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Neurotensin release in the periaqueductal grey during local morphine administration
287 NEUROTENSIN
RELEASE
GREY
LOCAL
DURING
IN THE
MORPHINE
PERIAQUEDUCTAL ADMINISTRATION
~CarI-Olav Stiiler~~KnJ Fried ~OlofBeck and SErnst Brodln. Departments of 1Physiologyand Pharmacology and 2Neurosclence,Karolinska Instltutet S-171 77 Stockholm, and department of 3Cl|nlcal Pharmacology, Karolinska Hospital, S-171 76 Stockholm, Sweden Introduction Local morphine administration in the perlaqueductal grey matter (PAG) can induce a potent antinoeiception (Yaksh et al. 1976). Electrical stimulation in the PAG and local administration of a variety of neurotransmltters and neuromodulators, have also antinociceptive effects. The neurotensin (NT)-induced analgesia, which is not naloxone reversible seems to involve a long lasting excitation of PAG-neurons and an increase of the firing rate of NRM-neurons with projections to the spinal cord (Behbehani and Pert 1984). Both morphine- and NT-indueed antinociceptlon can be blocked by lesions in the NRM. In the present study we investigated if morphine administered locally in the ventromedial PAG affects the release of NT-like immunoreactivity (NT-LI) in this region. Methods Male Sprague Dawley rats 300-400g were used. A microdialysls guide eannula (CMA 12/CMA microdialys, Stockholm, Sweden) was inserted 4 mm caudally to the bregma, 1.5 mm lateral to the mldline to a depth of 4.0 mm ventral to the dura. The probe was perfused at a rate of 7 pl/min with Krebs -Ringer solution (138 mM NaCI, $ mM KCI, 1 mM MgCI2, 1 mM CaCI2, U mM NaHCO3, 1 mM NaH2PO 4, 0.2 % bovine serum albumin (Sigma, USA), 0.2% glucose and 0.03% of the peptidase inhibitor bacitracin (Sigma, USA). The sampling of the dialysate (15 min fractions) was started one hour after insertion of the dialysis probe (CMA 12/CMA microdialys) with the membrane (0.5 mm in diameter and 2 mm length) in the ventromedial part of the PAG. The recovery of NT using this probe was tested in vitro with the probe descended in Krebs Ringer containing synthetic NT. During parts of the experiments (see results) morphine and naloxone were added to the perfusion medium at a concentration of 0.1 mM or 0.01 m M . Radioimmunoassay of NT was performed as previously described for the measurement of tachykinins (Lindefors et ah 1987) using the NT antiserum (8208-H) (Theodorsson-Norheim and Rosell 1983). The NT-LI in pooled neutral and acid extracts of the ventral PAG was characterized by reverse phase HPLC. Results The in vitro recovery of NT at room temperature and a flow rate of 7 pl/min and at a concentration of 1.8 nM sythetic NT (1-13) in the outer medium was 3%. HPLC of PAG-extracts revealed two NT-LI components out of whibh the major one coeluted with synthetic NT. The other component most likely represents a C-terminal fragment of NT. An NT-LI component with a similar elution position has previously been described in rat brain extracts. The addition of morphine ( 0.1 mM) to the perfusion fluid increased the NT-LI levels in the dialysates collected in the ventromedial PAG (Fig.l). Naloxone (0.1raM) in the perfusion fluid reversed the morphine induced increase of NT-LI. At a lower concentration of morphine (0.01 mM in the perfusion medium) (Fig.2), a slight increase of the NT-LI was observed. The addition of naloxone (0.01 mM), further increased the NT-LI concentration. Preliminary observations indicate that the enhancing effect of morphine on the NT-LI release can be observed only in the immediate vicinity of the aqueduct and the dorsal raphe nucleus. This is also the region where NTimmunofluorescence is most abundant (Shipley et ah 1987).
288 Discussion In conclusion, the p r e s e n t data Indicate that locally administered m o r p h i n e stimulates the release of NT in the v e n t r o m e d i a l PAG. F r o m the p r e s e n t d a t a , it might be suggested that a p a r t of the m o r p h i n e Induced analgesia Is partially mediated by a release of NT in the ventromedlal PAG. However, it seems unlikely that m o r p h i n e directly activates the release of NT. M o r p h i n e could instead modulate the activity of inhibitory n e u r o n s controlling the release of NT. The involvement of T-amino-butyrlc acid (GABA) containing n e u r o n s could be suggested, since systemically a d m i n i s t e r e d m o r p h i n e has been r e p o r t e d to reduce the extracellular GABA concentration in the P A G (Renno et al. 1991). The results obtained with m o r p h i n e and naloxone at 0.1 m M concentration i n d i c a t e that the m o r p h i n e effect is naloxone reversible. However, the increased NT-LI levels observed during perfusion with both m o r p h i n e and naloxone at 0.01mM concentration, m i g h t indicate a complex lnt.eraction between these substances in the PAG, which deserves to be studied f u r t h e r .
175
Fig, 1
*
175 -
E "6
E
125
"6
100
125 100
v
75 I
._J
naloxone(O.lrnM)
50
morphine(O.lmM)
25 0
Fig.2
,.
150
150
I k-Z
7550
noloxone(O.OlmM)
25
r'norphine(O.O 1raM)
i
0
30
60
Time
90 120 150 (rain)
0
o
30
60
90
120 150
Time (min)
References Yaksh, T., Young, C. and Rudy, T. Systematic examination of the rat brain sites sensitive to direct application of morphine: Observation of differential effects with the perlaqueductal gray. Brain Res. 114 (1976) 83-103. Behbehanl, M.M. and Pert A. A mechanism of the analgesic effect of neurotensln as revealed by behavloural and electrophyslologlcal techniques. Brain. Res. (1984) 34-42 Lindefors, N., Brodln, E. and Ungerstedt, U. Mlcrodlalysls combined with a sensitive radlolmmunoassay. A technique for studying In vivo release of neuropeptides. J. Pharmacoi. Methods. 17/4 (1987) 305-312. Theodorsson-Norhelm, E. and Rosell S. Characterization of human plasma neurotensln-llke lmmunoreactlvlty after fat ingestion. Regulatory Peptldes, 6 (1983) 207-212 Shlpley, M.T~ McLean, J.H. and Behbehanl, M.M. Heterogeneous distribution of neurotensln-llke immunoreactive neurons and fibers In the midbraln perlaqueductal gray of the rat. J Neurosci 7 (1987)2025-34. Renno, W.M~ Mullet, M.A. and Beitz AJ. Systemic morphine reduces GABA release in the lateral, but not the medial portion of the mldbraln perlaqueductal gray of the rat. Brain Res. 594 (1994) 221-232
RELEASE
GREY
LOCAL
DURING
IN THE
MORPHINE
PERIAQUEDUCTAL ADMINISTRATION
~CarI-Olav Stiiler~~KnJ Fried ~OlofBeck and SErnst Brodln. Departments of 1Physiologyand Pharmacology and 2Neurosclence,Karolinska Instltutet S-171 77 Stockholm, and department of 3Cl|nlcal Pharmacology, Karolinska Hospital, S-171 76 Stockholm, Sweden Introduction Local morphine administration in the perlaqueductal grey matter (PAG) can induce a potent antinoeiception (Yaksh et al. 1976). Electrical stimulation in the PAG and local administration of a variety of neurotransmltters and neuromodulators, have also antinociceptive effects. The neurotensin (NT)-induced analgesia, which is not naloxone reversible seems to involve a long lasting excitation of PAG-neurons and an increase of the firing rate of NRM-neurons with projections to the spinal cord (Behbehani and Pert 1984). Both morphine- and NT-indueed antinociceptlon can be blocked by lesions in the NRM. In the present study we investigated if morphine administered locally in the ventromedial PAG affects the release of NT-like immunoreactivity (NT-LI) in this region. Methods Male Sprague Dawley rats 300-400g were used. A microdialysls guide eannula (CMA 12/CMA microdialys, Stockholm, Sweden) was inserted 4 mm caudally to the bregma, 1.5 mm lateral to the mldline to a depth of 4.0 mm ventral to the dura. The probe was perfused at a rate of 7 pl/min with Krebs -Ringer solution (138 mM NaCI, $ mM KCI, 1 mM MgCI2, 1 mM CaCI2, U mM NaHCO3, 1 mM NaH2PO 4, 0.2 % bovine serum albumin (Sigma, USA), 0.2% glucose and 0.03% of the peptidase inhibitor bacitracin (Sigma, USA). The sampling of the dialysate (15 min fractions) was started one hour after insertion of the dialysis probe (CMA 12/CMA microdialys) with the membrane (0.5 mm in diameter and 2 mm length) in the ventromedial part of the PAG. The recovery of NT using this probe was tested in vitro with the probe descended in Krebs Ringer containing synthetic NT. During parts of the experiments (see results) morphine and naloxone were added to the perfusion medium at a concentration of 0.1 mM or 0.01 m M . Radioimmunoassay of NT was performed as previously described for the measurement of tachykinins (Lindefors et ah 1987) using the NT antiserum (8208-H) (Theodorsson-Norheim and Rosell 1983). The NT-LI in pooled neutral and acid extracts of the ventral PAG was characterized by reverse phase HPLC. Results The in vitro recovery of NT at room temperature and a flow rate of 7 pl/min and at a concentration of 1.8 nM sythetic NT (1-13) in the outer medium was 3%. HPLC of PAG-extracts revealed two NT-LI components out of whibh the major one coeluted with synthetic NT. The other component most likely represents a C-terminal fragment of NT. An NT-LI component with a similar elution position has previously been described in rat brain extracts. The addition of morphine ( 0.1 mM) to the perfusion fluid increased the NT-LI levels in the dialysates collected in the ventromedial PAG (Fig.l). Naloxone (0.1raM) in the perfusion fluid reversed the morphine induced increase of NT-LI. At a lower concentration of morphine (0.01 mM in the perfusion medium) (Fig.2), a slight increase of the NT-LI was observed. The addition of naloxone (0.01 mM), further increased the NT-LI concentration. Preliminary observations indicate that the enhancing effect of morphine on the NT-LI release can be observed only in the immediate vicinity of the aqueduct and the dorsal raphe nucleus. This is also the region where NTimmunofluorescence is most abundant (Shipley et ah 1987).
288 Discussion In conclusion, the p r e s e n t data Indicate that locally administered m o r p h i n e stimulates the release of NT in the v e n t r o m e d i a l PAG. F r o m the p r e s e n t d a t a , it might be suggested that a p a r t of the m o r p h i n e Induced analgesia Is partially mediated by a release of NT in the ventromedlal PAG. However, it seems unlikely that m o r p h i n e directly activates the release of NT. M o r p h i n e could instead modulate the activity of inhibitory n e u r o n s controlling the release of NT. The involvement of T-amino-butyrlc acid (GABA) containing n e u r o n s could be suggested, since systemically a d m i n i s t e r e d m o r p h i n e has been r e p o r t e d to reduce the extracellular GABA concentration in the P A G (Renno et al. 1991). The results obtained with m o r p h i n e and naloxone at 0.1 m M concentration i n d i c a t e that the m o r p h i n e effect is naloxone reversible. However, the increased NT-LI levels observed during perfusion with both m o r p h i n e and naloxone at 0.01mM concentration, m i g h t indicate a complex lnt.eraction between these substances in the PAG, which deserves to be studied f u r t h e r .
175
Fig, 1
*
175 -
E "6
E
125
"6
100
125 100
v
75 I
._J
naloxone(O.lrnM)
50
morphine(O.lmM)
25 0
Fig.2
,.
150
150
I k-Z
7550
noloxone(O.OlmM)
25
r'norphine(O.O 1raM)
i
0
30
60
Time
90 120 150 (rain)
0
o
30
60
90
120 150
Time (min)
References Yaksh, T., Young, C. and Rudy, T. Systematic examination of the rat brain sites sensitive to direct application of morphine: Observation of differential effects with the perlaqueductal gray. Brain Res. 114 (1976) 83-103. Behbehanl, M.M. and Pert A. A mechanism of the analgesic effect of neurotensln as revealed by behavloural and electrophyslologlcal techniques. Brain. Res. (1984) 34-42 Lindefors, N., Brodln, E. and Ungerstedt, U. Mlcrodlalysls combined with a sensitive radlolmmunoassay. A technique for studying In vivo release of neuropeptides. J. Pharmacoi. Methods. 17/4 (1987) 305-312. Theodorsson-Norhelm, E. and Rosell S. Characterization of human plasma neurotensln-llke lmmunoreactlvlty after fat ingestion. Regulatory Peptldes, 6 (1983) 207-212 Shlpley, M.T~ McLean, J.H. and Behbehanl, M.M. Heterogeneous distribution of neurotensln-llke immunoreactive neurons and fibers In the midbraln perlaqueductal gray of the rat. J Neurosci 7 (1987)2025-34. Renno, W.M~ Mullet, M.A. and Beitz AJ. Systemic morphine reduces GABA release in the lateral, but not the medial portion of the mldbraln perlaqueductal gray of the rat. Brain Res. 594 (1994) 221-232