40 times resulted in a long lasting increase of the EPSP elicited by stimulating LMAN recurrent collaterals. The long-term changes observed under their experimental conditions occurred only in 20-day-old zebra finches and not in the 60-day-old zebra finch group. A second study by Ding and Perkel [10] demonstrated an increase in the excitatory postsynaptic current in Area X by pairing 100 Hz electrical stimulation with depolarization of the postsynaptic neuron. The enhancement of the response was observed for approximately 20 min post tetani and required D1-like dopamine receptor activation. Unlike the study by Boettiger and Doupe, in this study the activity-
dependent plasticity was observed in adult zebra finches but not in birds younger than 37 days old. Plasticity within the zebra finch song circuitry predominates in the juvenile zebra finch during which the bird goes through a sensory acquisition phase where the bird memorizes the tutor’s song and a sensorimotor learning phase where the bird matches his own song production to that which was memorized. At the adult stage (>90 days post-hatch), the bird has a well-defined stereotyped song that is described as being ‘‘crystallized’’. Hence the sensory and motor plasticity is presumed diminished in adulthood, as the bird’s song no longer changes at this stage. While this lack of change is evident in the acoustic structure of song in normal adult zebra finches, the song syllable content and the amount of singing can still
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be altered under various conditions throughout life [3,15]. Moreover, artificially induced alterations in auditory feedback [19,28] and in the innervation of the syringeal muscles [46] produce significant changes in the adult song. The current study demonstrates that synaptic plasticity can be induced in the adult zebra finch brain under the right neuromodulatory influences. This study also reveals the possible existence of multiple subtypes of nAChRs in the zebra finch RA. Furthermore, it is apparent that the activation of different nAChR subtypes results in either LTP or LTD, suggesting the existence of bidirectional plasticity within the zebra finch RA. Bi-directional model of plasticity has been described by others based on their findings in rodent preparations [8,9,27]. One major criterion for such a phenomenon may be the calcium concentration. These studies describe a calcium dependent regulation of synaptic plasticity such that small increases in intracellular calcium concentrations due to tetanic stimulation lead to LTD and large increases lead to LTP [8,9,27]. In the absence of significant calcium increase or at intermediate levels of calcium influx, neither LTP nor LTD is induced. What we see in the present study may be a differential regulation of calcium influx due to differential activation of nAChR subtypes. In the absence of a pharmacological agent such as nicotine, the calcium influx due to depolarization and glutamate receptor activation as a result of a tetanic stimulation is insufficient to produce LTP. When a tetanic stimulation is applied in the presence of nicotine, the activation of nAChRs boosts the calcium influx to a level high enough to produce LTP. Blocking nAChRs such as alpha 7 receptors that are highly permeable to calcium [6,40] with the application of MLA, diminishes the calcium levels such that a tetanic stimulation leads to LTD, which is known to be induced at relatively low levels of intracellular calcium. Although alpha 7 containing nAChRs are susceptible to desensitization, the calcium influx that occurs during activation of these receptors may be sufficient to induce the cascade of events that lead to the LTP observed in our study. Alternatively, MLA at the concentration used in this study may be acting on more than one receptor subtype that leads to a net decrease in calcium levels that results in LTD. When nicotine is combined with the antagonist, DHhE, that more effectively blocks alpha 4 subunit containing receptors, part of the calcium influx is reduced. The levels of calcium under this experimental condition may be at an intermediate level and not low enough to produce LTD or high enough to produce LTP. When nicotine is combined with two antagonists, MLA and DHhE, the LTD does not appear to be enhanced. These results may suggest that under these experimental conditions, when multiple nAChRs are blocked, the calcium levels are further diminished and too low to produce LTD. Since in the presence of MLA alone tetanization appears to lead to LTD, it is possible that there may be a basal activation of nAChR by endogenously released ACh that is insufficient to produce LTP.
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Alternatively, the bi-phasic nature of the nicotine-induced plasticity that is emerging from our studies may be related to the fact that there are at least two different populations of neurons in the RA. Activation of one population of cells gives rise to LTP and activation of the other gives rise to LTD, and these differential effects might be due to different nAChR subtype distributions and densities in different cell populations. It is not clear from the current studies if multiple receptor subtypes are found on a single neuron or if different RA neurons have different nAChR subtypes. Immunohistochemical experiments conducted in our laboratory using monoclonal antibodies MAb 306 against alpha 7 and MAb 299 against alpha 4 receptors reveal that these antibodies bind to neurons in the RA of zebra finch (results not shown). It is also apparent from these studies that some neurons are more intensely stained than others indicating there is a heterogeneous population of RA neurons. These initial immunohisotchemical studies seem to lend support to the fact that, not all neurons within the RA contain nAChRs or nAChR of the same subtype, suggesting that there are different densities of these receptor subtypes in different populations of RA neurons. Our initial series of experiments at the single neuron level employing intracellular recording reveal that nicotine attenuates the AHP, increases the number of evoked action potentials, and increases the frequency of spontaneous activity in zebra finch neurons. A reduction in the amplitude of the AHP caused by activation of calcium-dependent potassium channels has been implicated in activity-dependent plasticity in other studies [7,34,43]. Experiments conducted on CA1 pyramidal neurons have shown that a tetanic stimulation that did not produce LTP, resulted in LTP when the AHP was inhibited [34]. This implies that reducing the AHP amplitude could decrease the threshold for LTP induction. The increase in excitability in the presence of nicotine observed at the single neuron level in our experiments may be a factor in the threshold for the induction of plasticity. These observations may suggest that such changes in excitability are part of the nAChR-mediated mechanism underlying synaptic plasticity. The zebra finch has a well-delineated neural circuit that produces a distinct learned behavior. Therefore it is a valuable model in which to study plasticity. It is evident that the influence of neuromodulators and developmental changes due to neuromodulatory innervations to song nuclei may be important for activity-dependent plasticity in zebra finches [10]. The fact that there is a cholinergic innervation to the song control system of the zebra finch makes it worthwhile to investigate if nAChR activation could lead to synaptic plasticity in the song nuclei. The results obtained thus far give evidence that nAChR are present in the RA of zebra finch and that nicotinic receptor activation is necessary to induce long-term potentiation in adult zebra finch slices. Selective inhibition of different nAChR reveals a bidirectional type of plasticity. Future studies to investigate the cellular mechanisms underlying these initial observa-
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tions on nicotine-induced long-lasting potentiation will contribute towards understanding the cholinergic mechanisms involved in song learning and synaptic plasticity in general.
Acknowledgements This work was supported by the M.R. Bauer Foundation and the National Institute on Deafness and Other Communication Disorders.
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Research report
Nicotine-mediated plasticity in robust nucleus of the archistriatum of the adult zebra finch Delanthi Salgado-Commissariat a,*, David B. Rosenfield a,b, Santosh A. Helekar a,c a
Speech and Language Center, Department of Neurology, Baylor College of Medicine, 6501 Fannin Street, NB 422, Houston, TX 77030, USA b Department of Otolaryngology, Baylor College of Medicine, Houston, TX 77030, USA c Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA Accepted 17 May 2004 Available online 24 June 2004
Abstract Activation of neuronal nicotinic acetylcholine receptors (nAChRs) modulates the induction of long-term potentiation (LTP), a possible cellular mechanism for learning. This study was undertaken to determine the effects of activation of nAChRs by nicotine on long-term plasticity in the songbird zebra finch, which is a valuable model to study synaptic plasticity and its implications to behavioral learning. Electrophysiological recordings in the robust nucleus of the archistriatum (RA) in adult zebra finch brain slices reveal that tetanic stimulation alone does not produce LTP. However, LTP is induced by such stimulation in the presence of nicotine. The nicotine-mediated LTP is blocked by dihydro-h-erythroidine (DHhE, 1 AM), an antagonist having a greater effect against nAChRs containing the alpha 4 subunit. In the presence of methyllcaconitine (MLA, 10 nM), an antagonist of nAChRs containing the alpha 7 subunit, a long-term depression (LTD) is unmasked, implicating a bi-directional type of plasticity in the zebra finch RA, which is modulated by differential activation of nAChR subtypes. Intracellular recordings from single neurons show a depression of the afterhyperpolarization (AHP) and an increase in frequency of evoked and spontaneous action potentials in the presence of nicotine. These results suggest that nicotinic cholinergic mechanisms may play a critical role in synaptic plasticity in the zebra finch song system and thereby influence song learning and plasticity. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Neural plasticity Keywords: Nicotine; Plasticity; Zebra finch; Songbird; Song nucleus; RA
1. Introduction Exposure to nicotine can lead to an enhancement of cognitive function [17,20]. There are many studies that have investigated the role of nicotine in long term changes in synaptic function and the majority of these studies have been conducted in the rodent hippocampus [11,12,16,23]. In
Abbreviations: nAChR: nicotinic acetylcholine receptor; ACh: acetylcholine; LTP: long-term potentiation; LTD: long-term depression; RA: robust nucleus of the archistriatum; HVC: higher vocal center; LMAN: lateral portion of the magnocellular nucleus of the anterior neostriatum; VP: ventral paleostriatum; OV: nucleus ovoidalis; AFP: anterior forebrain pathway; VMP: vocal motor pathway * Corresponding author. Tel.: +1-713-798-4671; fax: +1-713-7986417. E-mail address: [email protected] (D. Salgado-Commissariat). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.051
addition, it has been demonstrated that local infusion of nicotinic acetylcholine receptor (nAChR) antagonists impairs memory [13,31] while exogenous nicotine reverses lesion-impaired memory deficits [13]. The involvement of nAChRs in learning and memory has also been suggested through gene knock-out studies in mice [32]. However, no such role has yet been demonstrated in natural learning behaviors such as song learning in zebra finches. The effect of nicotine on long-term synaptic plasticity has not been studied in the zebra finch brain. Evidence for a link between the cholinergic system and the song control system of the zebra finch was suggested by the work of Ryan and Arnold [33] who demonstrated the presence of acetylcholinesterase (AChE) positive neurons in several song control nuclei including the robust nucleus of the archistriatum (RA), an important pre-motor nucleus in the zebra finch song system. This was later confirmed with
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studies by another group [47]. Evidence for the cholinergic participation in the song control circuitry has come from the work of Sakaguchi et al. Their studies reported the presence of acetylcholine (ACh) in the song control nuclei of the zebra finch. More interestingly, however, they showed that the ACh content was markedly increased in birds that were 30 –50 days post hatch which is the critical period for song learning [36]. The ACh content decreased in adult birds, however, it stayed higher than post-hatch day 30 levels. Sakaguchi et al. [37] also showed that the increase in ACh concentration paralleled enzyme activity of choline acetyltransferase (ChAT). This could suggest that the increase in ACh content in the song nuclei observed during the critical period of song learning might be related to plastic changes in the synapses of these nuclei. Coincident with these changes was an increase in the ACh agonist carbacholmediated increase in phosphoinositide turnover in synaptoneurosomes of the RA [35]. Subsequent studies showed that the cholinergic innervation to two song nuclei of the vocal motor pathway, higher vocal center (HVC) and RA, originates from the ventral paleostriatum (VP). This study also showed that there is an anterior – posterior topography of the cholinergic neurons in the VP that project to the HVC and RA [21]. The VP in turn receives auditory input from the nucleus ovoidalis (OV), which is a major relay in the main ascending auditory pathway [22]. In a different study by the same investigators it was shown that the VP is sexually dimorphic [38]. The size and density of the ChAT-immunoreactive somata in areas of the VP that project to the RA and HVC were significantly larger in males than in females. Since the female zebra finch never sings, the sexual dimorphism in the cholinergic neurons may be of significance to the sexual dimorphism in song behavior and plasticity. Autoradiographic evidence for the presence of nAChR [45] suggests that nAChRs are present in several of the song nuclei. A recent study on anesthetized zebra finches suggests that cholinergic basal forebrain may regulate auditory feedback in the birdsong system [41]. Despite the evidence of a link between the cholinergic system and song control system in the zebra finch there is a limited amount of data on the functional effects of activation of nAChRs in the zebra finch brain. The zebra finch brain has a well-defined song circuitry and well-delineated series of song nuclei that contribute to song learning and song production. The avian song system is composed of an anterior forebrain pathway (AFP) that is necessary for song learning and a vocal motor pathway (VMP) that is necessary for song production [4,5,14,30,39,42]. The RA, an important pre-motor nucleus of the VMP, receives inputs from the lateral portion of the magnocellular nucleus of the anterior neostriatum (LMAN) of the AFP. The RA, therefore, is at the site of convergence for two functionally distinct parts of the song system [4,14,29,30]. The activity of RA neurons and the modulation of synaptic plasticity may play a critical role in influencing the final motor output of the song system and song-related neuronal plasticity.
Since work from several laboratories has provided evidence for adult-phase song plasticity in the zebra finch, and because of our interest in studying this form of plasticity and its neuromodulation we wanted to determine whether longterm synaptic plasticity could be induced in the adult zebra finch RA. We wanted to study its modulation by activating nAChRs because the ACh content is known to persist at elevated levels in the adult zebra finch brain [36]. In this report we present the initial results of this study. This study could give us an insight into the importance of the cholinergic pathway in song learning in the zebra finch and explore the possibility of the role of nAChR activation in neurobehavioral mechanisms related to song learning and production.
2. Methods In our experiments we used adult male zebra finches (Taeniopygia guttata) that were 90 – 120 days post hatch. Birds were provided ad lib access to food and water and housed in the animal care facility of Baylor College of Medicine according to the guidelines of the Institutional Animal Care and Use Committee. Prior to the preparation of brain slices, birds were anesthetized by injecting xylazine (50 mg/kg body weight) and ketamine (25 mg/kg body weight) intramuscularly, and then decapitated. The brain was quickly removed and placed in ice cold, oxygenated (95% O2, 5% CO2) Artificial Cerebrospinal Fluid (ACSF) which contains (in mM): NaCl 124; KCl 3.5; CaCl2 2.5; MgCl2 1.3; NaHCO3 22; NaH2PO4 1.25; Glucose 10. The brain was transferred to a petri dish and gently placed on blotting paper while totally submerged in cold ACSF. Following this the brain was sectioned mid-sagittally to separate the two hemispheres, after which each hemisphere was sectioned such that a transverse block containing the RA could be obtained. Each block was then fixed to the stage of a vibroslice (Campden Instruments) using cyanoacrylate glue. The slices (400-Am thick) were immediately transferred to a beaker containing oxygenated ACSF maintained at room temperature. After a recovery period of approximately 2 h, a single slice was submerged in a recording chamber and trapped between two nylon meshes. The slices were superfused with oxygenated ACSF warmed to 32 jC using a solution in-line heater (Warner Instruments). Extracellular recording was achieved with borosilicate glass microelectrodes (Garner Glass Claremont, CA) filled with 2 M NaCl pulled to a final resistance of 0– 5 MV using a Brown and Flaming (P80/PC) electrode puller. The microelectrode was advanced onto the slice and visually placed on the RA using an MP-285 micromanipulator (Sutter Instrument). Intracellular recording of RA neurons was conducted with sharp electrodes filled with 3 M potassium acetate and resistance in the range of 120– 160 MV. A tungsten bipolar stimulating electrode (FHC Bowdoinham, ME) was placed on LMAN fiber tracts entering
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the RA. Intracellular/Extracellular responses were evoked by stimulating these afferent inputs. Signals were amplified using an intracellular/extracellular electrometer IE-210 (Warner Instrument). Signals were digitized by an axon instrument digidata 1321A. The software package pClamp 8 (Axon Instruments) was used for data acquisition and data analysis. The amplitude of the extracellularly recorded evoked response was determined by measuring the voltage difference between the negative and positive peaks. The amplitude of the afterhyperpolarization (AHP) of intracellularly recorded action potential was measured relative to the action potential threshold voltage. The firing frequency of evoked action potentials was determined by calculating the instantaneous firing frequency of the first two action potentials evoked with a depolarizing current stimulation. Under control conditions, a depolarizing current stimulus that evoked at least two action potentials were administered. The firing frequency in the presence of a pharmacological agent was determined by applying the same intensity current stimulus as that used during control. Spontaneous firing rates were determined by calculating the frequency between two or more action potentials during spontaneous membrane potential records. In the LTP experiments, a baseline recording of at least 20 min was conducted before administering a tetanic stimulation. Pharmacological agents, when used, were administered 10 min prior to tetanization, unless otherwise noted in the results section. The data from these experiments are expressed as mean ( F SEM) percentage of the response immediately prior to tetanic stimulation (0 min). Changes in the post-tetanic response was assessed by comparing the percent change in response at 60 min post tetani compared to the response at 0 min. Differences in the post-tetanic response between the various treatment groups were determined by comparing the percent response at 60 min. Statistical analysis between two groups was conducted using the Student’s t-test with the software package KyPlot (Kyence). For multiple comparisons we used one-way analysis of variance (ANOVA) followed by the Student Newman-Keuls post hoc test when necessary (Prism version 4.01, GraphPad Software). A p value < 0.05 was considered statistically significant.
3. Results The in vitro slice physiology experiments reveal that bath application of nicotine has significant effects on long-term plasticity in the zebra finch RA. These experiments were conducted on transverse slices (400-Am thick) of adult male zebra finches. Extracellular potentials were recorded from the RA by stimulating LMAN fibers entering the RA. Studies from the laboratories of Mooney and Perkel [24,26,44] have shown using intracellular and patch clamp recording methods, a glutamate receptor sensitive component to the EPSP recorded from RA with LMAN fiber stimulation. In line with this finding we demonstrate that the
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Fig. 1. Extracellularly recorded potentials from RA of a zebra finch. Responses were evoked by stimulating LMAN fibers entering the RA. After a control recording, the brain slice was superfused with ACSF containing the glutamate receptor antagonists, APV (50 AM) and CNQX (10 AM) for 10 min after which the slice was washed with drug-free ACSF for approximately 10 min. ‘‘a’’ depicts measurement of the amplitude of the extracellular response. * Indicates stimulus artifact that has been blanked out for clarity.
extracellular potentials evoked with LMAN fiber stimulation were depressed in the presence of APV (2-amino-5phosphonopentanoic acid) and CNQX (6-cyano-7-nitroquinoxaline-2, 3-dione). From experiments on seven different zebra finch slices, the mean amplitude (in mV) reduced from 0.53 F 0.06 to 0.2 F 0.03. The tracings from one of these experiments are shown in Fig. 1. Although under our experimental conditions the extracellular response was not completely inhibited with glutamate receptor blockade, the decrease in response was statistically significant compared to control (one-way ANOVA and Newman-Keuls post hoc test [ F(2,20) = 14.25, p < 0.001], n = 7). Next we performed tetanic stimulation of LMAN fibers using a protocol (20 pulses at 100 Hz) that has been shown to produce LTP in other preparations [11,12]. However in our preparation, this stimulation protocol did not elicit a long-term potentiation of the response. The response at 30 min post-tetani and 60 min post-tetani were 99 F 4.54% and 91 F 7.68% of the response at 0 min (n = 7, Fig. 2). The changes in response at these time points were not significantly different from each other [ F(2,20) = 0.92, p = 0.41]. Since nicotine application has been shown to enhance or induce LTP in other preparations, we applied nicotine (10 AM) to the superfusate to determine if this would result in an enhancement of the pretetanic response. In the presence of nicotine, the tetanic stimulation of 20 pulses at 100 Hz induced a long-term potentiation of the response (n = 7, Fig. 3). The results from seven different brain slices, show an average potentiation of 182 F 28.8% 1 h after administering a tetanic stimulation. Statistical analysis of the percent response at 30 and 60 min post-tetani revealed that the enhancement of the response is significantly different [ F(2,20) = 5.323, p < 0.05]. The Newman-Keuls post hoc test revealed that the responses at 30 and 60 min are significantly different from 0 min. Although it appears that nicotine by itself attenuates the amplitude of the extracellular potential, this effect of nicotine was not observed in all experiments with nicotine. Irrespective of this nicotine-induced effect on the amplitude of the extracellular potential evoked prior to a tetanic stimulation observed in some of our experiments, the nicotine-mediated
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Fig. 2. The effects of a tetanic stimulation on the extracellular response in zebra finch RA. In this and following figures the changes in the peak amplitude of the response is plotted as the percent of the pre-tetanic response immediately preceding the tetani (0 min). Baseline recordings were conducted for at least 20 min prior to applying a tetanic stimulus (‘‘T’’) to LMAN fibers entering the RA. In this and the following three figures, the bottom panel show representative traces from a single experiment of evoked extracellular potentials immediately prior to the tetanic stimulation, immediately after the tetanic stimulation and one hour after the tetanic stimulation.
LTP was elicited. To demonstrate this, a subset of these experiments have been plotted separately and added to Fig. 3 (Nic3, n = 3, Fig. 3). In these three experiments, there was no change in the baseline recording in the presence of nicotine. Yet, tetanic stimulation resulted in LTP. Here too a statistical analysis of the percent response at 30 and 60 min post-tetani revealed that the enhancement of the response is significantly different to 0 min [ F(2,8) = 73.08, p < 0.001]. Since extracellular potentials evoked in the RA with LMAN stimulation were not completely inhibited with glutamate receptor antagonists, we tested the effects of APV/CNQX on LTP in the presence of nicotine. As shown in Fig. 3, in these experiments, APV/CNQX when added to the superfusate reduced the percent response from 179.67 F 38.78 (at 20 min) to 98.00 F 5.67 (at 10 min). There was no further reduction in the response when nicotine was included into the superfusate. A tetanic stimulation following this combination of APV/CNQX and nicotine in the superfusate did not lead to LTP, indicating that LTP induced in the presence of nicotine reflects the LTP of glutamate receptor-mediated synaptic response, and possibly also the involvement of these receptors in induction of LTP (Nic + APV/CNQX, n = 3, Fig. 3). Statistical analysis of data in Fig. 3 revealed that the percent response at 60 min post-tetani is significantly different between the four groups
[ F(3,19) = 14.17, p < 0.05]. The Newman-Keuls post hoc test revealed that the nicotine group and the subset of nicotine experiments (Nic3) were significantly different from the control and the Nic + APV/CNQX groups. The LTP that was induced in the presence of nicotine was absent during the co-application of nicotine and dihydro-h-erythroidine (DHhE), a nAChR antagonist that shows greater potency against receptors containing the alpha 4 subunit (n = 6, Fig. 4). Statistical analysis revealed that there was no significant difference between the responses at 0, 30 and 60 min [ F(2,16) = 1.174, p = 0.34]. Although the concentration of 1 AM DHhE that was used in our experiments should predominantly block alpha 4 subunit containing receptors, its action on receptors containing the alpha 3 subunit can not be completely ruled out since it has been shown that DHhE at 1 AM blocks 30% of the alpha 3 nAChR-mediated current in rodent hippocampal neurons [1]. We tested another nAChR antagonist, methyllycaconitine (MLA, 10 nM) known to more effectively block alpha 7 subunit containing receptors [1]. Here too, the nicotinic receptor-dependent LTP was inhibited (n = 6, Fig. 5). Although the decrease in the percent response at 30 min and 60 min (73 F 10.81%)
Fig. 3. The effects of a tetanic stimulation on the extracellular response in zebra finch RA in the presence of nicotine (10 AM) or the combination of nicotine and APV/CNQX. In the data sets ‘‘Nicotine’’,‘‘Nic3’’ and ‘‘Nic + APV/CNQX’’, ‘‘D’’ indicates where nicotine was added to the superfusate in the absence (‘‘Nicotine’’,‘‘Nic3’’) or presence (‘‘Nic + APV/ CNQX’’) of APV/CNQX. ‘‘D1’’ applies only to the data set ‘‘Nic + APV/ CNQX and indicates where APV/CNQX was added to the superfusate. ‘‘Nic3’’ represents a subset of experiments from the ‘‘Nicotine’’ group that have been plotted separately to demonstrate that even in the absence of a nicotine-mediated depression of basal response, LTP is elicited. ‘‘Control’’ is the same data from Fig. 2, re-plotted for comparison. ‘‘T’’ indicates tetanic stimulation. The bottom panel shows a representative trace from the ‘‘Nicotine’’ data set.
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analysis revealed a significant difference between the control group and nicotine group. In addition to this, a nicotinemediated increase in spontaneous activity was also observed (n = 5, Fig. 6D). The frequency of spontaneous action potentials significantly increased from 24.27 F 5.15 to 38.88 F 3.39 Hz ( p < 0.05). The increase in neuronal excitability in response to nicotine suggested by these findings might contribute to the induction of LTP in presence of nicotine.
4. Discussion
Fig. 4. Inhibition of the nicotine-induced long-term potentiation with a nAChR antagonist. In these experiments a combination (‘‘D’’) of nicotine and nAChR antagonist DHhE (1 AM) was added to the superfusate 10 minutes before the tetanus (‘‘T’’).
was not statistically significant from the response at 0 min [ F(2,17) = 3.09, p = 0.08] it appears that in the presence of MLA, a long-term depression (LTD) was unmasked. Also included in Fig. 5 are the effects of the combination of the two antagonists MLA and DHhE with nicotine and MLA alone (Nic + MLA + DHhE, n = 3, MLA, n = 4, Fig. 5). A one-way ANOVA of the percent response at 60 min revealed no significant difference between these three groups [ F(2,12) = 1.26, p = 0.33]. The combination of the above results seems to suggest that in the zebra finch RA, there exists a bi-directional type of plasticity, which is dependent on the differential activation of nAChRs. To determine the effects of nicotine on membrane properties and excitability of single neurons within the RA, intracellular recordings of single neurons within the RA was conducted. These experiments revealed that nicotine increased the number of action potential spikes to a given depolarization current stimulus (Fig. 6A). As shown in Fig. 6C, the firing frequency of the first two action potentials evoked by a depolarizing current significantly increased from 42.05 F 4.1 to 64.9 F 8.2 Hz, p < 0.05, n = 6). Concomitantly, the amplitude of the AHP was significantly reduced to 62.28 F 10.1% of control levels. These data are shown in Fig. 6B (n = 6). The amplitude of the AHP during nicotine and wash has been plotted as percent response of control AHP amplitude. One-way ANOVA revealed a significant difference between these three groups [ F(2,13) = 5.73, p < 0.05]. The Newman-Keuls post hoc
This study demonstrates, for the first time, the effects of nicotine on long-term synaptic plasticity in the adult zebra finch brain slice preparation. We demonstrate that tetanic stimulation by itself does not induce LTP in the nucleus RA in vitro; however, activation of nAChRs by nicotine during tetanization causes its induction. It is important to study long-term synaptic plasticity and its neuromodulation in zebra finch song nuclei because the avian song system undergoes a remarkable amount of developmental and adult-phase song-related plasticity [18,25]. Despite evidence for the involvement of songbird neural circuitry in songrelated plasticity publications reporting long-term synaptic potentiation in song nuclei are scarce. A study by Boettiger and Doupe demonstrates the presence of long-term synaptic plasticity in the song nucleus LMAN of the zebra finch [2]. In their study, they delivered a postsynaptic depolarizing
Fig. 5. The effects of a tetanic stimulation on the extracellular response in zebra finch RA in the presence of another nAChR antagonist, MLA (10 nM). The combination of nicotine and MLA (‘‘Nic + MLA’’), nicotine, MLA and DHhE (‘‘Nic + MLA + DHhE’’), or MLA alone (‘‘MLA’’) was added (‘‘D’’) to the superfusate 10 min before tetanic stimulation (‘‘T’’). The bottom panel shows a representative trace from the ‘‘Nic + MLA’’ data set.
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Fig. 6. Intracellular recordings to determine the effect of nicotine on single neurons in male zebra finch RA. Panel A is an example of an increase in evoked action potentials in the presence of nicotine. Panel B is the summary of data showing a significant decrease in percent AHP amplitude in the presence of nicotine. Panel C shows an increase in the frequency of evoked action potentials in six different neurons. Panel D is an example of an increase in spontaneous action potentials in the presence of nicotine. * Indicates significant difference from control, p < 0.05.
current paired with the stimulation of recurrent axon collateral inputs that interconnect neurons within the LMAN nucleus. This protocol repeated 40 times resulted in a long lasting increase of the EPSP elicited by stimulating LMAN recurrent collaterals. The long-term changes observed under their experimental conditions occurred only in 20-day-old zebra finches and not in the 60-day-old zebra finch group. A second study by Ding and Perkel [10] demonstrated an increase in the excitatory postsynaptic current in Area X by pairing 100 Hz electrical stimulation with depolarization of the postsynaptic neuron. The enhancement of the response was observed for approximately 20 min post tetani and required D1-like dopamine receptor activation. Unlike the study by Boettiger and Doupe, in this study the activity-
dependent plasticity was observed in adult zebra finches but not in birds younger than 37 days old. Plasticity within the zebra finch song circuitry predominates in the juvenile zebra finch during which the bird goes through a sensory acquisition phase where the bird memorizes the tutor’s song and a sensorimotor learning phase where the bird matches his own song production to that which was memorized. At the adult stage (>90 days post-hatch), the bird has a well-defined stereotyped song that is described as being ‘‘crystallized’’. Hence the sensory and motor plasticity is presumed diminished in adulthood, as the bird’s song no longer changes at this stage. While this lack of change is evident in the acoustic structure of song in normal adult zebra finches, the song syllable content and the amount of singing can still
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be altered under various conditions throughout life [3,15]. Moreover, artificially induced alterations in auditory feedback [19,28] and in the innervation of the syringeal muscles [46] produce significant changes in the adult song. The current study demonstrates that synaptic plasticity can be induced in the adult zebra finch brain under the right neuromodulatory influences. This study also reveals the possible existence of multiple subtypes of nAChRs in the zebra finch RA. Furthermore, it is apparent that the activation of different nAChR subtypes results in either LTP or LTD, suggesting the existence of bidirectional plasticity within the zebra finch RA. Bi-directional model of plasticity has been described by others based on their findings in rodent preparations [8,9,27]. One major criterion for such a phenomenon may be the calcium concentration. These studies describe a calcium dependent regulation of synaptic plasticity such that small increases in intracellular calcium concentrations due to tetanic stimulation lead to LTD and large increases lead to LTP [8,9,27]. In the absence of significant calcium increase or at intermediate levels of calcium influx, neither LTP nor LTD is induced. What we see in the present study may be a differential regulation of calcium influx due to differential activation of nAChR subtypes. In the absence of a pharmacological agent such as nicotine, the calcium influx due to depolarization and glutamate receptor activation as a result of a tetanic stimulation is insufficient to produce LTP. When a tetanic stimulation is applied in the presence of nicotine, the activation of nAChRs boosts the calcium influx to a level high enough to produce LTP. Blocking nAChRs such as alpha 7 receptors that are highly permeable to calcium [6,40] with the application of MLA, diminishes the calcium levels such that a tetanic stimulation leads to LTD, which is known to be induced at relatively low levels of intracellular calcium. Although alpha 7 containing nAChRs are susceptible to desensitization, the calcium influx that occurs during activation of these receptors may be sufficient to induce the cascade of events that lead to the LTP observed in our study. Alternatively, MLA at the concentration used in this study may be acting on more than one receptor subtype that leads to a net decrease in calcium levels that results in LTD. When nicotine is combined with the antagonist, DHhE, that more effectively blocks alpha 4 subunit containing receptors, part of the calcium influx is reduced. The levels of calcium under this experimental condition may be at an intermediate level and not low enough to produce LTD or high enough to produce LTP. When nicotine is combined with two antagonists, MLA and DHhE, the LTD does not appear to be enhanced. These results may suggest that under these experimental conditions, when multiple nAChRs are blocked, the calcium levels are further diminished and too low to produce LTD. Since in the presence of MLA alone tetanization appears to lead to LTD, it is possible that there may be a basal activation of nAChR by endogenously released ACh that is insufficient to produce LTP.
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Alternatively, the bi-phasic nature of the nicotine-induced plasticity that is emerging from our studies may be related to the fact that there are at least two different populations of neurons in the RA. Activation of one population of cells gives rise to LTP and activation of the other gives rise to LTD, and these differential effects might be due to different nAChR subtype distributions and densities in different cell populations. It is not clear from the current studies if multiple receptor subtypes are found on a single neuron or if different RA neurons have different nAChR subtypes. Immunohistochemical experiments conducted in our laboratory using monoclonal antibodies MAb 306 against alpha 7 and MAb 299 against alpha 4 receptors reveal that these antibodies bind to neurons in the RA of zebra finch (results not shown). It is also apparent from these studies that some neurons are more intensely stained than others indicating there is a heterogeneous population of RA neurons. These initial immunohisotchemical studies seem to lend support to the fact that, not all neurons within the RA contain nAChRs or nAChR of the same subtype, suggesting that there are different densities of these receptor subtypes in different populations of RA neurons. Our initial series of experiments at the single neuron level employing intracellular recording reveal that nicotine attenuates the AHP, increases the number of evoked action potentials, and increases the frequency of spontaneous activity in zebra finch neurons. A reduction in the amplitude of the AHP caused by activation of calcium-dependent potassium channels has been implicated in activity-dependent plasticity in other studies [7,34,43]. Experiments conducted on CA1 pyramidal neurons have shown that a tetanic stimulation that did not produce LTP, resulted in LTP when the AHP was inhibited [34]. This implies that reducing the AHP amplitude could decrease the threshold for LTP induction. The increase in excitability in the presence of nicotine observed at the single neuron level in our experiments may be a factor in the threshold for the induction of plasticity. These observations may suggest that such changes in excitability are part of the nAChR-mediated mechanism underlying synaptic plasticity. The zebra finch has a well-delineated neural circuit that produces a distinct learned behavior. Therefore it is a valuable model in which to study plasticity. It is evident that the influence of neuromodulators and developmental changes due to neuromodulatory innervations to song nuclei may be important for activity-dependent plasticity in zebra finches [10]. The fact that there is a cholinergic innervation to the song control system of the zebra finch makes it worthwhile to investigate if nAChR activation could lead to synaptic plasticity in the song nuclei. The results obtained thus far give evidence that nAChR are present in the RA of zebra finch and that nicotinic receptor activation is necessary to induce long-term potentiation in adult zebra finch slices. Selective inhibition of different nAChR reveals a bidirectional type of plasticity. Future studies to investigate the cellular mechanisms underlying these initial observa-
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tions on nicotine-induced long-lasting potentiation will contribute towards understanding the cholinergic mechanisms involved in song learning and synaptic plasticity in general.
Acknowledgements This work was supported by the M.R. Bauer Foundation and the National Institute on Deafness and Other Communication Disorders.
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