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Neurotensin self-injection in the ventral tegmental area
Brain Research, 403 (1987) 147-150 Elsevier
147
BRE 22013
Neurotensin self-injection in the ventral tegmental area Paul W. Glimcher*, Adrienne A. Giovino** and Bart|ey G. Hoebe! Department of Psychology, Princeton University, Princeton, NJ 08544 (U.S.A. )
(Accepted 16 September 1986) Key words: Neurotensin; Reward: Self-injection; Ventral tegmental area
Earlier work with the conditioned place-preference paradigm suggested that neurotensin (NT) acts as a behavioral reinforcer when microinjected into the ventral tegmental area (VTA) of the midbrain. We report here that animals will perform an operant task to obtain microinfusions of NT into the VTA. Rats reliably pressed a lever to obtain NT infusions while neglecting an identical but inactive lever. Substitution of saline for NT initiated response extinction; following the reintroduction of NT, reliable responding resumed. These results extend earlier work suggesting that NT in the VTA can be a positive reinforcer. Previous work d e m o n s t r a t e d that the tridecapeptide, neurotensin (NT), g e n e r a t e d a conditioned place-preference following microinfusion into the ventral tegmental area ( V T A ) 3. This suggested that NT might be a p o t e n t reinforcer in the V T A as has been shown for opiate peptides 2'11. In o r d e r to crossvalidate NT's ability to function as a reinforcer, we used self-injection to test its rewarding effects. The reasons for predicting that N T would be a reinforcer in the V T A have been discussed elsewhere 3. In brief, specific NT receptors are present in the V T A ~'-9A2-15 and NT injected iontophoretically has been shown to activate d o p a m i n e ( D A ) cell bodies j. The mesolimbie D A system is thought to play a role in l o c o m o t o r and reward-related behaviors 4. In agreement with this suggestion, V T A infusions of NT increase spontaneous l o c o m o t o r activity 5. O u r work with the place-preference p a r a d i g m showed that the whole peptide, NTj_j3 injected into the V T A generated a conditioned place-preference and that the effect was biochemically specific. NTj_I3 and NTM1 were effective, but the shorter fragments NTI_ 8 and NTs_I3 were not 3. The question for the present study was whether or not rats would self-inject NTl_13. S p r a g u e - D a w l e y rats weighing between 275 and 325 g were anesthetized with chloropent and stereo-
taxically implanted with 22 gauge stainless steel guide cannulas that e n d e d 1.0 mm dorsal to the V T A site of injection. Coordinates for the injection site were selected based on previous work and the stereotaxic atlas of Pellegrino and Cushman jc~. The cannula tip was placed 2.2 mm anterior to the interaural line, 0.6 mm lateral to the midsagittal sinus and 6.5 mm vertical and ventral to dura. Cannulas were kept clear with 28 gauge stylettes which were removed only for experimental sessions. After a one-week postoperative period, animals were handled, and a 28 gauge injector was inserted which p r o t r u d e d 1.0 mm beyond the guide cannula to reach the V T A (A 2.2., L 0.6, V 7.5); it was inserted momentarily to remove tissue at the injection site and thus minimize mechanical tissue damage during experimental sessions. Three days later the self-injection procedure began. Sessions were conducted at the beginning of the dark phase of a 12 h light/12 h dark cycle. The test cage was a 37 × 19 × 60 cm Skinner box with a 3 x 4 cm lever at each end. O n e lever activated a syringe pump. The other did nothing but o p e r a t e a counter and was used as a control for non-specific activity. The experimental cage was enclosed in a dark, sound-attenuated chamber.
* Present address: Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104. U.S.A. ** Present address: Department of Psychology, Concordia University, Montreal, Que., Canada. Correspondence: B.G. Hoebel, Department of Psychology, Princeton University, Princeton. NJ 08544. U.S.A.
14,~ SELF-INJECTION OF NT DURING 4 HOURS
40 S
i~--o,
NEUROTENSIN VS. BLANK LEVER 4 Z 30 O
3
~
2
u) O 0 Z
5'
20
0 if)
0
Ul
MINUTES
Fig. 2. Some animals respond in bursts, but on the average, infusion of NT occurs throughout 4-h experimental sessions I mean responses per 10-rain interval, f~)rall 8 animals).
Ul _J
Z
-7
¢¢ ttl
,,-,
~g
Z
=
5
Fig. 1. Animals consistentl) select a lever associated with VTA infusions of NT and neglect a blank lever ( mean of lever presses per session for 8 rats shown. P < 0.0001 ~. Experimental sessions for each animal were separated by 3 days to allow recovery from tissue changes at the injection site caused by the self-injection procedure. At the beginning of a session, the stylette was removed and replaced by a 28 gauge injector which projected 1.0 mm beyond the guide cannula. One unsignaled injection of drug was administered, and the chamber was closed. The lever which operated the syringe pump was the same for every test and delivered 50 + 15 nl containing 2.5 _+ 0.3 ~g of NT (Bachem, U . S . A . ) during 0.5 s. After each 4-h session was completed, the injector was removed, the stylette reinserted, and the animal returned to its home cage. Animals which failed to acquire the operant response after 6 sessions were sacrificed for histological examination. Of an initial 20 animals, 8 developed stable rates of lever-pressing. These animals were maintained on this regimen for as long as 10 sessions (1 month). Of the 8 animals that learned to self-inject NT, 6 were used in the second stage of the experiment in which NT was replaced by saline. During 3 sessions. presses on the active lever yielded only saline infusions. Then NT was again offered as the operant rein-
forcer for the last 3 sessions. Animals were sacrificed with anesthetic and perfused with formalin. Brains were sectioned at 40 tim, mounted, and stained with Cresyl violet for histological localization of the cannula tip. Fig. 1 shows the mean rate of lever-pressing for all 8 animals that learned to respond consistently for NT. Animals clearly distinguished between the active and blank levers by selecting t h e l e v e r for N T significantly more than the blank lever ( P < 0.0001. paired t-test). Fig. 2 ~s group data demonstrating that. on the average, animals responded throughout the 4-h period. Individual records show that animals typically pressed in bursts (Fig. 3). Fig, 4 gives data for the 6 animals offered saline in place of NT. Mean lever-pressing rates show that animals stopped pressing the saline lever and seldom pressed the inactive lever either. The decrease in lever-pressing for saline was significant when compared to NT (P < 0.02, paired t-test). Response rates returned to near normal levels when NT was reintroduced. Rats selected the lever associated with NT infusions in preference to the blank lever. This controlled for random locomotor effects, The failure of saline infusions to support operant responding demonstrates that NT, not the vehicle, was acting as the reinforcer. Therefore, spurious environmental or volume effects associated with the infusions were not responsible for the reinforcing effects of NT in this paradigm. Of the animals that failed to self-administer NT. 4
149 RAT 67 SESSION 1
_=
:li I !,,
l,,
,,,,,,
I ,I,,,,,, I
,
I
,
iii
Ill
j
]
SELF--INJECTION
~3o
SESS~OH 4
'I
SESSIO'N
s l"
,11
II . . . . a
VS. SALINE
,q.
.......
S~SS~ON 3
~>
NEUROTENSIN S~SION 2
l,,
4
i,
,,,
II
,,
,,,,
2
s
>~D"10 ,,
J
TI M E (hr)
Fig. 3. Data for an individual animal showing responses per 10min interval on the NT lever during six 4-h test sessions.
NT MEAN BASELINE 3 RUNS
were histologically analyzed and showed cannula placement outside the V T A ; the 8 positive responders had cannula placements within the borders of the V T A described before 3. Proximity to the third ventricle did not a p p e a r to be a relevant factor. We conclude that the NT was p r o b a b l y acting locally in the VTA. It is not clear why the animals self-injected such large amounts of NT relative to endogenous levels in the brain. Perhaps the exogenous peptide is quickly d e g r a d e d after it is injected, or is of low physiological activity when c o m p a r e d to the endogenous
m SALINE
NT RECOVERY
3 ~
3
RUNS
N=6
Fig. 4. Six animals offered saline in place of NT show a decrease in rate of bar-pressing which is reversed by the reintroduction of NT as the operant reinforcer (P < 0.02). Following NT reintroduction, lever-pressing rates are almost indistinguishable from NT baseline rates.
ligand. In summary, V T A micro-infusions of NT can be a positive reinforcer in an o p e r a n t paradigm. Combined with earlier data showing a conditioned placepreference with whole NT and the fragment NTI_I~,
different ways. These results parallel the well-established ability of the opiates to generate reinforcement in the V T A with both paradigms 2'1~. A t present it is not possible to d e m o n s t r a t e conclusively that the reinforcing effect of NT is a normal physiological p h e n o m e n o n . Specific NT agonists, antagonists and aminopeptidases are required for further tests of this hypothesis. The present results suggest that reinforcement p r o d u c e d by local micro-injection of NT into the V T A is a robust p h e n o m e n o n .
but not with the fragments NTI_ls or NT~_133. the results suggest that specific receptors in the V T A can generate positive reinforcement in response to exogenously applied NT. The two experiments crossvalidate each other by measuring reinforcement in
A portion of this work was presented at the Meeting of the Society for Neurosciences, 1983, abstract 35.6. The research was s u p p o r t e d by PHS Grant MH-35740.
1 Andrade, R. and Aghajanian, G,K., Neurotensin selectively activates dopaminergic neurons of the substantia nigra, Soc. Neurosci. Abstr., 7 (1981) 553. 2 Bozarth. M.A. and Wise, R.A., Intracranial self-administration of morphine into the ventral tegmental area in rats, Life. Sci., 28 (1981) 551-555. 3 Glimcher, P.W., Margolin, D.M., Giovino, A.A. and Hoebel, B.G., Neurotensin: a new 'reward peptide', Brain Research, 291 (1984) 119-124. 4 Hoebel, B.G. and Novin, D. (Eds.), The Neural Basis" of Feeding and Reward, Haer Institute, Brunswick, Me, 1982. 5 Kalivas, P.W., Nemeroff, C.B. and Prange, A.J., Increase in spontaneous motor activity following infusion of neurotensin into the ventral tegmental area, Brain Research, 229 (198l) 525-529. 6 Kitagi, P,, Carraway, R., Van Rietschoten, J., Granier, C., Morgat, J.L., Menez, A., Leeman, S. and Freychet, P., Neurotensin specific binding to synaptic membranes from
7 8
9 10 11 12
rat braim Proc. Natl. Acad. Sci. U.S.A., 74 (1970) 1846-1850. Kataoka, K., Mizuno, N. and Frohman, L.A., Regional distribution of immunoreactive neurotensin in monkey brain, Brain Res. Bull., 4 (1979) 57-60. Lazarus, L.H., Brown, M.R. and Perrin, M.H., Distribution, localization and characteristics of neurotensin binding sites in the rat brain, Neuropharmacology, 16 (1977) 625-629. Palacios, J.M. and Kuhar, M.J., Neurotensin receptors are located on dopamine-containing neurons in rat midbrain, Nature (London), 294 (1981) 587-589. Pellegrino, L.J. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Appleton-Century-Crofts, New York, 1967. Phillips, A.G. and LePiane, F.G,, Reinforcing effects of morphine microinjection into the ventral tegmental area, Pharmacol, Biochem. Behav., 12 (1980) 965-968. Uhl, G.R., Bennett, J.P. and Snyder, S.H., Neurotensin, a
15(~ central nervous system peptide: apparent receptor binding in brain membranes, Brain Research, 130 (1977) 299-313. 13 Uhl, G.R., Kuhar, M.J. and Snyder, S.H., Neurotensin: immunohistochemical localization in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4059-4063. 14 Uhl, G.R. and Snyder, S.H., Neurotensin receptor bind-
ing: regional and sub-cellular distributions favor transmitter role, Eur. J. Pharmacol., 41 (1977) 89--91. 15 Uhl, G.R., Goodman, R.R. and Snyder. S.H., Neurotensin-containing cell bodies, fibers and nerve terminals in the brainstem of the rat immunohistochemical mapping, Brain Research, 167 (1979) 77-92.
147
BRE 22013
Neurotensin self-injection in the ventral tegmental area Paul W. Glimcher*, Adrienne A. Giovino** and Bart|ey G. Hoebe! Department of Psychology, Princeton University, Princeton, NJ 08544 (U.S.A. )
(Accepted 16 September 1986) Key words: Neurotensin; Reward: Self-injection; Ventral tegmental area
Earlier work with the conditioned place-preference paradigm suggested that neurotensin (NT) acts as a behavioral reinforcer when microinjected into the ventral tegmental area (VTA) of the midbrain. We report here that animals will perform an operant task to obtain microinfusions of NT into the VTA. Rats reliably pressed a lever to obtain NT infusions while neglecting an identical but inactive lever. Substitution of saline for NT initiated response extinction; following the reintroduction of NT, reliable responding resumed. These results extend earlier work suggesting that NT in the VTA can be a positive reinforcer. Previous work d e m o n s t r a t e d that the tridecapeptide, neurotensin (NT), g e n e r a t e d a conditioned place-preference following microinfusion into the ventral tegmental area ( V T A ) 3. This suggested that NT might be a p o t e n t reinforcer in the V T A as has been shown for opiate peptides 2'11. In o r d e r to crossvalidate NT's ability to function as a reinforcer, we used self-injection to test its rewarding effects. The reasons for predicting that N T would be a reinforcer in the V T A have been discussed elsewhere 3. In brief, specific NT receptors are present in the V T A ~'-9A2-15 and NT injected iontophoretically has been shown to activate d o p a m i n e ( D A ) cell bodies j. The mesolimbie D A system is thought to play a role in l o c o m o t o r and reward-related behaviors 4. In agreement with this suggestion, V T A infusions of NT increase spontaneous l o c o m o t o r activity 5. O u r work with the place-preference p a r a d i g m showed that the whole peptide, NTj_j3 injected into the V T A generated a conditioned place-preference and that the effect was biochemically specific. NTj_I3 and NTM1 were effective, but the shorter fragments NTI_ 8 and NTs_I3 were not 3. The question for the present study was whether or not rats would self-inject NTl_13. S p r a g u e - D a w l e y rats weighing between 275 and 325 g were anesthetized with chloropent and stereo-
taxically implanted with 22 gauge stainless steel guide cannulas that e n d e d 1.0 mm dorsal to the V T A site of injection. Coordinates for the injection site were selected based on previous work and the stereotaxic atlas of Pellegrino and Cushman jc~. The cannula tip was placed 2.2 mm anterior to the interaural line, 0.6 mm lateral to the midsagittal sinus and 6.5 mm vertical and ventral to dura. Cannulas were kept clear with 28 gauge stylettes which were removed only for experimental sessions. After a one-week postoperative period, animals were handled, and a 28 gauge injector was inserted which p r o t r u d e d 1.0 mm beyond the guide cannula to reach the V T A (A 2.2., L 0.6, V 7.5); it was inserted momentarily to remove tissue at the injection site and thus minimize mechanical tissue damage during experimental sessions. Three days later the self-injection procedure began. Sessions were conducted at the beginning of the dark phase of a 12 h light/12 h dark cycle. The test cage was a 37 × 19 × 60 cm Skinner box with a 3 x 4 cm lever at each end. O n e lever activated a syringe pump. The other did nothing but o p e r a t e a counter and was used as a control for non-specific activity. The experimental cage was enclosed in a dark, sound-attenuated chamber.
* Present address: Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104. U.S.A. ** Present address: Department of Psychology, Concordia University, Montreal, Que., Canada. Correspondence: B.G. Hoebel, Department of Psychology, Princeton University, Princeton. NJ 08544. U.S.A.
14,~ SELF-INJECTION OF NT DURING 4 HOURS
40 S
i~--o,
NEUROTENSIN VS. BLANK LEVER 4 Z 30 O
3
~
2
u) O 0 Z
5'
20
0 if)
0
Ul
MINUTES
Fig. 2. Some animals respond in bursts, but on the average, infusion of NT occurs throughout 4-h experimental sessions I mean responses per 10-rain interval, f~)rall 8 animals).
Ul _J
Z
-7
¢¢ ttl
,,-,
~g
Z
=
5
Fig. 1. Animals consistentl) select a lever associated with VTA infusions of NT and neglect a blank lever ( mean of lever presses per session for 8 rats shown. P < 0.0001 ~. Experimental sessions for each animal were separated by 3 days to allow recovery from tissue changes at the injection site caused by the self-injection procedure. At the beginning of a session, the stylette was removed and replaced by a 28 gauge injector which projected 1.0 mm beyond the guide cannula. One unsignaled injection of drug was administered, and the chamber was closed. The lever which operated the syringe pump was the same for every test and delivered 50 + 15 nl containing 2.5 _+ 0.3 ~g of NT (Bachem, U . S . A . ) during 0.5 s. After each 4-h session was completed, the injector was removed, the stylette reinserted, and the animal returned to its home cage. Animals which failed to acquire the operant response after 6 sessions were sacrificed for histological examination. Of an initial 20 animals, 8 developed stable rates of lever-pressing. These animals were maintained on this regimen for as long as 10 sessions (1 month). Of the 8 animals that learned to self-inject NT, 6 were used in the second stage of the experiment in which NT was replaced by saline. During 3 sessions. presses on the active lever yielded only saline infusions. Then NT was again offered as the operant rein-
forcer for the last 3 sessions. Animals were sacrificed with anesthetic and perfused with formalin. Brains were sectioned at 40 tim, mounted, and stained with Cresyl violet for histological localization of the cannula tip. Fig. 1 shows the mean rate of lever-pressing for all 8 animals that learned to respond consistently for NT. Animals clearly distinguished between the active and blank levers by selecting t h e l e v e r for N T significantly more than the blank lever ( P < 0.0001. paired t-test). Fig. 2 ~s group data demonstrating that. on the average, animals responded throughout the 4-h period. Individual records show that animals typically pressed in bursts (Fig. 3). Fig, 4 gives data for the 6 animals offered saline in place of NT. Mean lever-pressing rates show that animals stopped pressing the saline lever and seldom pressed the inactive lever either. The decrease in lever-pressing for saline was significant when compared to NT (P < 0.02, paired t-test). Response rates returned to near normal levels when NT was reintroduced. Rats selected the lever associated with NT infusions in preference to the blank lever. This controlled for random locomotor effects, The failure of saline infusions to support operant responding demonstrates that NT, not the vehicle, was acting as the reinforcer. Therefore, spurious environmental or volume effects associated with the infusions were not responsible for the reinforcing effects of NT in this paradigm. Of the animals that failed to self-administer NT. 4
149 RAT 67 SESSION 1
_=
:li I !,,
l,,
,,,,,,
I ,I,,,,,, I
,
I
,
iii
Ill
j
]
SELF--INJECTION
~3o
SESS~OH 4
'I
SESSIO'N
s l"
,11
II . . . . a
VS. SALINE
,q.
.......
S~SS~ON 3
~>
NEUROTENSIN S~SION 2
l,,
4
i,
,,,
II
,,
,,,,
2
s
>~D"10 ,,
J
TI M E (hr)
Fig. 3. Data for an individual animal showing responses per 10min interval on the NT lever during six 4-h test sessions.
NT MEAN BASELINE 3 RUNS
were histologically analyzed and showed cannula placement outside the V T A ; the 8 positive responders had cannula placements within the borders of the V T A described before 3. Proximity to the third ventricle did not a p p e a r to be a relevant factor. We conclude that the NT was p r o b a b l y acting locally in the VTA. It is not clear why the animals self-injected such large amounts of NT relative to endogenous levels in the brain. Perhaps the exogenous peptide is quickly d e g r a d e d after it is injected, or is of low physiological activity when c o m p a r e d to the endogenous
m SALINE
NT RECOVERY
3 ~
3
RUNS
N=6
Fig. 4. Six animals offered saline in place of NT show a decrease in rate of bar-pressing which is reversed by the reintroduction of NT as the operant reinforcer (P < 0.02). Following NT reintroduction, lever-pressing rates are almost indistinguishable from NT baseline rates.
ligand. In summary, V T A micro-infusions of NT can be a positive reinforcer in an o p e r a n t paradigm. Combined with earlier data showing a conditioned placepreference with whole NT and the fragment NTI_I~,
different ways. These results parallel the well-established ability of the opiates to generate reinforcement in the V T A with both paradigms 2'1~. A t present it is not possible to d e m o n s t r a t e conclusively that the reinforcing effect of NT is a normal physiological p h e n o m e n o n . Specific NT agonists, antagonists and aminopeptidases are required for further tests of this hypothesis. The present results suggest that reinforcement p r o d u c e d by local micro-injection of NT into the V T A is a robust p h e n o m e n o n .
but not with the fragments NTI_ls or NT~_133. the results suggest that specific receptors in the V T A can generate positive reinforcement in response to exogenously applied NT. The two experiments crossvalidate each other by measuring reinforcement in
A portion of this work was presented at the Meeting of the Society for Neurosciences, 1983, abstract 35.6. The research was s u p p o r t e d by PHS Grant MH-35740.
1 Andrade, R. and Aghajanian, G,K., Neurotensin selectively activates dopaminergic neurons of the substantia nigra, Soc. Neurosci. Abstr., 7 (1981) 553. 2 Bozarth. M.A. and Wise, R.A., Intracranial self-administration of morphine into the ventral tegmental area in rats, Life. Sci., 28 (1981) 551-555. 3 Glimcher, P.W., Margolin, D.M., Giovino, A.A. and Hoebel, B.G., Neurotensin: a new 'reward peptide', Brain Research, 291 (1984) 119-124. 4 Hoebel, B.G. and Novin, D. (Eds.), The Neural Basis" of Feeding and Reward, Haer Institute, Brunswick, Me, 1982. 5 Kalivas, P.W., Nemeroff, C.B. and Prange, A.J., Increase in spontaneous motor activity following infusion of neurotensin into the ventral tegmental area, Brain Research, 229 (198l) 525-529. 6 Kitagi, P,, Carraway, R., Van Rietschoten, J., Granier, C., Morgat, J.L., Menez, A., Leeman, S. and Freychet, P., Neurotensin specific binding to synaptic membranes from
7 8
9 10 11 12
rat braim Proc. Natl. Acad. Sci. U.S.A., 74 (1970) 1846-1850. Kataoka, K., Mizuno, N. and Frohman, L.A., Regional distribution of immunoreactive neurotensin in monkey brain, Brain Res. Bull., 4 (1979) 57-60. Lazarus, L.H., Brown, M.R. and Perrin, M.H., Distribution, localization and characteristics of neurotensin binding sites in the rat brain, Neuropharmacology, 16 (1977) 625-629. Palacios, J.M. and Kuhar, M.J., Neurotensin receptors are located on dopamine-containing neurons in rat midbrain, Nature (London), 294 (1981) 587-589. Pellegrino, L.J. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Appleton-Century-Crofts, New York, 1967. Phillips, A.G. and LePiane, F.G,, Reinforcing effects of morphine microinjection into the ventral tegmental area, Pharmacol, Biochem. Behav., 12 (1980) 965-968. Uhl, G.R., Bennett, J.P. and Snyder, S.H., Neurotensin, a
15(~ central nervous system peptide: apparent receptor binding in brain membranes, Brain Research, 130 (1977) 299-313. 13 Uhl, G.R., Kuhar, M.J. and Snyder, S.H., Neurotensin: immunohistochemical localization in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4059-4063. 14 Uhl, G.R. and Snyder, S.H., Neurotensin receptor bind-
ing: regional and sub-cellular distributions favor transmitter role, Eur. J. Pharmacol., 41 (1977) 89--91. 15 Uhl, G.R., Goodman, R.R. and Snyder. S.H., Neurotensin-containing cell bodies, fibers and nerve terminals in the brainstem of the rat immunohistochemical mapping, Brain Research, 167 (1979) 77-92.