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Neurotensin induces an inward current in rat mesencephalic dopaminergic neurons
192
Neuro.wience: Letlct~s. ',53 ( i99_'1j i 92 i 9t~ .c) 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved ()304-3940/93/$ (t6.0t')
NSL 09456
Neurotensin induces an inward current in rat mesencephalic dopaminergic neurons Nicola B. Mercuri a, Francesca Stratta a, Paolo Calabresi a and Giorgio Bernardi a'b ~Clinica Neurologica Dipartimento Sanith Pubblica, Universit~ di Roma Tor Vergata, Rome (Italy) and bIRCCS, Clinica S. Lucia, Rome (Italy) (Received 23 October 1992; Revised version received 8 January 1993; Accepted 18 January 1993)
Key words: Substantia nigra; Ventral tegmental area; Electrophysiological recording; Current- and voltage-clamp Neurotensin (0.3-3/aM) depolarized the membrane and increased the firing discharge of dopaminergic ceils in slices of the rat mesencephalon. Under voltage-clamp, at holding potentials from -50 to -60 mV (near the resting membrane potential), neurotensin produced a sustained inward shift in the holding current. This inward current was reduced with the hyperpolarization of the membrane to -125 mV. It was resistant to tetrodotoxin, but it was diminished following the perfusion with low sodium (choline chloride substitution) solution, tt persisted in low calcium [0-0.5 mM). Changes in the intraceltular concentration of chloride did not affect neurotensin-induced current. The neurotensin-induced inward current did not reverse at hyperpolafized potentials in 10.5 mM cxtraeellular K ÷. It was also seen in the presence of the potassium channel blockers tetraethylamm onium (10-20 raM), barium (1 m M), apamine (1 p M ) and 4-aminopyridine (1-1.5 mM). Also the extracellular application of cesium (1-5 raM) had no effect on the cellular responsiveness to neurotensin. The action of neurotensin appears to be mediated, at least partially, by a TTX-insensitive but voltage-dependent inward current carried by sodium. The non-dopaminergic cells of the substantia nigra and ventral tegmental area were not affected by neurotensin.
Neurotensin is a tridecapeptide colocalized with dopamine in the dopaminergic neurons of the mesencephalon [16]. It is released in a CaZ+-dependent manner in the brain [5]. Local application of neurotensin in the substantia nigra and ventral tegmental area has been shown to increase the release of dopamine in these structures and in the striatum, globus pallidus and nucleus accumbens [1, 8, 12, 13]. Furthermore, receptors for neurotensin are present on the soma, dendrites and terminals of the dopaminergic cells [14, 18]. In agreement with the presence of neurotensin receptor localization on dopaminergic cells, it has been reported that the density of neurotensin binding sites is reduced in the brain of parkinsonian patients [20]. This paper reports the results of intracellular electrophysiological experiments performed in current- and voltage-clamp modes which attempt to identify the membrane properties responsible for the excitatory effects of neurotensin on dopaminergic neurons of the rat substantia nigra pars compacta and the ventral tegmental area (VTA). We conclude that neurotensin acts directly on Correspondence. N.B. Mercuri, Clinica Neurologica, Dipartimento Sanita' Pubblica, Universita' di Roma Tor Vergata, Via O. Raimondo 8, 00173 Rome, Italy. Fax: (39) (6) 7233063.
dopamine neurons to increase a voltage-dependent inward current. Slices of the ventral mesencephalon obtained from albino Wistar rats (150-350 g) were prepared as described previously [9], transferred to a recording chamber, and continuously superfused at a rate of 2.5 ml,min-1, with a solution maintained at 35°C and equilibrated with 95% 0 2 / 5 % CO2. The standard solution contained (mM): NaC1 126, KCI 2.5, NaH2PO 4 1.2, MgC12 1.2, CaC12 2.4, glucose 10, NaHCO 3 26. Intracellular recording electrodes were filled with 2 M KC1 or 2 M potassium acetate and had a resistance of 30-100 MO. A single electrode voltage-clamp amplifier was used (Axoclamp 2A) for both voltage and current recordings. Numerical data were expressed as mean _+ S.E.M. Data were obtained from 21 neurons of the ventral tegmental area and 46 cells of the substantia nigra pars compacta. The majority of these cells (43/67) fired action potentials spontaneously in vitro at a rate ranging between 0.2 and 9 Hz. They showed a pronounced voltage and time dependent inward rectification with the hyperpolarization of the membrane and responded to dopamine application (10-30 gM, n=22/22) with a hyperpolarization or an outward current. These properties are characteristics of the 'principal' cells [3, 6, 7, 9, 11] which
193
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Fig. 1. Effects of neurotensin on dopamine cells. A: the chart record shows the excitatory effects produced by 300 nM neurotensin, applied for the period indicated by the bar, on a substantia nigra zona compacta cell. In order to show individual action potentials, the speed of the chart recorder was changed (see the time bars below). The full amplitude of the action potentials is not reproduced due to the low frequency of the pen recorder. B: the application of neurotensin on a different neuron produced an inward current. Time calibration in A (1 min) is also valid for B. C: averages (3 runs) of hyperpolarizing current pulses (4 s; 10, 20, 25 mV) before, during and after neurotensin (1 ktM) application on a different substantia nigra dopaminergic cell. Note the inward shift of the holding current and the reversible decrease in the apparent input conductance caused by neurotensin. Holding potential -62 mV.
contain dopamine. No differences in the electrical properties and pharmacological responses were seen in VTA and compacta neurons; therefore, data from these neurons were pooled. Neurotensin (0.3-3 /IM) increased the spontaneous firing rate of the dopaminergic cells without altering the regular discharge pattern, which was due to a reversible depolarization of the cell membrane (Fig. 1A). When hyperpolarizing current was injected to prevent firing, neurotensin (1 ¢tM) caused a depolarization of 4-14 mV (n=22). The amplitude and the duration of the afterhyperpolarization following the action potential decreased during the neurotensin-induced depolarization. How-
ever, repolarizing the cell to the initial level by intracellular current injection restored the original shape of the afterpotentials. Under voltage-clamp (holding potential from - 5 0 to - 6 0 mV), neurotensin produced a slow and sustained inward current which underlies the depolarization (Fig. 1B). The amplitude of this current was concentration-dependent, averaging 3 4 + 8 pA (n=15) and 70 + 6 pA (n=36) with 0.3 and 1/IM neurotensin, respectively. This effect ofneurotensin occurred within 1 2 min from the start of the superfusion, and peaked in 2 4 min. The recovery was observed 7 30 min after washout. The cellular responses to neurotensin could be evoked repeatedly on the same cell, but in several cases they decreased in amplitude with the successive applications of the peptide and did not recover even after washing of the cell for more than 1 h. Neurotensin (1/~M) decreased the apparent input conductance in 12 out of 17 cells by about 24 + 6% of control (Fig. 1C). In the remaining 5 neurons, no changes were observed. Current voltage relations were constructed under voltage-clamp by measuring membrane currents in control conditions and during the application of neurotensin. Neurotensin caused in some neurons (6 out of 10) a voltage-dependent decrease of the slope conductance particularly evident in the range of holding potentials from -55 to -75 mV. The response was inward in all tested potentials (between -40 and -125 mV) (7 out of 7 cells). The magnitude of the inward current decreased with the hyperpolarization of the membrane, (Fig. 2) but it did not reverse even al -125 mV. In order to investigate the ionic nature of this current different tests were conducted. The neurotensin-induced response was not blocked by TTX (1 ~M) (not shown). The inward current was greatly depressed when normal solution was exchanged with one containing only 27 51% of control sodium by choline substitution (n=8) (Fig. 3A). We were unable to obtain reliable I V plots of the neurotensin-induced current during perfusion with low sodium, because the current produced by the peptide in low sodium was very small. The effect of neurotensin persisted in solution with 0-0.5 mM calcium containing 15-20 mM magnesium (4 cells) (Fig. 3B), but it was irreversibly reduced after a persistent exposure to low calcium for longer than 8 min and also after the addition of cobalt (1-2 mM in the medium (3 cells)). The increase of extracellular potassium from 2.5 to 10.5 mM did not modify the polarity of the neurotensin-induced current which was inward over the range of potentials examined. The amplitude of the inward current induced by 1 /~M neurotensin in 10.5 mM extracellular potassium at - 5 0 to - 6 0 mV was of 70 + 9 pA, n=4. The application of TEA (10-20 mM, 4 cells) and barium ( 1 raM, 3 cells) did not significantly (P > 0.05, Student's t-test) affect the responses to neurotensin. Apamin (1 /IM, 2 ceils) or
194
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Fig. 2. (I V relationship for the response of dopaminergic cells to neurotensin. A: current-voltage plots obtained before and during the application of neurotensin (1 ~M). The plots were constructed by depolarizing the neurons from hyperpolarized regions of the membrane potential at a rate of 1 mV-s-J while measuring membrane currents. Neurotensin caused an inward current throughout the voltage range. Note that the extracellular potassium concentration was 2.5 mM. B: voltage sensitivity of the neurotensin-induced inward current. Data were obtained from 6 dopaminergic cells. Note that the amplitude of the inward current increased at depolarized and decreased at hyperpolarized potentials.
4-aminopyridine (4-AR 1.5-3 mM, 2 cells) did not depress the neurotensin-induced responses (not shown). To eliminate the time- and voltage-dependent inward rectification of the membrane, extracellular cesium (1-5 mM) was perfused on the cells. Under this condition, neurotensin still produced an inward current. When electrodes filled with potassium acetate were used, muscimol, a GABAA agonist which opens C1- channels, produced an outward current while neurotensin caused an inward current (not shown). The actions ofneurotensin were not affected by (-)-sulpiride (1 ¢tM), a dopamine D2 antagonist and scopolamine (3/IM), a muscarinic antagonist. We also recorded from 4 and 6 non-dopaminergic cells in
the ventral tegmental area and in the substantia nigra, respectively [3, 6, 7]. Neurotensin (1 /2M) had no effect on these cells (not shown). Using intracellular recordings in current- and voltageclamp modes we have shown that neurotensin depolarizes the dopaminergic neurons of the rat mesencephalon, while the non-dopaminergic cells are not affected by the peptide. This confirms previous electrophysiotogical experiments with extracellular and intracellular recordings (see ref. 17 for a review), describing an excitatory effect of neurotensin only on the dopamine-containing cells. Since the effect of neurotensin persisted in media containing tetrodotoxin and with external calcium concentration of 0-0.5 mM plus 15 20 mM magnesium, the peptide acts postsynaptically and its effect is probably neither dependent on calcium nor on TTX-sensitive sodium channels. Accordingly, Szigethy and Beaudet [18] have already demonstrated with autoradiographic and immunohistochemical techniques that the neurotensin binding sites in the ventral mesencephalon are only located on the dopaminergic cells. The voltage-clamp results show that neurotensin produces an activation of a sustained and voltage-dependent inward current accompanied by a decrease in slope conductance. Although the reversal potential for the neurotensin-induced current could not be determined, experiments using channel blockers and ionic substitution suggest that the inward current may be carried by sodium. An increased permeability to ion channels is also suggested by the increased noise of the current trace, during the effect of neurotensin (Fig. 1B). An alternative explanation for the Na + dependance of the neurotensin-induced response is that the lowering of the extracellular sodium concentration may change the affinity for neurotensin receptors, or might interfere with the intracellutar processes that are activated by the neuropeptide. The neurotensin-induced current was not dependent on chloride because changes in the intracellular concentrations of CI- ions, obtained through KC1 or potassium acetatefilled electrodes, did not cause any change in amplitude or polarity of the response. An influx of calcium ions does not play a significant role in the neurotensin-induced current as demonstrated by lowering extracellular calcium and adding magnesium. The diminishing effect of prolonged exposure to low calcium may indicate that intracellular calcium is important in the response to neurotensin. Alternatively, the blocking effect of cobalt could be due to a non-selective depressant action of this cation [4]. A major effect of neurotensin on classical potassium conductances is discounted, because the inward response did not become outward at the potassium equilibrium potential and also because the neurotensin-induced response was not
195
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N e u r o t e n s i n (1 .uM)
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2 min Fig. 3. Effects of lowering external sodium and calcium. A: the inward current produced by neurotensin decreased in amplitude when external NaCI was replaced by choline chloride. Holding potential -58 mV. B: the cellular response to neurotensin was attenuated but not abolished in calcium free plus 20 mM extracellular MgCI 2. Holding potential -57 inV.
greatly depressed during the pharmacological blockade of potassium channels. The neurotensin-induced current that we describe here is reminiscent of that produced by cholinergic agonists in lobster cardiac ganglion neurons [2]. Furthermore, the sodium-dependent and voltagegated inward current activated by neurotensin resembles the one recently described for vasopressin on facial motoneurons [15]. The amino acid sequence of the neurotensin receptor has been recently determined [19] and it is presently unclear what signaling pathway is involved in the excitatory effect of neurotensin on dopaminergic cells. The relatively slow time course of the effect supports the involvement of a second messenger system. The decrease in amplitude of the neurotensin-induced response with repeated applications could be due to receptor desensitization or to receptor uncoupling with intracellular mechanisms. In conclusion, the present electrophysiological data support the hypothesis that the interplay of dopamine inhibition [9] with neurotensin and glutamate [10, 11] excitation may tune the firing of the dopamine-containing cells during different states of neuronal activity. We thank G. Gattoni and M. Tolu for their excellent technical assistance.
1 Faggin, B.M., Zubieta, J.K., Rezvani, A.H. and Cubeddu, L.X., Neurotensin-induced dopamine release in vivo and in vitro from substantia nigra and nucleus caudate, J. Pharmacol. Exp. Then, 252 (1990) 817-825. 2 Frescbi, J.E. and Livengood, D.R., Membrane current underlying muscarinic cholinergic excitation of motoneurons in lobster cardiac ganglion, J. Neurophysiol., 62 (1989) 984~ 995. 3 Grace, A.A. and Onn, S.P., Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. J. Neurosci., 9 (1989) 3463 3481. 4 Gerber, U. and Gahwiler, B.H., Cobalt blocks postsynaptic responses induced by neurotransmitters in the hippocampus in vitro, Neurosci. Lett., 134 (1991) 53 56. 5 lversen, L.L., Iversen, S.D., Bloom, F., Douglas, C., Brown, M. and Vale, W., Calcium-dependent release of somatostatin and neurotensin from rat brain 'in vitro', Nature, 273 (1978) 161 163. 6 Johnson, S.W. and North, R.A., Two types of neurone in the rat ventral tegmental area and their synaptic inputs, J. Physiol., 450 (1992) 455M,68. 7 Lacey, M.G., Mercuri, N.B. and North, R.A., Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids, J. Neurosci., 9 (1989) 1233-1241. 8 Laitinen, K,, Crawley, J.N., Mefford, I.N. and De Witte, P., Neurotensin and cholecystokinin microinjected into the ventral tegmental area modulate microdyalisate concentrations of dopamine and metabolites in the posterior nucleus accumbens, Brain Res., 523 (1990) 342 346. 9 Mercuri, N.B., Calabresi, R and Bernardi, G.. The mechanism of
196
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11
12
13
14
15
amphetamine-induced inhibition of rat substantia nigra compacta neurones investigated with intracellular recording in vitro. Br. J. Pharmacol., 98 (1989) 127-134. Mercuri, N.B., Stratta, F., Calabresi, P. and Bernardi, G., A voltage-clamp analysis of NMDA-induced responses on dopaminergic neurons of the rat substantia nigra zona compacta and ventral tegmental area, Brain Res., 593 (1992) 51 56. Mereu, G., Costa, E., Armstrong, D.M. and Vicini, S., Glutamate receptor subtypes mediate excitatory synaptic currents of dopamine neurons in midbrain slices, J. Neurosci., 11 (1991) 1359 1366. Myers, R.D. and Lee, T.F., In vivo release of dopamine during perfusion of neurotensin in substantia nigra of unrestrained rat, Peptides, 4 (1983) 955-961. Napier, T.C., Gay, D.A., Hulebak, K.L. and Bresse, G.R., Behavioural and biochemical assessment of time-related changes in globus pallidus and striatal dopamine induced by intranigrally administered neurotensin, Peptides, 6 (1985) 1057-1068. Palacios, J.M. and Kuhar, M.J., Neurotensin receptors are found on dopaminergic-containing neurons in the rat brain: an autoradiographic study, Nature, 294 (1981) 587 589. Raggenbass, M.. Goumaz, M., Sermasi, E., Tribbollet, E. and
16
17
18
19
20
Dreifuss, J.J., Vasopressin generates a persistent voltage-dependent sodium current in a mammalian motoneuron. J. Neurosci., II (1991) 1609-1616. Seroogy, K.B., Mehta, A. and Fallon, J.H., Neurotensin and cho[ecystokinin coexistence within neurons of the ventral mesencephaIon: projections to forebrain, Exp. Brain Res., 68 (1987) 277 289. Stowe, Z.N. and Nemeroff, C.B., The electrophysiological actions of neurotensin in the central nervous system, Life Sci., 49 (1991) 987 1002. Szigethy, E. and Beaudet, A., Correspondence between high affinity ~251-Neurotensin binding sites and dopaminergic neurons in the rat substantia nigra and ventral tegmental area: a combined radioautographic and immunohistochemical light microscopy study, J. Comp. Neurol., 279 (1989) 128--137. Tanaka, K., Masu, M. and Nakanishi, S., Structure and functional expression of the cloned rat neurotensin receptor, Neuron, 4 (1990) 847 854. Uhl, G.R., Whitehouse, P.J., Price, D.L., Tourtellotte, W.W. and Kuhar, M.J., Parkinson's disease: depletion of substantia nigra neurotensin receptors, Brain Res., 308 (1984) 186-190.
Neuro.wience: Letlct~s. ',53 ( i99_'1j i 92 i 9t~ .c) 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved ()304-3940/93/$ (t6.0t')
NSL 09456
Neurotensin induces an inward current in rat mesencephalic dopaminergic neurons Nicola B. Mercuri a, Francesca Stratta a, Paolo Calabresi a and Giorgio Bernardi a'b ~Clinica Neurologica Dipartimento Sanith Pubblica, Universit~ di Roma Tor Vergata, Rome (Italy) and bIRCCS, Clinica S. Lucia, Rome (Italy) (Received 23 October 1992; Revised version received 8 January 1993; Accepted 18 January 1993)
Key words: Substantia nigra; Ventral tegmental area; Electrophysiological recording; Current- and voltage-clamp Neurotensin (0.3-3/aM) depolarized the membrane and increased the firing discharge of dopaminergic ceils in slices of the rat mesencephalon. Under voltage-clamp, at holding potentials from -50 to -60 mV (near the resting membrane potential), neurotensin produced a sustained inward shift in the holding current. This inward current was reduced with the hyperpolarization of the membrane to -125 mV. It was resistant to tetrodotoxin, but it was diminished following the perfusion with low sodium (choline chloride substitution) solution, tt persisted in low calcium [0-0.5 mM). Changes in the intraceltular concentration of chloride did not affect neurotensin-induced current. The neurotensin-induced inward current did not reverse at hyperpolafized potentials in 10.5 mM cxtraeellular K ÷. It was also seen in the presence of the potassium channel blockers tetraethylamm onium (10-20 raM), barium (1 m M), apamine (1 p M ) and 4-aminopyridine (1-1.5 mM). Also the extracellular application of cesium (1-5 raM) had no effect on the cellular responsiveness to neurotensin. The action of neurotensin appears to be mediated, at least partially, by a TTX-insensitive but voltage-dependent inward current carried by sodium. The non-dopaminergic cells of the substantia nigra and ventral tegmental area were not affected by neurotensin.
Neurotensin is a tridecapeptide colocalized with dopamine in the dopaminergic neurons of the mesencephalon [16]. It is released in a CaZ+-dependent manner in the brain [5]. Local application of neurotensin in the substantia nigra and ventral tegmental area has been shown to increase the release of dopamine in these structures and in the striatum, globus pallidus and nucleus accumbens [1, 8, 12, 13]. Furthermore, receptors for neurotensin are present on the soma, dendrites and terminals of the dopaminergic cells [14, 18]. In agreement with the presence of neurotensin receptor localization on dopaminergic cells, it has been reported that the density of neurotensin binding sites is reduced in the brain of parkinsonian patients [20]. This paper reports the results of intracellular electrophysiological experiments performed in current- and voltage-clamp modes which attempt to identify the membrane properties responsible for the excitatory effects of neurotensin on dopaminergic neurons of the rat substantia nigra pars compacta and the ventral tegmental area (VTA). We conclude that neurotensin acts directly on Correspondence. N.B. Mercuri, Clinica Neurologica, Dipartimento Sanita' Pubblica, Universita' di Roma Tor Vergata, Via O. Raimondo 8, 00173 Rome, Italy. Fax: (39) (6) 7233063.
dopamine neurons to increase a voltage-dependent inward current. Slices of the ventral mesencephalon obtained from albino Wistar rats (150-350 g) were prepared as described previously [9], transferred to a recording chamber, and continuously superfused at a rate of 2.5 ml,min-1, with a solution maintained at 35°C and equilibrated with 95% 0 2 / 5 % CO2. The standard solution contained (mM): NaC1 126, KCI 2.5, NaH2PO 4 1.2, MgC12 1.2, CaC12 2.4, glucose 10, NaHCO 3 26. Intracellular recording electrodes were filled with 2 M KC1 or 2 M potassium acetate and had a resistance of 30-100 MO. A single electrode voltage-clamp amplifier was used (Axoclamp 2A) for both voltage and current recordings. Numerical data were expressed as mean _+ S.E.M. Data were obtained from 21 neurons of the ventral tegmental area and 46 cells of the substantia nigra pars compacta. The majority of these cells (43/67) fired action potentials spontaneously in vitro at a rate ranging between 0.2 and 9 Hz. They showed a pronounced voltage and time dependent inward rectification with the hyperpolarization of the membrane and responded to dopamine application (10-30 gM, n=22/22) with a hyperpolarization or an outward current. These properties are characteristics of the 'principal' cells [3, 6, 7, 9, 11] which
193
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Fig. 1. Effects of neurotensin on dopamine cells. A: the chart record shows the excitatory effects produced by 300 nM neurotensin, applied for the period indicated by the bar, on a substantia nigra zona compacta cell. In order to show individual action potentials, the speed of the chart recorder was changed (see the time bars below). The full amplitude of the action potentials is not reproduced due to the low frequency of the pen recorder. B: the application of neurotensin on a different neuron produced an inward current. Time calibration in A (1 min) is also valid for B. C: averages (3 runs) of hyperpolarizing current pulses (4 s; 10, 20, 25 mV) before, during and after neurotensin (1 ktM) application on a different substantia nigra dopaminergic cell. Note the inward shift of the holding current and the reversible decrease in the apparent input conductance caused by neurotensin. Holding potential -62 mV.
contain dopamine. No differences in the electrical properties and pharmacological responses were seen in VTA and compacta neurons; therefore, data from these neurons were pooled. Neurotensin (0.3-3 /IM) increased the spontaneous firing rate of the dopaminergic cells without altering the regular discharge pattern, which was due to a reversible depolarization of the cell membrane (Fig. 1A). When hyperpolarizing current was injected to prevent firing, neurotensin (1 ¢tM) caused a depolarization of 4-14 mV (n=22). The amplitude and the duration of the afterhyperpolarization following the action potential decreased during the neurotensin-induced depolarization. How-
ever, repolarizing the cell to the initial level by intracellular current injection restored the original shape of the afterpotentials. Under voltage-clamp (holding potential from - 5 0 to - 6 0 mV), neurotensin produced a slow and sustained inward current which underlies the depolarization (Fig. 1B). The amplitude of this current was concentration-dependent, averaging 3 4 + 8 pA (n=15) and 70 + 6 pA (n=36) with 0.3 and 1/IM neurotensin, respectively. This effect ofneurotensin occurred within 1 2 min from the start of the superfusion, and peaked in 2 4 min. The recovery was observed 7 30 min after washout. The cellular responses to neurotensin could be evoked repeatedly on the same cell, but in several cases they decreased in amplitude with the successive applications of the peptide and did not recover even after washing of the cell for more than 1 h. Neurotensin (1/~M) decreased the apparent input conductance in 12 out of 17 cells by about 24 + 6% of control (Fig. 1C). In the remaining 5 neurons, no changes were observed. Current voltage relations were constructed under voltage-clamp by measuring membrane currents in control conditions and during the application of neurotensin. Neurotensin caused in some neurons (6 out of 10) a voltage-dependent decrease of the slope conductance particularly evident in the range of holding potentials from -55 to -75 mV. The response was inward in all tested potentials (between -40 and -125 mV) (7 out of 7 cells). The magnitude of the inward current decreased with the hyperpolarization of the membrane, (Fig. 2) but it did not reverse even al -125 mV. In order to investigate the ionic nature of this current different tests were conducted. The neurotensin-induced response was not blocked by TTX (1 ~M) (not shown). The inward current was greatly depressed when normal solution was exchanged with one containing only 27 51% of control sodium by choline substitution (n=8) (Fig. 3A). We were unable to obtain reliable I V plots of the neurotensin-induced current during perfusion with low sodium, because the current produced by the peptide in low sodium was very small. The effect of neurotensin persisted in solution with 0-0.5 mM calcium containing 15-20 mM magnesium (4 cells) (Fig. 3B), but it was irreversibly reduced after a persistent exposure to low calcium for longer than 8 min and also after the addition of cobalt (1-2 mM in the medium (3 cells)). The increase of extracellular potassium from 2.5 to 10.5 mM did not modify the polarity of the neurotensin-induced current which was inward over the range of potentials examined. The amplitude of the inward current induced by 1 /~M neurotensin in 10.5 mM extracellular potassium at - 5 0 to - 6 0 mV was of 70 + 9 pA, n=4. The application of TEA (10-20 mM, 4 cells) and barium ( 1 raM, 3 cells) did not significantly (P > 0.05, Student's t-test) affect the responses to neurotensin. Apamin (1 /IM, 2 ceils) or
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Fig. 2. (I V relationship for the response of dopaminergic cells to neurotensin. A: current-voltage plots obtained before and during the application of neurotensin (1 ~M). The plots were constructed by depolarizing the neurons from hyperpolarized regions of the membrane potential at a rate of 1 mV-s-J while measuring membrane currents. Neurotensin caused an inward current throughout the voltage range. Note that the extracellular potassium concentration was 2.5 mM. B: voltage sensitivity of the neurotensin-induced inward current. Data were obtained from 6 dopaminergic cells. Note that the amplitude of the inward current increased at depolarized and decreased at hyperpolarized potentials.
4-aminopyridine (4-AR 1.5-3 mM, 2 cells) did not depress the neurotensin-induced responses (not shown). To eliminate the time- and voltage-dependent inward rectification of the membrane, extracellular cesium (1-5 mM) was perfused on the cells. Under this condition, neurotensin still produced an inward current. When electrodes filled with potassium acetate were used, muscimol, a GABAA agonist which opens C1- channels, produced an outward current while neurotensin caused an inward current (not shown). The actions ofneurotensin were not affected by (-)-sulpiride (1 ¢tM), a dopamine D2 antagonist and scopolamine (3/IM), a muscarinic antagonist. We also recorded from 4 and 6 non-dopaminergic cells in
the ventral tegmental area and in the substantia nigra, respectively [3, 6, 7]. Neurotensin (1 /2M) had no effect on these cells (not shown). Using intracellular recordings in current- and voltageclamp modes we have shown that neurotensin depolarizes the dopaminergic neurons of the rat mesencephalon, while the non-dopaminergic cells are not affected by the peptide. This confirms previous electrophysiotogical experiments with extracellular and intracellular recordings (see ref. 17 for a review), describing an excitatory effect of neurotensin only on the dopamine-containing cells. Since the effect of neurotensin persisted in media containing tetrodotoxin and with external calcium concentration of 0-0.5 mM plus 15 20 mM magnesium, the peptide acts postsynaptically and its effect is probably neither dependent on calcium nor on TTX-sensitive sodium channels. Accordingly, Szigethy and Beaudet [18] have already demonstrated with autoradiographic and immunohistochemical techniques that the neurotensin binding sites in the ventral mesencephalon are only located on the dopaminergic cells. The voltage-clamp results show that neurotensin produces an activation of a sustained and voltage-dependent inward current accompanied by a decrease in slope conductance. Although the reversal potential for the neurotensin-induced current could not be determined, experiments using channel blockers and ionic substitution suggest that the inward current may be carried by sodium. An increased permeability to ion channels is also suggested by the increased noise of the current trace, during the effect of neurotensin (Fig. 1B). An alternative explanation for the Na + dependance of the neurotensin-induced response is that the lowering of the extracellular sodium concentration may change the affinity for neurotensin receptors, or might interfere with the intracellutar processes that are activated by the neuropeptide. The neurotensin-induced current was not dependent on chloride because changes in the intracellular concentrations of CI- ions, obtained through KC1 or potassium acetatefilled electrodes, did not cause any change in amplitude or polarity of the response. An influx of calcium ions does not play a significant role in the neurotensin-induced current as demonstrated by lowering extracellular calcium and adding magnesium. The diminishing effect of prolonged exposure to low calcium may indicate that intracellular calcium is important in the response to neurotensin. Alternatively, the blocking effect of cobalt could be due to a non-selective depressant action of this cation [4]. A major effect of neurotensin on classical potassium conductances is discounted, because the inward response did not become outward at the potassium equilibrium potential and also because the neurotensin-induced response was not
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N e u r o t e n s i n (1 .uM)
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2 min Fig. 3. Effects of lowering external sodium and calcium. A: the inward current produced by neurotensin decreased in amplitude when external NaCI was replaced by choline chloride. Holding potential -58 mV. B: the cellular response to neurotensin was attenuated but not abolished in calcium free plus 20 mM extracellular MgCI 2. Holding potential -57 inV.
greatly depressed during the pharmacological blockade of potassium channels. The neurotensin-induced current that we describe here is reminiscent of that produced by cholinergic agonists in lobster cardiac ganglion neurons [2]. Furthermore, the sodium-dependent and voltagegated inward current activated by neurotensin resembles the one recently described for vasopressin on facial motoneurons [15]. The amino acid sequence of the neurotensin receptor has been recently determined [19] and it is presently unclear what signaling pathway is involved in the excitatory effect of neurotensin on dopaminergic cells. The relatively slow time course of the effect supports the involvement of a second messenger system. The decrease in amplitude of the neurotensin-induced response with repeated applications could be due to receptor desensitization or to receptor uncoupling with intracellular mechanisms. In conclusion, the present electrophysiological data support the hypothesis that the interplay of dopamine inhibition [9] with neurotensin and glutamate [10, 11] excitation may tune the firing of the dopamine-containing cells during different states of neuronal activity. We thank G. Gattoni and M. Tolu for their excellent technical assistance.
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