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Nitric oxide-induced inhibition on striatal cells and excitation on globus pallidus neurons: a microiontophoretic study in the rat
Neuroscience Letters 343 (2003) 101–104 www.elsevier.com/locate/neulet
Nitric oxide-induced inhibition on striatal cells and excitation on globus pallidus neurons: a microiontophoretic study in the rat Pierangelo Sardo, Giuseppe Ferraro, Giuseppe Di Giovanni, Vittorio La Grutta* Dipartimento di Medicina Sperimentale – Sezione di Fisiologia umana, Universita` degli Studi di Palermo, Corso Tukory, 129-90134 Palermo, Italy Received 22 January 2003; received in revised form 10 March 2003; accepted 12 March 2003
Abstract Single units were recorded in the striatum and in the globus pallidus (GP) of urethane-anesthetized rats under microiontophoretic administration of either Nv-nitro-L -arginine methyl ester (L -NAME, inhibitor of nitric oxide synthase), or 3-morpholino-sydnoniminhydrocloride (SIN-1, nitric oxide, NO donor). A steady baseline firing of sporadically discharging striatal neurons (basal firing rate , 0.1 spikes/s) was evoked by a pulsed microiontophoretic ejection of glutamate. On striatal neurons, microiontophoretic application of SIN-1 induced a current-dependent inhibition (11/13), whereas L -NAME administration produced a clear excitation (9/9). On GP cells, the administration of SIN-1 had excitatory effects (10/15), whereas the administration of L -NAME reduced the neuronal activity (6/6). We hypothesize that NO could exert an intrinsic regulatory action on the activity of both striatal and GP cells. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basal ganglia; Striatum; Globus pallidus; Nitric oxide; Microiontophoresis
The striatum (CPN, caudate-putamen nucleus in the rat), and the globus pallidus (GP) are involved in the basal ganglia indirect pathway [10]. Through its projections, the striatum exerts a strong inhibitory action on GP neuronal activity. In turn, a small amount of GP fibers join striatal interneurons [2]. A number of neurotransmitter interactions have been evidenced to play a role in this circuit, mainly at the CPN level. Besides the action of classical neurotransmitters (e.g. glutamate, GABA, dopamine, acetylcholine), recent in vitro evidences [4] suggest a role for the nitric oxide (NO) [1,6] in the modulation of basal ganglia activity. NO is an atypical gaseous neurotransmitter, widely distributed in the rat brain and functionally coupled with glutamatergic neurotransmission [3,5,8,16]. Both striatum and GP receive glutamatergic fibers, the former from cerebral cortex and thalamus, the latter mainly from subthalamic nucleus [9,10]. Moreover, even if both structures have been recently showed to contain NOproducing interneurons [3,15], little in vivo evidence exists of a role of this neurotransmitter on either CPN or GP cells’ activity. In a preliminary study in vivo, we showed an excitatory influence exerted by the pharmacological block* Corresponding author. Tel.: þ 39-91-651-2070; fax: þ39-91-652-0701. E-mail address: [email protected] (V. La Grutta)
ade of NO synthesis on striatal neuronal activity [12]; such an action was not generalized to all cells, being restricted to neurons exhibiting a sporadic discharge activity and presumed to be the electrophysiological phenotype of striatal output neurons. Because of the systemic pharmacological approach used, the question remained open if the observed effect was due to an intrinsic striatal modulation of neuronal activity, or if it could be ascribed to a more general action of NO at different levels along the circuit. To extend these findings, in this study we directly studied the effects exerted by the microiontophoretic application of drugs modifying NO neurotransmission on the activity of striatal sporadically discharging cells. Moreover, with the aim of further exploring the action of NO neurotransmission in basal ganglia circuitry, we also studied the effects of the same drugs on GP single units, in the light of a potential action exerted by NO also in the GP context. Such a role is suggested by the presence of NO-producing interneurons in the GP, whose synaptic endings are in contact with striatal terminals [3] and could induce a modulation of CPN action on GP cells. NO neurotransmission was manipulated by using Nv-nitro-L -arginine methyl ester (L -NAME), an inhibitor of neuronal nitric oxide synthase, and 3-morpholino-sydnonimin-hydrocloride (SIN-1), a NO donor [14]. All procedures were carried out under the Italian laws on
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00350-1
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animal experimentation and the National Institute of Health Guide for the Care and Use of Laboratory Animals. Male Wistar rats (n ¼ 18; 230– 260 g) anesthetized with urethane (1.2 g/kg, intraperitoneal; Sigma Chemical Co., St Louis, MO, USA) were positioned in a stereotaxic apparatus. Body temperature (37 – 38 8C), heart rate and pupillary diameter were monitored. Five-barrel glass microelectrodes were stereotaxically [11] lowered to the CPN (7.6 – 10 mm anterior (A) to the interaural line, 3.5 –4.5 mm lateral (L) to the midline, 3 – 7.5 mm ventral (H) to the cortical surface) or to the GP (A 7.2– 7.8 mm, L 3 –3.8 mm, H 5– 7 mm). The recording barrel (1.4 – 2.0 MV) contained 2M NaCl with 1% Pontamine Sky Blue (Sigma). The side barrels contained a 2M NaCl solution (for automatic balancing), L -glutamic monosodium salt (GLU, 100 mM in 1 mM NaCl, pH 8), SIN-1 (40 mM, pH 4.5) and L -NAME (50 mM, pH 6.5), respectively. All drugs were purchased from Sigma. Retaining currents of 8 – 10 nA (positive for GLU, negative for L -NAME and SIN-1 solutions) were applied to drug barrels (20 – 70 MV). In order to exclude direct effects of the pH of drug solutions on neuronal activity, in five animals preliminary tests were performed by passing currents of the same intensity and polarity as for drugs ejection through barrels containing buffered saline solutions, with pH and concentrations identical to drug solutions. No effects were observed. Detailed recording procedures have been previously reported [12]. Briefly, electrical signals were amplified, band-pass filtered (300 – 1500 Hz), monitored by audio and video, digitally converted and discriminated (Experimenter’s Workbench Software V6.0; Datawave Technologies, Longmont, CO, USA). Discriminated waveforms were stored on a computer with their temporal markers, for off-line analysis. Variations of neuronal firing rate were detected on-line by means of a ratemeter histogram of neuronal activity. When spikes generated by a neuron were well isolated (signal to noise ratio of 3:1 or greater), the baseline activity was recorded for 10 min or more before drug administration. In CPN recordings, in order to obtain a steady control activity, sporadically discharging neurons were repetitively activated by pulsed iontophoretic administrations of GLU (20 –35 nA, 30 s pulse duration, 40 s inter-pulse interval duration) [7]. Striatal cells were tested for drugs only when GLU-induced activity was reproducible across pulses. During each drug administration (duration 22.5 min), the ejection current was increased every 2.5 min (from 20 to 100 nA, step 10 nA), so allowing two GLU pulses during each current step. In GP recordings, due to the sustained spontaneous activity of the cells, no activation procedure was necessary. For these neurons, each drug administration (20 – 100 nA, step 20 nA) lasted 60 s and followed the preceding one after complete recovery of neuronal firing. At the end of each experiment, the recording site was marked by the ejection of pontamine sky blue (20 mA current, 10 min) and the animals received an overdose of pentobarbital. After transcardial perfusion with saline, followed by 10% buffered formalin, the brain
was removed, frozen, sectioned at 50 mm and stained with cresyl violet for verification of recording sites. Off-line analyses of spike waveforms, firing rates and discharge patterns were performed for each unit. Individual ratemeter histograms (10 s bin width) were analyzed by means of a non-parametric Mann – Whitney U-test to detect any statistically significant treatment-related change in neuronal firing. Interspike interval histograms (1 ms bin width) were calculated for each unit. Inasmuch as the measured control values of neuronal activity were not normally distributed, we used the non-parametric Mann –Whitney U-test also for statistical comparisons in the studied neuronal population with the null hypothesis being rejected at a probability level (P) of less than 0.05. All results are expressed as mean ^ standard deviation. All sporadically discharging (, 0.1 spikes/s; n ¼ 13) striatal cells recorded had biphasic (2 /þ ) potentials [12,13, 17]. Unless activated by GLU pulsed ejection, these neurons could remain quiescent for many seconds or minutes, displaying very long interspike intervals and typical waveform features [12]. During control GLU pulses the mean neuronal firing was 11.56 ^ 4.08 spikes/s. For all neurons, neither the repetitive pulsed application of GLU induced a decrease in the firing rate across pulses, nor an increase of ejection current was necessary to maintain a steady control level of neuronal activity during the 10 min (or more) epoch preceding the pharmacological treatment. A representative neuronal response to drug treatments is reported in Fig. 1A. Local application of SIN-1 caused a clear, currentdependent decrease of the GLU-induced activity of most sporadically discharging striatal neurons (11/13 cells, Fig. 2A), with a maximum of inhibition at 100 nA (2 79.96 ^ 15.14%, P , 0:0001). At the end of drug ejection, the GLU-induced activity suddenly returned to control levels. Conversely, iontophoretic application of L NAME induced a current-dependent increase in GLUevoked firing of all sporadically discharging striatal neurons tested (9/9 cells, Fig. 2A). Also this effect reached a maximal intensity at currents of 100 nA (þ 53.21 ^ 32.53%, P , 0:05). In GP recordings, all neurons (n ¼ 15) exhibited spontaneous, sustained discharge activity, with a mean firing rate of 18.55 ^ 6.69 spikes/s and a symmetric unimodal distribution of interspike intervals. Recorded impulses had diphasic (2 /þ ) waveforms, a mean total duration of 1.30 ^ 0.17 ms and a mean peak to peak amplitude of 1.94 ^ 0.68 mV. A representative neuronal response to drug treatments is reported in Fig. 1B. The local administration of SIN-1 caused a currentdependent, statistically significant increase of the mean firing rate of most GP neurons (10/15 cells, Fig. 2B) with a peak at 100 nA (þ 70.00 ^ 43.80%, P , 0:05). Six out of the responding neurones also received the iontophoretic administration of L -NAME. In all cells a current-dependent, statistically significant decrease of the mean firing rate was observed (Fig. 2B) with a maximal response at 100 nA (2 61.95 ^ 25.84%, P , 0:01).
P. Sardo et al. / Neuroscience Letters 343 (2003) 101–104
Fig. 1. Ratemeter histograms (bin width 10 s) illustrating the effects of microiontophoretic application (20–100 nA, step 10 nA) of SIN-1 and L NAME on the activity of representative CPN (A); and GP (B) neurons. In A, GLU pulses (30 nA) lasted 30 s, with a 40 s inter-pulse interval.
Our present data extend previous observations about the role exerted by NO on the activity of striatal neurons in the rat; in fact, in a recent paper we described that sporadically discharging neurons are significantly excited following the systemic administration of an inhibitor of the NO synthase (7-nitro-indazole), suggesting that NO neurotransmission exerts a tonic depression upon sporadically discharging neurons [12]. This effect could depend on the release of NO by interneurons intrinsic to the striatum [4,9], but the systemic administration of the drug did not allow us to make this statement conclusively. In the present study, the results show a current-dependent decrease in GLU-induced excitation of most sporadically discharging neurons under SIN-1 administration and, conversely, a clear and reproducible excitation of the cells studied under L -NAME administration. Thus, the data shows that variations of the intrinsic NO levels can induce marked changes in the firing rate of striatal sporadically discharging neurons. In the light of the general agreement about a projection role for these neurons [17], an intrinsic striatal nitrergic activity, potentially depressing the activity of striatal output cells, can be
103
Fig. 2. Responses of all studied striatal (A); and GP (B) neurons to microiontophoretic administration (100 nA) of SIN-1 and L -NAME. Each point (triangle or circle) represents the baseline mean firing rate of a neuron plotted against the mean firing rate of the same cell during the ejection of SIN-1 (triangles) or L -NAME (circles). The points representing the responses of the same neuron, when separately tested for the two drugs, are connected by dashed lines. Unmatched triangles represent responses to SIN-1 administration of neurons which were not subsequently tested for L NAME. Points positioned on the straight line indicates neurons not responding to the pharmacological treatment.
hypothesized. Such a modulatory action could be either phasic or tonic in nature. In fact, on one side, the report of a direct influence of striatal NO levels in modulating the cortical hyperactivity-induced long-term depression of striatal medium spiny neurons [4] highlights a phasic role of NO. On the other side, our data suggest a further modality of intervention of the NO on the activity of striatal output neurons, based on a tonic control of the firing activity: in fact, even if in our experiments the activity of striatal neurons was enhanced by means of a pulsed GLU ejection, potentially mimicking the role of cortical drive in depressing the neuronal activity, we never observed a decrease in the extracellular firing rate over prolonged periods. On this basis, the observed effects could express the existence of a tonic striatal NO level regulating the output
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activity, potentially acting through the increase of GABAergic neurotransmission at both pre- and post-synaptic level [1]. Moreover, our data suggest the existence of a tonic excitatory role exerted by NO upon the activity of GP neurones. In fact, the microiontophoretic application of L NAME induced a decrease in neuronal firing rate, whereas the local administration of SIN-1 induced a current dependent increase of the firing activity of GP neurones. The possible intrinsic nature of such a modulatory action is supported by the existence of local NO-producing interneurones in the GP, whose synaptic pattern suggest a role in modulating output cells’ activity; in fact, their synaptic terminals are anatomically close to both striatal inhibitory and subthalamic excitatory endings [3]. In the light of our results, the suggested modulatory role of NO could be interpreted as promoting the discharge activity of GP cells. In conclusion, our data suggest a role of NO in controlling the level of activity of both striatal and GP neurons. Interestingly, the microiontophoretic application of the same drugs induced opposite effects on the cells belonging to these structures: in fact, striatal neurons were depressed by local NO increased levels, whereas GP units were excited by the same conditions. These observations suggest a role exerted by NO neurotransmission on the functional status of the basal ganglia indirect pathway, potentially able to disentangle the activity of GP neurons from the inhibitory control exerted by striatal output cells.
[3]
[4]
[5]
[6] [7]
[8]
[9]
[10]
[11] [12]
[13]
Acknowledgements This work was supported by grants from the Italian Ministry for University, Scientific, and Technological Research.
[14]
[15]
References [16] [1] J.S. Bains, A.V. Ferguson, Nitric oxide regulates NMDA-driven GABAergic inputs to type I neurons of rat paraventricular nucleus, J. Physiol. 499 (1997) 733 –746. [2] M.D. Bevan, P.A.C. Booth, S.A. Eaton, J.P. Bolam, Selective
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innervation of neostriatal interneurons by a subclass of neuron in the globus pallidus of the rat, J. Neurosci. 18 (1998) 9438–9452. E.A. Bolan, K.N. Gracy, J. Chan, R.R. Trifiletti, V.M. Pickel, Ultrastructural localization of nitrityrosine within the caudateputamen nucleus and the globus pallidus of normal rat brain, J. Neurosci. 20 (2000) 4798–4808. P. Calabresi, P. Gubellini, D. Centonze, G. Sancesario, M. Morello, M. Giorgi, A. Pisani, G. Bernardi, A critical role of the nitric oxide/ cGMP pathway in corticostriatal long-term depression, J. Neurosci. 19 (1999) 2489–2499. J. De Vente, D.A. Hopkins, M. Markerink-Van Ittersum, P.C. Emson, H.H.H.W. Schmidt, H.W.M. Steinbusch, Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain, Neuroscience 87 (1998) 207 –241. J. Garthwaite, Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci. 14 (1991) 60–67. X.T. Hu, F.J. White, Dopamine enhances glutamate-induced excitation of rat striatal neurons by cooperative activation of D1 and D2 class receptors, Neurosci. Lett. 224 (1997) 61 –65. K. Iwase, K. Iyama, K. Akagi, S. Yano, K. Fukunaga, E. Miyamoto, M. Mori, M. Takiguchi, Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization, Mol. Brain Res. 53 (1998) 1– 12. A. Parent, L. Hazrati, Functional anatomy of the basal ganglia. I. The cortico-basal ganglia-thalamo-cortical loop, Brain Res. Rev. 20 (1995) 91– 127. A. Parent, L. Hazrati, Functional anatomy of the basal ganglia. II. The place of subthalamic nucleus and external pallidum in the basal ganglia circuitry, Brain Res. Rev. 20 (1995) 128–154. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1998. P. Sardo, G. Ferraro, G. Di Giovanni, S. Galati, V. La Grutta, Inhibition of nitric oxyde synthase influences the activity of striatal neurons in the rat, Neurosci. Lett. 325 (2002) 179–182. L.R. Skirboll, B.S. Bunney, The effects of acute and chronic haloperidol treatment on spontaneously firing neurons in the caudate nucleus of the rat, Life Sci. 25 (1979) 1419– 1434. E. Southam, J. Garthwaite, Comparative effects of some nitric oxide donors on cyclic GMP levels in rat cerebellar slices, Neurosci. Lett. 130 (1991) 107–111. S.R. Vincent, O. Johansson, T. Hokfelt, L. Skirboll, R.P. Elde, L. Terenius, J. Kimmel, M. Goldstein, NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities, J. Comp. Neurol. 217 (1983) 252 –263. S.R. Vincent, H. Kimura, Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience 46 (1992) 755–784. C.J. Wilson, The generation of natural firing patterns in neostriatal neurons, in: G.W. Arbuthnott, P.C. Emson (Eds.), Progress in Brain Research, 99, Elsevier, Amsterdam, 1993, pp. 277–297.
Nitric oxide-induced inhibition on striatal cells and excitation on globus pallidus neurons: a microiontophoretic study in the rat Pierangelo Sardo, Giuseppe Ferraro, Giuseppe Di Giovanni, Vittorio La Grutta* Dipartimento di Medicina Sperimentale – Sezione di Fisiologia umana, Universita` degli Studi di Palermo, Corso Tukory, 129-90134 Palermo, Italy Received 22 January 2003; received in revised form 10 March 2003; accepted 12 March 2003
Abstract Single units were recorded in the striatum and in the globus pallidus (GP) of urethane-anesthetized rats under microiontophoretic administration of either Nv-nitro-L -arginine methyl ester (L -NAME, inhibitor of nitric oxide synthase), or 3-morpholino-sydnoniminhydrocloride (SIN-1, nitric oxide, NO donor). A steady baseline firing of sporadically discharging striatal neurons (basal firing rate , 0.1 spikes/s) was evoked by a pulsed microiontophoretic ejection of glutamate. On striatal neurons, microiontophoretic application of SIN-1 induced a current-dependent inhibition (11/13), whereas L -NAME administration produced a clear excitation (9/9). On GP cells, the administration of SIN-1 had excitatory effects (10/15), whereas the administration of L -NAME reduced the neuronal activity (6/6). We hypothesize that NO could exert an intrinsic regulatory action on the activity of both striatal and GP cells. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basal ganglia; Striatum; Globus pallidus; Nitric oxide; Microiontophoresis
The striatum (CPN, caudate-putamen nucleus in the rat), and the globus pallidus (GP) are involved in the basal ganglia indirect pathway [10]. Through its projections, the striatum exerts a strong inhibitory action on GP neuronal activity. In turn, a small amount of GP fibers join striatal interneurons [2]. A number of neurotransmitter interactions have been evidenced to play a role in this circuit, mainly at the CPN level. Besides the action of classical neurotransmitters (e.g. glutamate, GABA, dopamine, acetylcholine), recent in vitro evidences [4] suggest a role for the nitric oxide (NO) [1,6] in the modulation of basal ganglia activity. NO is an atypical gaseous neurotransmitter, widely distributed in the rat brain and functionally coupled with glutamatergic neurotransmission [3,5,8,16]. Both striatum and GP receive glutamatergic fibers, the former from cerebral cortex and thalamus, the latter mainly from subthalamic nucleus [9,10]. Moreover, even if both structures have been recently showed to contain NOproducing interneurons [3,15], little in vivo evidence exists of a role of this neurotransmitter on either CPN or GP cells’ activity. In a preliminary study in vivo, we showed an excitatory influence exerted by the pharmacological block* Corresponding author. Tel.: þ 39-91-651-2070; fax: þ39-91-652-0701. E-mail address: [email protected] (V. La Grutta)
ade of NO synthesis on striatal neuronal activity [12]; such an action was not generalized to all cells, being restricted to neurons exhibiting a sporadic discharge activity and presumed to be the electrophysiological phenotype of striatal output neurons. Because of the systemic pharmacological approach used, the question remained open if the observed effect was due to an intrinsic striatal modulation of neuronal activity, or if it could be ascribed to a more general action of NO at different levels along the circuit. To extend these findings, in this study we directly studied the effects exerted by the microiontophoretic application of drugs modifying NO neurotransmission on the activity of striatal sporadically discharging cells. Moreover, with the aim of further exploring the action of NO neurotransmission in basal ganglia circuitry, we also studied the effects of the same drugs on GP single units, in the light of a potential action exerted by NO also in the GP context. Such a role is suggested by the presence of NO-producing interneurons in the GP, whose synaptic endings are in contact with striatal terminals [3] and could induce a modulation of CPN action on GP cells. NO neurotransmission was manipulated by using Nv-nitro-L -arginine methyl ester (L -NAME), an inhibitor of neuronal nitric oxide synthase, and 3-morpholino-sydnonimin-hydrocloride (SIN-1), a NO donor [14]. All procedures were carried out under the Italian laws on
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00350-1
102
P. Sardo et al. / Neuroscience Letters 343 (2003) 101–104
animal experimentation and the National Institute of Health Guide for the Care and Use of Laboratory Animals. Male Wistar rats (n ¼ 18; 230– 260 g) anesthetized with urethane (1.2 g/kg, intraperitoneal; Sigma Chemical Co., St Louis, MO, USA) were positioned in a stereotaxic apparatus. Body temperature (37 – 38 8C), heart rate and pupillary diameter were monitored. Five-barrel glass microelectrodes were stereotaxically [11] lowered to the CPN (7.6 – 10 mm anterior (A) to the interaural line, 3.5 –4.5 mm lateral (L) to the midline, 3 – 7.5 mm ventral (H) to the cortical surface) or to the GP (A 7.2– 7.8 mm, L 3 –3.8 mm, H 5– 7 mm). The recording barrel (1.4 – 2.0 MV) contained 2M NaCl with 1% Pontamine Sky Blue (Sigma). The side barrels contained a 2M NaCl solution (for automatic balancing), L -glutamic monosodium salt (GLU, 100 mM in 1 mM NaCl, pH 8), SIN-1 (40 mM, pH 4.5) and L -NAME (50 mM, pH 6.5), respectively. All drugs were purchased from Sigma. Retaining currents of 8 – 10 nA (positive for GLU, negative for L -NAME and SIN-1 solutions) were applied to drug barrels (20 – 70 MV). In order to exclude direct effects of the pH of drug solutions on neuronal activity, in five animals preliminary tests were performed by passing currents of the same intensity and polarity as for drugs ejection through barrels containing buffered saline solutions, with pH and concentrations identical to drug solutions. No effects were observed. Detailed recording procedures have been previously reported [12]. Briefly, electrical signals were amplified, band-pass filtered (300 – 1500 Hz), monitored by audio and video, digitally converted and discriminated (Experimenter’s Workbench Software V6.0; Datawave Technologies, Longmont, CO, USA). Discriminated waveforms were stored on a computer with their temporal markers, for off-line analysis. Variations of neuronal firing rate were detected on-line by means of a ratemeter histogram of neuronal activity. When spikes generated by a neuron were well isolated (signal to noise ratio of 3:1 or greater), the baseline activity was recorded for 10 min or more before drug administration. In CPN recordings, in order to obtain a steady control activity, sporadically discharging neurons were repetitively activated by pulsed iontophoretic administrations of GLU (20 –35 nA, 30 s pulse duration, 40 s inter-pulse interval duration) [7]. Striatal cells were tested for drugs only when GLU-induced activity was reproducible across pulses. During each drug administration (duration 22.5 min), the ejection current was increased every 2.5 min (from 20 to 100 nA, step 10 nA), so allowing two GLU pulses during each current step. In GP recordings, due to the sustained spontaneous activity of the cells, no activation procedure was necessary. For these neurons, each drug administration (20 – 100 nA, step 20 nA) lasted 60 s and followed the preceding one after complete recovery of neuronal firing. At the end of each experiment, the recording site was marked by the ejection of pontamine sky blue (20 mA current, 10 min) and the animals received an overdose of pentobarbital. After transcardial perfusion with saline, followed by 10% buffered formalin, the brain
was removed, frozen, sectioned at 50 mm and stained with cresyl violet for verification of recording sites. Off-line analyses of spike waveforms, firing rates and discharge patterns were performed for each unit. Individual ratemeter histograms (10 s bin width) were analyzed by means of a non-parametric Mann – Whitney U-test to detect any statistically significant treatment-related change in neuronal firing. Interspike interval histograms (1 ms bin width) were calculated for each unit. Inasmuch as the measured control values of neuronal activity were not normally distributed, we used the non-parametric Mann –Whitney U-test also for statistical comparisons in the studied neuronal population with the null hypothesis being rejected at a probability level (P) of less than 0.05. All results are expressed as mean ^ standard deviation. All sporadically discharging (, 0.1 spikes/s; n ¼ 13) striatal cells recorded had biphasic (2 /þ ) potentials [12,13, 17]. Unless activated by GLU pulsed ejection, these neurons could remain quiescent for many seconds or minutes, displaying very long interspike intervals and typical waveform features [12]. During control GLU pulses the mean neuronal firing was 11.56 ^ 4.08 spikes/s. For all neurons, neither the repetitive pulsed application of GLU induced a decrease in the firing rate across pulses, nor an increase of ejection current was necessary to maintain a steady control level of neuronal activity during the 10 min (or more) epoch preceding the pharmacological treatment. A representative neuronal response to drug treatments is reported in Fig. 1A. Local application of SIN-1 caused a clear, currentdependent decrease of the GLU-induced activity of most sporadically discharging striatal neurons (11/13 cells, Fig. 2A), with a maximum of inhibition at 100 nA (2 79.96 ^ 15.14%, P , 0:0001). At the end of drug ejection, the GLU-induced activity suddenly returned to control levels. Conversely, iontophoretic application of L NAME induced a current-dependent increase in GLUevoked firing of all sporadically discharging striatal neurons tested (9/9 cells, Fig. 2A). Also this effect reached a maximal intensity at currents of 100 nA (þ 53.21 ^ 32.53%, P , 0:05). In GP recordings, all neurons (n ¼ 15) exhibited spontaneous, sustained discharge activity, with a mean firing rate of 18.55 ^ 6.69 spikes/s and a symmetric unimodal distribution of interspike intervals. Recorded impulses had diphasic (2 /þ ) waveforms, a mean total duration of 1.30 ^ 0.17 ms and a mean peak to peak amplitude of 1.94 ^ 0.68 mV. A representative neuronal response to drug treatments is reported in Fig. 1B. The local administration of SIN-1 caused a currentdependent, statistically significant increase of the mean firing rate of most GP neurons (10/15 cells, Fig. 2B) with a peak at 100 nA (þ 70.00 ^ 43.80%, P , 0:05). Six out of the responding neurones also received the iontophoretic administration of L -NAME. In all cells a current-dependent, statistically significant decrease of the mean firing rate was observed (Fig. 2B) with a maximal response at 100 nA (2 61.95 ^ 25.84%, P , 0:01).
P. Sardo et al. / Neuroscience Letters 343 (2003) 101–104
Fig. 1. Ratemeter histograms (bin width 10 s) illustrating the effects of microiontophoretic application (20–100 nA, step 10 nA) of SIN-1 and L NAME on the activity of representative CPN (A); and GP (B) neurons. In A, GLU pulses (30 nA) lasted 30 s, with a 40 s inter-pulse interval.
Our present data extend previous observations about the role exerted by NO on the activity of striatal neurons in the rat; in fact, in a recent paper we described that sporadically discharging neurons are significantly excited following the systemic administration of an inhibitor of the NO synthase (7-nitro-indazole), suggesting that NO neurotransmission exerts a tonic depression upon sporadically discharging neurons [12]. This effect could depend on the release of NO by interneurons intrinsic to the striatum [4,9], but the systemic administration of the drug did not allow us to make this statement conclusively. In the present study, the results show a current-dependent decrease in GLU-induced excitation of most sporadically discharging neurons under SIN-1 administration and, conversely, a clear and reproducible excitation of the cells studied under L -NAME administration. Thus, the data shows that variations of the intrinsic NO levels can induce marked changes in the firing rate of striatal sporadically discharging neurons. In the light of the general agreement about a projection role for these neurons [17], an intrinsic striatal nitrergic activity, potentially depressing the activity of striatal output cells, can be
103
Fig. 2. Responses of all studied striatal (A); and GP (B) neurons to microiontophoretic administration (100 nA) of SIN-1 and L -NAME. Each point (triangle or circle) represents the baseline mean firing rate of a neuron plotted against the mean firing rate of the same cell during the ejection of SIN-1 (triangles) or L -NAME (circles). The points representing the responses of the same neuron, when separately tested for the two drugs, are connected by dashed lines. Unmatched triangles represent responses to SIN-1 administration of neurons which were not subsequently tested for L NAME. Points positioned on the straight line indicates neurons not responding to the pharmacological treatment.
hypothesized. Such a modulatory action could be either phasic or tonic in nature. In fact, on one side, the report of a direct influence of striatal NO levels in modulating the cortical hyperactivity-induced long-term depression of striatal medium spiny neurons [4] highlights a phasic role of NO. On the other side, our data suggest a further modality of intervention of the NO on the activity of striatal output neurons, based on a tonic control of the firing activity: in fact, even if in our experiments the activity of striatal neurons was enhanced by means of a pulsed GLU ejection, potentially mimicking the role of cortical drive in depressing the neuronal activity, we never observed a decrease in the extracellular firing rate over prolonged periods. On this basis, the observed effects could express the existence of a tonic striatal NO level regulating the output
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activity, potentially acting through the increase of GABAergic neurotransmission at both pre- and post-synaptic level [1]. Moreover, our data suggest the existence of a tonic excitatory role exerted by NO upon the activity of GP neurones. In fact, the microiontophoretic application of L NAME induced a decrease in neuronal firing rate, whereas the local administration of SIN-1 induced a current dependent increase of the firing activity of GP neurones. The possible intrinsic nature of such a modulatory action is supported by the existence of local NO-producing interneurones in the GP, whose synaptic pattern suggest a role in modulating output cells’ activity; in fact, their synaptic terminals are anatomically close to both striatal inhibitory and subthalamic excitatory endings [3]. In the light of our results, the suggested modulatory role of NO could be interpreted as promoting the discharge activity of GP cells. In conclusion, our data suggest a role of NO in controlling the level of activity of both striatal and GP neurons. Interestingly, the microiontophoretic application of the same drugs induced opposite effects on the cells belonging to these structures: in fact, striatal neurons were depressed by local NO increased levels, whereas GP units were excited by the same conditions. These observations suggest a role exerted by NO neurotransmission on the functional status of the basal ganglia indirect pathway, potentially able to disentangle the activity of GP neurons from the inhibitory control exerted by striatal output cells.
[3]
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Acknowledgements This work was supported by grants from the Italian Ministry for University, Scientific, and Technological Research.
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