- Email: [email protected]
l -aspartate effects on single neurons and interactions with glutamate in striatal slice preparation from chicken brain
brain research 1474 (2012) 1–7
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report L-aspartate
effects on single neurons and interactions with glutamate in striatal slice preparation from chicken brain Da´vid Bala´zs, Andra´s Csillag, Ga´bor Gerbern + ´ utca 58, Budapest, Hungary Department of Anatomy, Histology and Embryology, Semmelweis University, Tuzolto
art i cle i nfo
ab st rac t
Article history:
There is an accumulating evidence for a transmitter role of L-aspartate (L-Asp) in various
Accepted 24 July 2012
brain regions. Recent studies from our laboratory have indicated that L-Asp is present in
Available online 31 July 2012
excitatory synapses of the striatum/nucl. accumbens of domestic chicks where it is co-
Keywords:
released with L-glutamate (L-Glu) from axon terminals. Here we provide data on the
Avian brain
postsynaptic effects of L-Asp alongside with L-Glu in striatal slices from chicken (1- to
Striatum
10-day-old) using visually guided patch-clamp technique.
Aspartate
L-Asp
and L-Glu produced similar dose-dependent inward currents and an increase in
Glutamate
spontaneous synaptic activity in all of the recorded striatal neurons. In the presence of
Excitatory amino acid
TTX both the NMDA receptor antagonist D-AP5 and the AMPA/kainate receptor antagonist
Patch clamp
CNQX reduced and the co-application of these two antagonists almost abolished the postsynaptic effects of L-Asp and L-Glu in a reversible manner. Testing the interactions of L-Asp
and L-Glu in these striatal neurons we found that co-application of L-Asp and L-Glut
produced significantly larger inward currents than L-Asp or L-Glut alone. Our data are the first to demonstrate that L-Asp can induce postsynaptic effects on the chicken striatal neurons. These effects are mediated by both NMDA and non-NMDA type ionotropic glutamate receptors and are similar to those evoked by L-Glu. In addition our results show that co-application of L-Asp and L-Glut facilitates each other’s effect, which is at least in part an excitatory amino acid (EAA) transporter dependent process. This phenomenon may explain the biological importance of the two EAAs with apparently similar postsynaptic activities in the same brain region. & 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Aspartate (Asp) has been described as a putative neurotransmitter in some systems (Baughman and Gilbert 1980; Fagg and Foster 1983; Gundersen and Storm-Mathisen 2000; Nadler et al., 1976; Wiklund et al., 1982). However, its definitive role as a neurotransmitter awaits further confirmation. The distribution of aspartatergic neurons in rat brain has been n
Corresponding author. Fax: þ36 1 215 5158. E-mail address: [email protected] (G. Gerber).
0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.07.049
described previously (Aoki et al., 1987; Ottersen and StormMathisen 1985). Several studies have shown that Asp was present in synaptic vesicles and released exocytotically from presynaptic terminals (Gundersen et al., 1998; Gundersen et al., 2004), possibly by Ca2þ dependent corelease of aspartate (Asp) and glutamate (Glu). It has been demonstrated that both excitatory amino acids (EAAs) are released from the rat striatum as part of corticostriatal neurotransmission (Girault
2
brain research 1474 (2012) 1–7
et al., 1986) and an evoked release was also reported in the domestic chicken (Zachar et al., 2012). There is also an evidence for the involvement of NMDA and non-NMDA receptors in excitatory synaptic transmission in the chick striatum (Matsushima et al., 2001). Until very recently, Asp immunoreactive neurons and terminals, as distinct from those immunoreactive to Glu have not yet been identified and characterized in avian brain structures. Then, Adam and Csillag (2006) demonstrated by ultrastructural immunocytochemistry the single or colocalized occurrence of Asp and Glu in specific synaptic boutons of the chicken medial striatum (MSt)/nucl. accumbens. Unlike Glu, Asp was found to act as a pure NMDA agonist, at least in rat brain hippocampal neuronal cultures (Patneau and Mayer 1990). However, in rat brain slices from suprachiasmatic nucleus, both NMDA and non-NMDA activation was demonstrated (Brautigan and Eagles 1998), whereas other studies even raised the possibility of an Asp receptor of unknown type (Yuzaki et al., 1996). The intermediate medial mesopallium–arcopallium-MSt system (earlier nomenclature: IMHV-archistriatum-lobus parolfactorius) for current nomenclature of avian brain see Reiner et al. (2004) is thought to play a pivotal role in memory formation during passive avoidance learning (Csillag 1999; Csillag et al., 2008) and in the behavioral control of reinforcement learning (Izawa et al., 2003) in domestic chicks. The striatal afferents originate from the arcopallium and posterior amygdaloid pallium (PoA). The afferents terminate in the MSt, forming varicosities with asymmetrical axospinous synapses (Csillag et al., 1997). More recently, a distinct nucleus accumbens has been identified in birds, including the domestic chicken (Balint and Csillag 2007), in the ventrobasal part of the former MSt. Given the known occurrence of L-Asp in avian striatal synaptic terminals, postsynaptic effects of this excitatory amino acid in these systems are yet to be demonstrated. The aim of the present study was to investigate the effects of Asp and Glu on membrane currents of single neurons in slices from the striatum/nucl. accumbens of young posthatch domestic chicks using voltage clamp technique.
2.
Results
2.1. Electrophysiological properties of the chicken striatal neurons A total of 41 neurons were investigated from 1- to 10-day-old domestic chicks striatal slices using whole-cell patch-clamp recordings (Fig. 1). The average membrane potential and input resistance of the recorded striatal neurons were (mean7s.e.m.): -56.371.3 mV, 461.2722.1 MO n¼ 39. Four biocytin labeled neurons were successfully visualized following electrophysiological recordings. The labeled neurons presented the characteristic dendritic arborization pattern of a medium spiny neuron (Fig. 1B).
Fig. 1 – (A) An example of a coronal chicken brain slice (left image) the medial striatum/accumbens area is marked by dotted line (MSt/Acc) and a visually identified striatal neuron with a patch-clamp electrode (right image). (B) One of the recorded biocytin labeled neuron from the same region. (scale bar 20 lm).
inward current and increased the spontaneous synaptic activity in a dose-dependent manner. The threshold dose under these conditions was around 250 mM. Introducing TTX (0.5–1 mM) to the perfusing solution prevented the increase in spontaneous synaptic activity evoked by either L-Glu or L-Asp, however, the inward current evoked by these excitatory amino acids persisted in all cases (Fig. 2B). In all subsequent experiments TTX (0.5–1 mM) was used to minimize presynaptic effects. The inward currents evoked by L-Asp or L-Glu were reduced by ionotropic glutamate receptor antagonists. The NMDA receptor antagonist AP5 substantially reduced and the coapplication of AP5 and the non-NMDA type glutamate ionotropic receptor antagonist CNQX almost completely blocked the L-Asp or L-Glu evoked inward current in a reversible manner (Fig. 3). The effectiveness of bath-applied AP5 (50–100 mM) or CNQX (10–50 mM) to reduce inward currents was similar with both agonists.
2.3. 2.2.
Interactions of L-Asp and L-Glu
Effects of L-Asp and L-Glu
All of the tested neurons were sensitive to bath applied L-Glu and L-Asp (Fig. 2A). In normal Krebs solution at a holding potential of -60 mV both excitatory amino acids evoked an
Co-application of L-Asp and L-Glu was tested in 15 striatal neurons. Application of 2 mM L-Asp or L-Glu alone evoked inward currents of 305.7723.9 pA and 270.6723.5 pA amplitude respectively with no significant difference. However, in 9
brain research 1474 (2012) 1–7
3
Fig. 2 – Dose-dependent inward currents induced by L-Asp and L-Glu in the chicken brain. (A) Representative examples of the effects of bath application of L-Asp on membrane currents. Drug applications are marked by the horizontal bars. (2-day-old chick). (B) Dose–response curve of L-Asp and L-Glu recorded from chick striatal neurons in the presence of TTX (0.5 lM). Each data point represents 3 to13 measurements on different preparations.
Fig. 3 – The inward currents evoked by L-Asp or L-Glu are reduced by ionotropic glutamate receptor antagonists. (A) In the presence of TTX (1 lM) the NMDA receptor antagonist AP5 substantially reduced and the co-application of AP5 and the nonNMDA type glutamate receptor antagonist CNQX almost completely blocked the L-Asp evoked inward current in a reversible manner. 2-day-old chick. (B) L-Asp and L-Glu evoked inward currents with similar amplitudes. These currents were reduced approximately by 50% following bath-application of AP5 or CNQX in a parallel manner. 1-day-old chick. (C) Summary data showing dose-dependent inhibition of L-Asp and L-Glu evoked currents by AP5 (C; n ¼ 3) and CNQX (D; n¼ 3).
4
brain research 1474 (2012) 1–7
Fig. 4 – Co-application of L-Asp and L-Glu produced substantially larger inward currents than L-Asp or L-Glu alone. (A) Representative examples of the potentiating effect of bath application of L-Asp and L-Glu on the inward membrane currents recorded from a striatal neuron. (2-day-old chick). (B) Summary data showing the facilitating effect of L-Asp and L-Glu in 2 mM and 1þ1 mM concentration in 9 of 15 tested chick striatal neurons. po0.001.
of the 15 neurons co-application of 1 mM L-Asp and 1 mM L-Glu, a total of 2 mM, produced significantly larger inward currents (404.6717.6 pA) than each agonist alone in 2 mM concentration (Fig. 4). po0.001, paired Student’s t-test.
2.4. The role of glutamate transporters in L-Asp and L-Glu interaction To investigate the possible involvement of glutamate transporters in the L-Asp and L-Glu evoked membrane responses and the observed interaction between these two agonists, we used the competitive, non-transportable blocker of excitatory amino acid transporters DL-threo-b-Benzyloxyaspartic acid (DL-TBOA) (Shimamoto et al., 1998). Bath application of 50 mM DL-TBOA produced no significant changes in membrane currents or input resistance in any of the tested neurons (n¼12). However, the peak amplitude of the inward currents evoked by 500 mM L-Asp or L-Glu were significantly increased in the presence of 50 mM DL-TBOA (Fig. 5; L-Asp cont.: 128.8756.5 pA, in DL-TBOA: 265.1777.2 pA, n¼ 12, po0.05; L-Glu cont.: 143.4751.5 pA, in DL-TBOA: 313.2790.7 pA, n¼ 12, po0.01). On the other hand, the facilitatory effect of coapplication of L-Asp (250 mM) and L-Glu (250 mM) was abolished (266.8769.1 pA) by DL-TBOA (50 mM; Fig.5).
3.
Discussion
Here we provide evidence for the first time that L-Asp can activate postsynaptic neurons in the MSt/nucl. accumbens of
young posthatch domestic chicks substantiating the recent demonstration of the occurrence of Asp in specific synaptic boutons of the chicken MSt/nucl. accumbens (Adam and Csillag 2006). In addition we provided evidence for a facilitatory interaction between L-Asp and L-Glu in the same neurons. Asp was found to be present in specific neuronal perikarya in the amygdala-equivalent region of chick brain (arcopallium, posterior amygdaloid pallium), many of which contained Asp only, while in the rest of neurons Asp colocalised with Glu (Adam and Csillag 2006). In the same study, Asp was detected in several axon terminals synapsing with medial striatal dendrites or spines, either with or without colocalising with Glu. The synapses clearly belonged to the excitatory type, displaying asymmetrical synaptic specialization. To identify Asp as a neurotransmitter in the chicken brain one has to prove that besides its presence in the synaptic vesicles it is released in a Ca2þ -dependent manner by exocytosis and activates postsynaptic receptors. Evoked synaptic release of both L-Asp and L-Glu has recently been demonstrated in the striatum of domestic chicken (Zachar et al., 2012). The chick striatum contains a population of spiny neurons as well as ‘‘aspiny neurons’’ with pallidum-like morphology and an extraordinary variety of intrinsic electrophysiological properties like burstiness, sag, inward rectification and ability to sustain firing (Farries et al., 2005). The population of the cells we recorded showed no significant difference in basic membrane properties, however, we did not test them for bursting activities or other electrophysiological characteristics. All of these cells responded to bath-applied L-Asp and L-Glu.
brain research 1474 (2012) 1–7
5
Fig. 5 – Blocking of EAA-transporters increases the L-Asp or L-Glu evoked inward currents and eliminates the facilitatory interactions of co-applied L-Asp and L-Glu. (A) In the presence of TTX (0.5 lM) and DL-TBOA (50 lM) the inward currents evoked by L-Asp (500 lM) or L-Glu (500 lM) were significantly increased but no facilitating effect was seen with the coapplication of L-Asp (250 lM) and L-Glu (250 lM). (Vm¼50 mV, 6-day-old chick). (B) Summary data showing the increases of L-Asp and L-Glu evoked inward currents induced by DL-TBOA(50 lM) in the presence of TTX (0.5 lM) in chick striatal neurons. n ¼12; po0.05.
The sensitivity of these neurons was similar to both EAAs. In contrast to Glu, Asp has little, if any, affinity for AMPA receptors which mediate fast transmission at the vast majority of CNS excitatory synapses, or for metabotropic glutamate receptors as reported for cultured neurons (Patneau and Mayer 1990). However, non-NMDA activation by L-Asp has also been demonstrated in rat hippocampal slices (Brautigan and Eagles 1998). In any case, L-Asp is considered as a selective activator of NMDA receptors at least in the mammalian brain (Frauli et al., 2006; Patneau and Mayer 1990). In the present study, the potent NMDA receptor antagonist D-AP5 and the AMPA/kainate receptor antagonist CNQX had very similar antagonistic effect on the inward currents evoked by L-Asp and L-Glu in the MSt/nucl. accumbens neurons from chicken brain slices. These results indicate an identical/similar target site for both EAAs in these neurons. Blockade of EAA-transporters significantly increased both L-Asp and L-Glu evoked currents in a similar manner. This has confirmed that the inward current evoked by L-Asp could not be ascribed solely to an influence on EAA-transporters, i.e. acting through L-Glu indirectly. Co-application of L-Asp and L-Glu, however, showed a clear facilitation in the majority of the tested neurons, and this facilitation was abolished by
blocking EAA-transporters, indicating their role in the observed Asp-Glu interaction. Earlier, such facilitatory interaction between Glu and Asp has been demonstrated only in lobster neuromuscular junction (Constanti and Nistri 1978; Constanti and Nistri 1979; Shank and Freeman 1975). Although an L-Aspinduced reduction in L-Glu uptake by EAA-transporters might explain the synergistic effect of L-Asp and L-Glu in chicken striatum, other mechanisms such as partially different sites of action, which might be the extrasynaptic NR1-NR2B receptors in the case of Asp (for review see (Nadler 2011), or sensitization of postsynaptic receptors to Glu (Lombardi et al., 1993) also need further investigation.
4.
Experimental procedure
4.1.
Electrophysiology
Experiments were carried out in neurons of the striatum/ nucl. accumbens of domestic chicks (1- to 10-day-old). Animals were decapitated and transverse striatal slices (300–450 mm thick) were cut in an oxygenated (95% O2, 5%
6
brain research 1474 (2012) 1–7
CO2) Krebs solution (4 1C) on a vibratome and incubated in a chamber at 3671 1C for at least 1 h. A single slice was then transferred to the recording chamber of an upright microscope (Olympus BX 51 WI) and continuously perfused with Krebs solution containing (in mM): NaCl 128.0; KCl 1.9; KH2PO4 1.2; MgSO4 1.3; CaCl2 2.4; NaHCO3 26.0; glucose 10.0, adjusted to pH 7.4 and supplemented when appropriate with 0.5–1 mM tetrodotoxin (TTX, Alomone Diagnostics) at a rate of about 3 ml/min. Cells were subsequently visually identified (Fig. 1) beneath a long-working distance water-immersion objective (Olympus XLUMPlan 20x, NA 0.95) with differential interference contrast (DIC) optics using a digital camera (F-view II). Striatal neurons within 50–100 mm of the surface of the slice were recorded in the whole-cell configuration of a patch-clamp amplifier (Axopatch 200B) at the holding potential of 60 mV using pipettes (8–12 MO) filled with a solution containing (in mM): K-Gluconate (120.0), KCl (20.0), MgCl2 (2.0), HEPES (10.0), EGTA (0.5), Mg-ATP (2.0), GTP-tris (0.5), adjusted to 7.18–7.23 pH with KOH. In some experiment 0.1% biocytin (Sigma) was added to the pipette solution in order to visualize the morphology of the recorded neurons. Liquid junction potentials were not compensated. Stimulation and acquisition of electrical signals was done using pClamp version 9 acquisition system and software. Measurements were made of resting membrane potential, input resistance (from the slope of I–V relationship), and the peak amplitude of membrane current responses to L-Asp (0.2–4 mM) or L-Glu (0.2–4 mM). Drugs were applied by bath-perfusion. All experiments were done at room temperature.
4.2.
Intracellular labeling
At the end of recording, the slices were placed in 0.1 M phosphate buffer (PBS; pH 7.3), containing 4% formaldehyde for 20 h at 4 1C. After rinsing the slices 3 times for 15 min in PBS they were placed in 3% H2O2 containing PBS to suppress endogenous peroxidase activity. Following rinses 3 times in PBS as above slices were incubated in 0.1 M PBS with 2% bovine albumin (Sigma A-2153) and Vector ABC solutions (Vector Pk-4000) for 48 h at 4 1C After rinsing thoroughly 3 times with PBS a standard diaminobenzidine (DAB) protocol was used for the final peroxidase reaction: 0.06% NiCl, 0.05% DAB, and 0.06% H2O2 staining for 20 min.
4.3.
Statistical analysis
Values are means 7S.E.M. (standard error of means). Statistical significance was evaluated by paired Student’s t-test. po0.05 was considered significant.
4.4.
Materials
The following compounds were purchased from: L-Glutamic acid monosodium salt hydrate and L-Aspartic acid sodium salt monohydrate from Sigma; D-AP5, CNQX disodium salt, DL-TBOA and Tetrodotoxin citrate from Tocris Bioscience.
Acknowledgments This work was supported by the Hungarian National Scientific Research Fund (OTKA73219).
r e f e r e n c e s
´ da´m, A ´ .S., Csillag, A., 2006. Differential distribution of L-asparA tate- and L-glutamate-immunoreactive structures in the arcopallium and medial striatum of the domestic chick (Gallus domesticus). J. Comp. Neurol. 498, 266–276. Aoki, E., Semba, R., Kato, K., Kashiwamata, S., 1987. Purification of specific antibody against aspartate and immunocytochemical localization of aspartergic neurons in the rat brain. Neurosci. 21, 755–765. Ba´lint, E., Csillag, A., 2007. Nucleus accumbens subregions: hodological and immunohistochemical study in the domestic chick (Gallus domesticus). Cell Tissue Res. 327, 221–230. Baughman, R.W., Gilbert, C.D., 1980. Aspartate and glutamate as possible neurotransmitters of cells in layer 6 of the visual cortex. Nature 287, 848–850. Brautigan, R.A., Eagles, D.A., 1998. Activation of NMDA and nonNMDA receptors by L-aspartate in the suprachiasmatic nucleus of the rat. Cell Signal 10, 85–90. Constanti, A., Nistri, A., 1978. A study of the interactions between glutamate and aspartate at the lobster neuromuscular junction. Br. J. Pharmacol. 62, 495–505. Constanti, A., Nistri, A., 1979. Further observations on the interaction between glutamate and aspartate on lobster muscle. Br. J. Pharmacol. 65, 287–301. Csillag, A., Sze´kely, A.D., Stewart, M.G, 1997. Synaptic terminals immunolabelled against glutamate in the lobus parolfactorius of domestic chicks (Gallus domesticus) in relation to afferents from the archistriatum. Brain Res. 750, 171–179. Csillag, A., 1999. Striato-telencephalic and striato-tegmental circuits: relevance to learning in domestic chicks. Behav. Brain Res. 98, 227–236. ´ da´m, A ´ ., Zachar, G., 2008. The organisation Csillag, A., Ba´lint, E., A of the basal ganglia in the domestic chick (Gallus domesticus): Anatomical localisation of DARPP-32 in relation to glutamate. Brain Res. Bull 76, 183–191. Fagg, G.E., Foster, A.C., 1983. Amino acid neurotransmitters and their pathways in the mammalian central nervous system. Neuroscience 9, 701–719. Farries, M.A., Meitzen, J., Perkel, D.J., 2005. Electrophysiological properties of neurons in the basal ganglia of the domestic chick: conservation and divergence in the evolution of the avian basal ganglia. J. Neurophysiol. 94 (454–67), 16 Epub 2005 March. Frauli, M., Neuville, P., Vol, C., Pin, J.-P., Pre´zeau, L., 2006. Among the twenty classical l-aminoacids, only glutamate directly activates metabotropic glutamate receptors. Neuropharmacol. 50, 245–253. Girault, J.A., Barbeito, L., Spampinato, U., Gozlan, H., Glowinski, J., Besson, M.J., 1986. In vivo release of endogenous amino acids from the rat striatum: further evidence for a role of glutamate and aspartate in corticostriatal neurotransmission. J. Neurochem. 47, 98–106. Gundersen, V., Chaudhry, F.A., Bjaalie, J.G., Fonnum, F., Ottersen, O.P., Storm-Mathisen, J., 1998. Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus. J. Neurosci. 18, 6059–6070. Gundersen, V., Storm-Mathisen, J., 2000. Chapter II aspartateneurochemical evidence for a transmitter role. In: Handbook
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of Chemical Neuroanatomy, Glutamate vol.18, Ottersen O.P. and Storm-Mathisen J. eds. Elsevier, pp.45–62. Gundersen, V., Talgøy Holten, A., Storm-Mathisen, J., 2004. GABAergic synapses in hippocampus exocytose aspartate on to NMDA receptors: quantitative immunogold evidence for cotransmission. Mol. Cell. Neurosci. 26, 156–165. Izawa, E., Zachar, G., Yanagihara, S., Matsushima, T., 2003. Localized lesion of caudal part of lobus parolfactorius caused impulsive choice in the domestic chick: evolutionarily conserved function of ventral striatum. J. Neurosci. 23, 1894–1902. Lombardi, G., Alesiani, M., Leonardi, P., Cherici, G., Pellicciari, R., Moroni, F., 1993. Pharmacological characterization of the metabotropic glutamate receptor inhibiting D-[3H]-aspartate output in rat striatum. Br. J. Pharmacol. 110, 1407–1412. Matsushima, T., Izawa, E.-I., Yanagihara, S., 2001. D1-receptor dependent synaptic potentiation in the basal ganglia of quail chicks. NeuroReport 12, 2831–2837. Nadler, J.V., Vaca, K.W., White, W.F., Lynch, G.S., Cotman, C.W., 1976. Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents. Nature 260, 538–540. Nadler, J.V., 2011. Aspartate release and signalling in the hippocampus. Neurochem. Res. 36, 668–676. Ottersen, O.P., Storm-Mathisen, J., 1985. Different neuronal localization of aspartate-like and glutamate-like immunoreactivities in the hippocampus of rat, guinea-pig and senegalese baboon (Papio papio), with a note on the distribution of g-aminobutyrate. Neurosci. 16, 589–606. Patneau, D.K., Mayer, M.L., 1990. Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-Daspartate and quisqualate receptors. J. Neurosci. 10, 2385–2399.
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Reiner, A., Perkel, D.J., Bruce, L.L., Butler, A.B., Csillag, A., Kuenzel, W., Medina, L., Paxinos, G., Shimizu, T., Striedter, G., Wild, M., Ball, G.F., Durand, S., Gunturkun, O., Lee, D.W., Mello, C.V., Powers, A., White, S.A., Hough, G., Kubikova, L., Smulders, T.V., Wada, K., Dugas-Ford, J., Husband, S., Yamamoto, K., Yu, J., Siang, C., Jarvis, E.D., 2004. Revised nomenclature for avian telencephalon and some related brainstem nuclei. J. Comp. Neurol. 473, 377–414. Shank, R.P., Freeman, A.R., 1975. Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster. J. Neurobiol. 6, 289–303. Shimamoto, K., Lebrun, B., Yasuda-Kamatani, Y., Sakaitani, M., Shigeri, Y., Yumoto, N., Nakajima, T., 1998. DL-threo-b-Benzyloxyaspartate, A Potent Blocker of Excitatory Amino Acid Transporters. Mol. Pharmacol. 53, 195–201. Wiklund, L., Toggenburger, G., Cuenod, M., 1982. Aspartate: possible neurotransmitter in cerebellar climbing fibers. Science 216, 78–80. Yuzaki, M., Forrest, D., Curran, T., Connor, J.A., 1996. Selective activation of calcium permeability by aspartate in Purkinje cells. Science 273, 1112–1114. Csillag, A., + E., Zachar, G., Wagner, Z., Ta´bi, T., Ba´lint, E., Szo¨ko, 2012. Differential changes of extracellular aspartate and glutamate in the striatum of domestic chicken evoked by high potassium or distress: an in vivo microdialysis study. Neurochem. Res. 37, 1730–1737, http://dx.doi.org/10.1007/ s11064-012-0783-4.
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report L-aspartate
effects on single neurons and interactions with glutamate in striatal slice preparation from chicken brain Da´vid Bala´zs, Andra´s Csillag, Ga´bor Gerbern + ´ utca 58, Budapest, Hungary Department of Anatomy, Histology and Embryology, Semmelweis University, Tuzolto
art i cle i nfo
ab st rac t
Article history:
There is an accumulating evidence for a transmitter role of L-aspartate (L-Asp) in various
Accepted 24 July 2012
brain regions. Recent studies from our laboratory have indicated that L-Asp is present in
Available online 31 July 2012
excitatory synapses of the striatum/nucl. accumbens of domestic chicks where it is co-
Keywords:
released with L-glutamate (L-Glu) from axon terminals. Here we provide data on the
Avian brain
postsynaptic effects of L-Asp alongside with L-Glu in striatal slices from chicken (1- to
Striatum
10-day-old) using visually guided patch-clamp technique.
Aspartate
L-Asp
and L-Glu produced similar dose-dependent inward currents and an increase in
Glutamate
spontaneous synaptic activity in all of the recorded striatal neurons. In the presence of
Excitatory amino acid
TTX both the NMDA receptor antagonist D-AP5 and the AMPA/kainate receptor antagonist
Patch clamp
CNQX reduced and the co-application of these two antagonists almost abolished the postsynaptic effects of L-Asp and L-Glu in a reversible manner. Testing the interactions of L-Asp
and L-Glu in these striatal neurons we found that co-application of L-Asp and L-Glut
produced significantly larger inward currents than L-Asp or L-Glut alone. Our data are the first to demonstrate that L-Asp can induce postsynaptic effects on the chicken striatal neurons. These effects are mediated by both NMDA and non-NMDA type ionotropic glutamate receptors and are similar to those evoked by L-Glu. In addition our results show that co-application of L-Asp and L-Glut facilitates each other’s effect, which is at least in part an excitatory amino acid (EAA) transporter dependent process. This phenomenon may explain the biological importance of the two EAAs with apparently similar postsynaptic activities in the same brain region. & 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Aspartate (Asp) has been described as a putative neurotransmitter in some systems (Baughman and Gilbert 1980; Fagg and Foster 1983; Gundersen and Storm-Mathisen 2000; Nadler et al., 1976; Wiklund et al., 1982). However, its definitive role as a neurotransmitter awaits further confirmation. The distribution of aspartatergic neurons in rat brain has been n
Corresponding author. Fax: þ36 1 215 5158. E-mail address: [email protected] (G. Gerber).
0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.07.049
described previously (Aoki et al., 1987; Ottersen and StormMathisen 1985). Several studies have shown that Asp was present in synaptic vesicles and released exocytotically from presynaptic terminals (Gundersen et al., 1998; Gundersen et al., 2004), possibly by Ca2þ dependent corelease of aspartate (Asp) and glutamate (Glu). It has been demonstrated that both excitatory amino acids (EAAs) are released from the rat striatum as part of corticostriatal neurotransmission (Girault
2
brain research 1474 (2012) 1–7
et al., 1986) and an evoked release was also reported in the domestic chicken (Zachar et al., 2012). There is also an evidence for the involvement of NMDA and non-NMDA receptors in excitatory synaptic transmission in the chick striatum (Matsushima et al., 2001). Until very recently, Asp immunoreactive neurons and terminals, as distinct from those immunoreactive to Glu have not yet been identified and characterized in avian brain structures. Then, Adam and Csillag (2006) demonstrated by ultrastructural immunocytochemistry the single or colocalized occurrence of Asp and Glu in specific synaptic boutons of the chicken medial striatum (MSt)/nucl. accumbens. Unlike Glu, Asp was found to act as a pure NMDA agonist, at least in rat brain hippocampal neuronal cultures (Patneau and Mayer 1990). However, in rat brain slices from suprachiasmatic nucleus, both NMDA and non-NMDA activation was demonstrated (Brautigan and Eagles 1998), whereas other studies even raised the possibility of an Asp receptor of unknown type (Yuzaki et al., 1996). The intermediate medial mesopallium–arcopallium-MSt system (earlier nomenclature: IMHV-archistriatum-lobus parolfactorius) for current nomenclature of avian brain see Reiner et al. (2004) is thought to play a pivotal role in memory formation during passive avoidance learning (Csillag 1999; Csillag et al., 2008) and in the behavioral control of reinforcement learning (Izawa et al., 2003) in domestic chicks. The striatal afferents originate from the arcopallium and posterior amygdaloid pallium (PoA). The afferents terminate in the MSt, forming varicosities with asymmetrical axospinous synapses (Csillag et al., 1997). More recently, a distinct nucleus accumbens has been identified in birds, including the domestic chicken (Balint and Csillag 2007), in the ventrobasal part of the former MSt. Given the known occurrence of L-Asp in avian striatal synaptic terminals, postsynaptic effects of this excitatory amino acid in these systems are yet to be demonstrated. The aim of the present study was to investigate the effects of Asp and Glu on membrane currents of single neurons in slices from the striatum/nucl. accumbens of young posthatch domestic chicks using voltage clamp technique.
2.
Results
2.1. Electrophysiological properties of the chicken striatal neurons A total of 41 neurons were investigated from 1- to 10-day-old domestic chicks striatal slices using whole-cell patch-clamp recordings (Fig. 1). The average membrane potential and input resistance of the recorded striatal neurons were (mean7s.e.m.): -56.371.3 mV, 461.2722.1 MO n¼ 39. Four biocytin labeled neurons were successfully visualized following electrophysiological recordings. The labeled neurons presented the characteristic dendritic arborization pattern of a medium spiny neuron (Fig. 1B).
Fig. 1 – (A) An example of a coronal chicken brain slice (left image) the medial striatum/accumbens area is marked by dotted line (MSt/Acc) and a visually identified striatal neuron with a patch-clamp electrode (right image). (B) One of the recorded biocytin labeled neuron from the same region. (scale bar 20 lm).
inward current and increased the spontaneous synaptic activity in a dose-dependent manner. The threshold dose under these conditions was around 250 mM. Introducing TTX (0.5–1 mM) to the perfusing solution prevented the increase in spontaneous synaptic activity evoked by either L-Glu or L-Asp, however, the inward current evoked by these excitatory amino acids persisted in all cases (Fig. 2B). In all subsequent experiments TTX (0.5–1 mM) was used to minimize presynaptic effects. The inward currents evoked by L-Asp or L-Glu were reduced by ionotropic glutamate receptor antagonists. The NMDA receptor antagonist AP5 substantially reduced and the coapplication of AP5 and the non-NMDA type glutamate ionotropic receptor antagonist CNQX almost completely blocked the L-Asp or L-Glu evoked inward current in a reversible manner (Fig. 3). The effectiveness of bath-applied AP5 (50–100 mM) or CNQX (10–50 mM) to reduce inward currents was similar with both agonists.
2.3. 2.2.
Interactions of L-Asp and L-Glu
Effects of L-Asp and L-Glu
All of the tested neurons were sensitive to bath applied L-Glu and L-Asp (Fig. 2A). In normal Krebs solution at a holding potential of -60 mV both excitatory amino acids evoked an
Co-application of L-Asp and L-Glu was tested in 15 striatal neurons. Application of 2 mM L-Asp or L-Glu alone evoked inward currents of 305.7723.9 pA and 270.6723.5 pA amplitude respectively with no significant difference. However, in 9
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Fig. 2 – Dose-dependent inward currents induced by L-Asp and L-Glu in the chicken brain. (A) Representative examples of the effects of bath application of L-Asp on membrane currents. Drug applications are marked by the horizontal bars. (2-day-old chick). (B) Dose–response curve of L-Asp and L-Glu recorded from chick striatal neurons in the presence of TTX (0.5 lM). Each data point represents 3 to13 measurements on different preparations.
Fig. 3 – The inward currents evoked by L-Asp or L-Glu are reduced by ionotropic glutamate receptor antagonists. (A) In the presence of TTX (1 lM) the NMDA receptor antagonist AP5 substantially reduced and the co-application of AP5 and the nonNMDA type glutamate receptor antagonist CNQX almost completely blocked the L-Asp evoked inward current in a reversible manner. 2-day-old chick. (B) L-Asp and L-Glu evoked inward currents with similar amplitudes. These currents were reduced approximately by 50% following bath-application of AP5 or CNQX in a parallel manner. 1-day-old chick. (C) Summary data showing dose-dependent inhibition of L-Asp and L-Glu evoked currents by AP5 (C; n ¼ 3) and CNQX (D; n¼ 3).
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Fig. 4 – Co-application of L-Asp and L-Glu produced substantially larger inward currents than L-Asp or L-Glu alone. (A) Representative examples of the potentiating effect of bath application of L-Asp and L-Glu on the inward membrane currents recorded from a striatal neuron. (2-day-old chick). (B) Summary data showing the facilitating effect of L-Asp and L-Glu in 2 mM and 1þ1 mM concentration in 9 of 15 tested chick striatal neurons. po0.001.
of the 15 neurons co-application of 1 mM L-Asp and 1 mM L-Glu, a total of 2 mM, produced significantly larger inward currents (404.6717.6 pA) than each agonist alone in 2 mM concentration (Fig. 4). po0.001, paired Student’s t-test.
2.4. The role of glutamate transporters in L-Asp and L-Glu interaction To investigate the possible involvement of glutamate transporters in the L-Asp and L-Glu evoked membrane responses and the observed interaction between these two agonists, we used the competitive, non-transportable blocker of excitatory amino acid transporters DL-threo-b-Benzyloxyaspartic acid (DL-TBOA) (Shimamoto et al., 1998). Bath application of 50 mM DL-TBOA produced no significant changes in membrane currents or input resistance in any of the tested neurons (n¼12). However, the peak amplitude of the inward currents evoked by 500 mM L-Asp or L-Glu were significantly increased in the presence of 50 mM DL-TBOA (Fig. 5; L-Asp cont.: 128.8756.5 pA, in DL-TBOA: 265.1777.2 pA, n¼ 12, po0.05; L-Glu cont.: 143.4751.5 pA, in DL-TBOA: 313.2790.7 pA, n¼ 12, po0.01). On the other hand, the facilitatory effect of coapplication of L-Asp (250 mM) and L-Glu (250 mM) was abolished (266.8769.1 pA) by DL-TBOA (50 mM; Fig.5).
3.
Discussion
Here we provide evidence for the first time that L-Asp can activate postsynaptic neurons in the MSt/nucl. accumbens of
young posthatch domestic chicks substantiating the recent demonstration of the occurrence of Asp in specific synaptic boutons of the chicken MSt/nucl. accumbens (Adam and Csillag 2006). In addition we provided evidence for a facilitatory interaction between L-Asp and L-Glu in the same neurons. Asp was found to be present in specific neuronal perikarya in the amygdala-equivalent region of chick brain (arcopallium, posterior amygdaloid pallium), many of which contained Asp only, while in the rest of neurons Asp colocalised with Glu (Adam and Csillag 2006). In the same study, Asp was detected in several axon terminals synapsing with medial striatal dendrites or spines, either with or without colocalising with Glu. The synapses clearly belonged to the excitatory type, displaying asymmetrical synaptic specialization. To identify Asp as a neurotransmitter in the chicken brain one has to prove that besides its presence in the synaptic vesicles it is released in a Ca2þ -dependent manner by exocytosis and activates postsynaptic receptors. Evoked synaptic release of both L-Asp and L-Glu has recently been demonstrated in the striatum of domestic chicken (Zachar et al., 2012). The chick striatum contains a population of spiny neurons as well as ‘‘aspiny neurons’’ with pallidum-like morphology and an extraordinary variety of intrinsic electrophysiological properties like burstiness, sag, inward rectification and ability to sustain firing (Farries et al., 2005). The population of the cells we recorded showed no significant difference in basic membrane properties, however, we did not test them for bursting activities or other electrophysiological characteristics. All of these cells responded to bath-applied L-Asp and L-Glu.
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Fig. 5 – Blocking of EAA-transporters increases the L-Asp or L-Glu evoked inward currents and eliminates the facilitatory interactions of co-applied L-Asp and L-Glu. (A) In the presence of TTX (0.5 lM) and DL-TBOA (50 lM) the inward currents evoked by L-Asp (500 lM) or L-Glu (500 lM) were significantly increased but no facilitating effect was seen with the coapplication of L-Asp (250 lM) and L-Glu (250 lM). (Vm¼50 mV, 6-day-old chick). (B) Summary data showing the increases of L-Asp and L-Glu evoked inward currents induced by DL-TBOA(50 lM) in the presence of TTX (0.5 lM) in chick striatal neurons. n ¼12; po0.05.
The sensitivity of these neurons was similar to both EAAs. In contrast to Glu, Asp has little, if any, affinity for AMPA receptors which mediate fast transmission at the vast majority of CNS excitatory synapses, or for metabotropic glutamate receptors as reported for cultured neurons (Patneau and Mayer 1990). However, non-NMDA activation by L-Asp has also been demonstrated in rat hippocampal slices (Brautigan and Eagles 1998). In any case, L-Asp is considered as a selective activator of NMDA receptors at least in the mammalian brain (Frauli et al., 2006; Patneau and Mayer 1990). In the present study, the potent NMDA receptor antagonist D-AP5 and the AMPA/kainate receptor antagonist CNQX had very similar antagonistic effect on the inward currents evoked by L-Asp and L-Glu in the MSt/nucl. accumbens neurons from chicken brain slices. These results indicate an identical/similar target site for both EAAs in these neurons. Blockade of EAA-transporters significantly increased both L-Asp and L-Glu evoked currents in a similar manner. This has confirmed that the inward current evoked by L-Asp could not be ascribed solely to an influence on EAA-transporters, i.e. acting through L-Glu indirectly. Co-application of L-Asp and L-Glu, however, showed a clear facilitation in the majority of the tested neurons, and this facilitation was abolished by
blocking EAA-transporters, indicating their role in the observed Asp-Glu interaction. Earlier, such facilitatory interaction between Glu and Asp has been demonstrated only in lobster neuromuscular junction (Constanti and Nistri 1978; Constanti and Nistri 1979; Shank and Freeman 1975). Although an L-Aspinduced reduction in L-Glu uptake by EAA-transporters might explain the synergistic effect of L-Asp and L-Glu in chicken striatum, other mechanisms such as partially different sites of action, which might be the extrasynaptic NR1-NR2B receptors in the case of Asp (for review see (Nadler 2011), or sensitization of postsynaptic receptors to Glu (Lombardi et al., 1993) also need further investigation.
4.
Experimental procedure
4.1.
Electrophysiology
Experiments were carried out in neurons of the striatum/ nucl. accumbens of domestic chicks (1- to 10-day-old). Animals were decapitated and transverse striatal slices (300–450 mm thick) were cut in an oxygenated (95% O2, 5%
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CO2) Krebs solution (4 1C) on a vibratome and incubated in a chamber at 3671 1C for at least 1 h. A single slice was then transferred to the recording chamber of an upright microscope (Olympus BX 51 WI) and continuously perfused with Krebs solution containing (in mM): NaCl 128.0; KCl 1.9; KH2PO4 1.2; MgSO4 1.3; CaCl2 2.4; NaHCO3 26.0; glucose 10.0, adjusted to pH 7.4 and supplemented when appropriate with 0.5–1 mM tetrodotoxin (TTX, Alomone Diagnostics) at a rate of about 3 ml/min. Cells were subsequently visually identified (Fig. 1) beneath a long-working distance water-immersion objective (Olympus XLUMPlan 20x, NA 0.95) with differential interference contrast (DIC) optics using a digital camera (F-view II). Striatal neurons within 50–100 mm of the surface of the slice were recorded in the whole-cell configuration of a patch-clamp amplifier (Axopatch 200B) at the holding potential of 60 mV using pipettes (8–12 MO) filled with a solution containing (in mM): K-Gluconate (120.0), KCl (20.0), MgCl2 (2.0), HEPES (10.0), EGTA (0.5), Mg-ATP (2.0), GTP-tris (0.5), adjusted to 7.18–7.23 pH with KOH. In some experiment 0.1% biocytin (Sigma) was added to the pipette solution in order to visualize the morphology of the recorded neurons. Liquid junction potentials were not compensated. Stimulation and acquisition of electrical signals was done using pClamp version 9 acquisition system and software. Measurements were made of resting membrane potential, input resistance (from the slope of I–V relationship), and the peak amplitude of membrane current responses to L-Asp (0.2–4 mM) or L-Glu (0.2–4 mM). Drugs were applied by bath-perfusion. All experiments were done at room temperature.
4.2.
Intracellular labeling
At the end of recording, the slices were placed in 0.1 M phosphate buffer (PBS; pH 7.3), containing 4% formaldehyde for 20 h at 4 1C. After rinsing the slices 3 times for 15 min in PBS they were placed in 3% H2O2 containing PBS to suppress endogenous peroxidase activity. Following rinses 3 times in PBS as above slices were incubated in 0.1 M PBS with 2% bovine albumin (Sigma A-2153) and Vector ABC solutions (Vector Pk-4000) for 48 h at 4 1C After rinsing thoroughly 3 times with PBS a standard diaminobenzidine (DAB) protocol was used for the final peroxidase reaction: 0.06% NiCl, 0.05% DAB, and 0.06% H2O2 staining for 20 min.
4.3.
Statistical analysis
Values are means 7S.E.M. (standard error of means). Statistical significance was evaluated by paired Student’s t-test. po0.05 was considered significant.
4.4.
Materials
The following compounds were purchased from: L-Glutamic acid monosodium salt hydrate and L-Aspartic acid sodium salt monohydrate from Sigma; D-AP5, CNQX disodium salt, DL-TBOA and Tetrodotoxin citrate from Tocris Bioscience.
Acknowledgments This work was supported by the Hungarian National Scientific Research Fund (OTKA73219).
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