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The effect of morphine on responses of ventrolateral orbital cortex (VLO) neurons to colorectal distension in the rat
Brain Research 808 Ž1998. 101–105
Short communication
The effect of morphine on responses of ventrolateral orbital cortex ŽVLO . neurons to colorectal distension in the rat Shou-wei Yang, Kenneth A. Follett
)
DiÕision of Neurosurgery, The UniÕersity of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA Accepted 28 July 1998
Abstract In 49 halothane-anesthetized rats, we characterized the responses of single neurons in the ventrolateral orbital cortex ŽVLO. to a noxious visceral stimulus Žcolorectal balloon distension, CRD., and studied the effects of intravenous morphine on these responses using standard extracellular microelectrode recording techniques. One hundred and four neurons were isolated on the basis of spontaneous activity. Fifty-seven Ž55%. responded to CRD, of which 32% had excitatory and 68% had inhibitory responses. Neurons showed tendencies toward graded responses to graded CRD pressures Ž20–100 mmHg., with maximum excitation or inhibition occurring at 80 or 100 mmHg, respectively. Responses to noxious Žpinch, heat. and innocuous Žbrush, tap. cutaneous stimuli were studied in 80 of the VLO neurons isolated. Thirty-three Ž41%. of these neurons Ž21 CRD-responsive and 12 CRD-nonresponsive. had cutaneous receptive fields, of which 79% were large and bilateral, 18% were small and bilateral, 3% were small and ipsilateral. Ninety-four percent of these neurons responded only to noxious cutaneous stimulation, 6% responded to both noxious and innocuous stimulation. No neurons responded solely to innocuous stimulation. Cumulative doses of morphine Ž0.0625, 0.125 and 0.25 mgrkg i.v.. produced statistically significant dose-dependent attenuation of neuronal responses to CRD. Naloxone Ž0.4 mgrkg i.v.. reversed the effects of morphine. Morphine and naloxone had no significant effects on spontaneous activity. These data support the involvement of VLO neurons in visceral nociception. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Visceral; Analgesia; Supraspinal; Opiate
Neurons in ventrolateral orbital cortex ŽVLO. respond to noxious stimuli, suggesting a role in nociception w1,2,9,10,17x. Most experimental studies of nociceptive properties of VLO neurons have been based on superficial or cutaneous pain models w1,2,9,17x. While data derived from studies of cutaneous nociception are important in building our knowledge of pain mechanisms, visceral pain is of greater clinical significance. The role of VLO neurons in visceral nociception is unclear and has received relatively little attention. Orbital cortex receives afferent input from the splanchnic nerve conveyed by A-d, g-fibers w13x. These small diameter fibers can be activated by application of natural noxious stimuli to intraperitoneal structures w11x, suggesting that VLO may play a role in visceral nociception. Direct evidence for the involvement of VLO in visceral nociception is provided by the observations in rats
) Corresponding author. Fax: q1-319-353-6605; E-mail: [email protected]
w10x and cats w17x that neurons respond to the noxious visceral stimulation produced by distension of the colon and gallbladder, respectively. Colorectal balloon distension ŽCRD. is a particularly useful animal model because it mimics acute intestinal obstruction and distension, a clinically significant cause of visceral pain, and has been established as a reliable, quantifiable, reproducible physiologic model of visceral pain, and has been used extensively to study visceral pain mechanisms w16x. In the present study, we have evaluated the responses of VLO neurons to CRD in halothane-anesthetized animals. In addition, we have studied the effects of intravenous morphine on VLO neuron responses evoked by CRD. If VLO responses to CRD are nociceptive, then we anticipate those responses would be attenuated by analgesic administration. Experiments were performed on 49 halothane-anesthetized male Sprague–Dawley rats ŽBiolab, St. Paul, MN. weighing 280–420 g. The procedure has been described previously w10x, with the exception that halothane was used for anesthesia in this study. Rats were administered 1–1.5%
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 0 4 - X
S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105
102
halothane via mask during initial surgical preparation, including placement of a tracheal cannula, and anesthesia was maintained at 0.5–0.7% inhaled halothane via the tracheal cannula during experimental observations. As described previously w10,19x, standard extracellular microelectrode recording techniques were used to isolate single neurons. We characterized the responses of single neurons to inflation of the colorectal balloon to a pressure of 80 mmHg. Three types of units were recognized according to their responses to the noxious visceral stimulation: CRD-excited, CRD-inhibited, and CRD-nonresponsive units. Each unit’s activity was measured for 10 s before, 20 s during, and 20 s after CRD and was evaluated as a peristimulus–time histogram with 1-s bins. A unit was deemed to respond to CRD if its activity changed Žduring CRD. at least 10% from its predistension baseline frequency. Stimulus–response curves were developed through testing 20, 40, 60, and 100 mmHg CRD for those units which responded to 80 mmHg CRD and could be held for a sufficiently long period of time. The location of a cutaneous receptive field for each neuron was determined qualitatively in response to innocuous brush and tap and noxious pinch Žforceps. and heat Žophthalmic cautery probe.. The dose-related effects of morphine Ž0.0625, 0.125, 0.25, 0.5 and 1 mgrkg i.v., cumulative doses. on responses to 80 mmHg CRD were studied in 15 inhibited and 4 excited neurons. For eight inhibited and three excited neurons in which CRD-evoked discharges were suppressed completely by morphine, the effect of subsequent naloxone Ž0.4 mgrkg i.v.. was studied. Data are expressed as the mean " S.E.M. Stimulus–response curves and differences among the effects of morphine doses were assessed by analysis of variance, using post-tests with Bonferroni correction to compare groups. A p-value less than 0.05 was considered statistically significant. At the conclusion of each experiment, animals were sacrificed by overdose of saturated KCl solution, and locations of recorded neurons were determined histologically. Data reported are only for those neurons that were confirmed histologically to be within VLO.
1. Responses of VLO neurons to CRD One hundred and four neurons isolated on the basis of spontaneous activity were studied. Fifty-seven of the 104
neurons Ž55%. responded to CRD. Eighteen of the CRDresponsive neurons Ž32%. had excitatory responses to CRD, 39 Ž68%. had inhibitory responses. Spontaneous neuronal activity remained relatively consistent over time Ž7.3 " 0.4 Hz, n s 15, continuous observation for 40 min.. Spontaneous discharge activity was not related significantly to time ŽANOVA, p ) 0.8.. Responses to CRD were independent of changes in spontaneous activity. For neurons excited by CRD, graded increases in distension pressure from 20 to 100 mmHg produced peak neuronal activity at 80 mmHg Žaverage increase to 40% greater than baseline. ŽTable 1.. For inhibited neurons, graded increases in CRD pressures caused maximal inhibition at 100 mmHg Žactivity attenuated to 60% of baseline. ŽTable 1.. Both excited and inhibited neurons showed tendencies toward graded changes in activity related to graded changes in distension pressures, but ANOVA analyses of the stimulus–response curves demonstrated no statistically significant dependence of responses on distension pressures ŽTable 1.. Eighty VLO neurons Ž39 CRD-responsive and 41 CRD-nonresponsive. were tested for cutaneous receptive fields. Twenty-one CRD-responsive and 12 CRD-nonresponsive neurons had cutaneous receptive fields, of which one Ž3%. was small and ipsilateral, six Ž18%. were small and bilateral, and 26 Ž79%. were large and bilateral Žsometimes whole body. cutaneous receptive fields. Ninety-four percent of the neurons with cutaneous receptive fields responded only to noxious pinch and heat. Two neurons Ž6%. responded to both noxious and non-noxious cutaneous stimulation. No neuron responded exclusively to innocuous cutaneous stimulation.
2. Effects of morphine on CRD-evoked activity of VLO neurons Morphine had statistically significant dose-dependent effects on CRD-evoked activity ŽTable 2.. For inhibited neurons, the change produced by morphine was statistically significant for the 0.25 mgrkg dose Ž p - 0.05.. For excited neurons, a trend toward dose-dependent attenuation by morphine was evident, but the small sample size precludes statistical significance Ž p ) 0.05.. For both inhibited and excited neurons, naloxone restored CRD-evoked responses to a level not statistically different from premorphine. Spontaneous activity was not altered significantly by any dose of morphine or by naloxone ŽTable 2..
Table 1 CRD-evoked activities ŽHz. of VLO neurons Response to CRD
Number of neurons
CRD ŽmmHg. 20
40
60
80
100
ANOVA
Excited Inhibited
6 9
0.4 " 0.9 y1.1 " 0.6
1.8 " 0.8 y1.5 " 0.9
3.6 " 1.5 y2.2 " 0.9
4.3 " 1.3 y3.1 " 0.8
3.8 " 0.8 y3.2 " 0.9
F4,23 s 1.730 F4,35 s 1.110
p s 0.178 p s 0.367
Neuronal activity
Response to CRD
Number of neurons
Baseline
Evoked
Inhibited Excited Inhibited Excited
15 4 15 4
y3.1"0.7 5.1"1.7 9.4"1.7 12.6"6.1
Spontaneous
) p- 0.05, post-test with Bonferroni correction.
Morphine Žmgrkg i.v.. 0.0625 0.125
0.25
y2.2"0.8 4.1"3.1 9.8"2.3 13.1"6.9
y0.01"0.5) 1.9"0.6 9.1"2.6 13.3"5.3
y1.6"0.5 2.2"0.8 8.2"1.8 14.5"5.0
0.5
1
1.6"0.5
y0.2"0.6
14.3"6.6
10.4"3.4
Naloxone Ž0.4 mgrkg.
ANOVA
y2.4"0.7 2.6"0.8 10.5"3.2 7.4"3.4
F4,50 s 2.728 ps 0.039 F6,14 s1.635 ps 0.210 F4,51 s 0.126 p) 0.8 F6,15 s 0.1759 p) 0.8
S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105
Table 2 Effects of morphine on CRD-evoked and spontaneous activities ŽHz. of VLO neurons
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S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105
Our present data show that VLO neurons respond to physiologic noxious visceral stimulation, CRD, and these responses can be attenuated significantly by an intravenous analgesic Žmorphine.. These findings support a role of VLO in visceral nociception. Evidence has accumulated suggesting that VLO may be an important site for processing of nociceptive information. Cooper w5x has shown that application of local anesthetics to the VLO in rats decreases aversive responses to noxious stimulation; Tsubokawa et al. w18x have shown that electrical stimulation of C-fibers in the sciatic nerve or perfusion of bradykinin into the extremity in cats increases blood flow in VLO. In addition, it has been demonstrated that single neurons in VLO respond to noxious cutaneous w1,2,9,17x and visceral w10,17x stimulation. Further support for a role of VLO in visceral nociception is provided through noting its relationship to the Nucleus Submedius ŽSm. of medial thalamus. Anatomical studies in rats w4,20x and cats w6x have demonstrated that the Sm projects primarily to the VLO and that this cortical region projects reciprocally back to Sm. Sm has been demonstrated in numerous studies to be involved in nociception w3,7,8,12,15x. In a recent study w19x, we demonstrated that Sm neurons respond to noxious visceral stimulation ŽCRD.. Our present data show that the responses of VLO neurons to CRD have some physiologic properties similar to those we have observed for Sm neurons w19x, suggesting that the anatomical connections between VLO and Sm may have functional significance. In both sites, the CRD-responsive neurons demonstrate tendencies toward graded responses to graded CRD, and the responses can be attenuated by morphine in a dose-dependent fashion. Of the neurons which have cutaneous receptive fields, we have observed that in both Sm and VLO the majority Ž72% and 79%, respectively. demonstrate very large Žsometimes whole body. and usually bilateral cutaneous receptive fields. An additional 14% of Sm and 18% of VLO neurons have small receptive fields that are bilateral. Only 14% of Sm and 3% of VLO neurons have small contralateral or ipsilateral cutaneous receptive fields. These data suggest that both Sm and VLO neurons have little ability to encode stimulus location, and support a role of neurons of both sites in affective–motivational rather than discriminative aspect of nociception. We have observed that the majority of neurons in both Sm and VLO Ž90% and 94%, respectively. respond exclusively to noxious cutaneous stimulation. A few neurons in each site Ž10% in Sm, 6% in VLO. respond to both noxious and non-noxious cutaneous stimulation, but no Sm or VLO neurons respond exclusively to non-noxious stimulation. These characteristics of nociceptive-specific cutaneous responses indicate that Sm and VLO neurons have little ability to encode cutaneous stimulus intensity, and provide further support for the involvement of Sm and VLO neurons in affective–motivational aspects of nociception. Sm neurons demonstrate statistically significant
graded responses to graded CRD, suggesting the ability to encode stimulus intensities. In contrast, VLO neurons show a tendency toward graded responses to graded colorectal distension but the correlation is not statistically significant, suggesting a lesser ability to encode stimulus intensities. If VLO neurons encode the intensity of visceral stimulation, it may be based upon patterns of activity, since the grouped responses ŽTable 1. show a prominent trend toward greater responses at greater distension pressures. Based on the similarities of response properties of Sm and VLO neurons, and in view of data showing that VLO is the main efferent target of Sm in the cortex, it seems reasonable to postulate that Sm and VLO may be components of a pathway for the transmission of noxious visceral signals within brain, and this pathway might be important in subserving affective–motivational functions of nociception. Responses of VLO neurons differ from those of Sm neurons in several aspects. The proportion of neurons responding to CRD is lower in VLO than in Sm Ž55% and 83%, respectively.. Of the CRD-responsive neurons, a much higher proportion of inhibitory neurons has been noted in VLO Ž68%., contrasting with that in Sm Ž11%.. If the CRD-evoked responses of VLO neurons are nociceptive in nature, as we believe, then the responses should be attenuated by morphine. In the present study, responses of VLO neurons to noxious visceral stimulation were inhibited significantly by intravenous morphine in a dose-dependent fashion, providing additional support for the involvement of VLO in nociception. To our knowledge, this is the first report describing such an effect of morphine on the responses of VLO neurons to noxious visceral stimulation. In the VLO of rats, enkephalin-immunoreactive neurons have been demonstrated by McGinty et al. w14x. In the present study, we have observed that the effect of morphine on the evoked activities of VLO neurons can be reversed by intravenous naloxone, suggesting that the antinociceptive effect of morphine may be mediated by opioid receptors. The specific site at which morphine acts to attenuate VLO responses to noxious visceral stimulation is unknown, and could be at spinal andror supraspinal levels. In summary, our present data indicate that VLO neurons may be involved in visceral nociception. The data are most consistent with a role of VLO in affective–motivational aspects of nociception. Acknowledgements The authors thank Ms. Qiao-ling Cui for her assistance. References w1x M. Backonja, V. Miletic, Responses of neurons in the rat ventrolateral orbital cortex to phasic and tonic nociceptive stimulation, Brain Res. 557 Ž1991. 353–355.
S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105 w2x M. Backonja, B. Wang, V. Miletic, Responses of neurons in the ventrolateral orbital cortex to noxious cutaneous stimulation in a rat model of peripheral mononeuropathy, Brain Res. 639 Ž1994. 337– 340. w3x J.A. Coffield, V. Miletic, Responses of rat nucleus submedius neurons to enkephalins applied with micropressure, Brain Res. 630 Ž1993. 252–261. w4x J.A. Coffield, K.K. Bowen, V. Miletic, Retrograde tracing of projections between the nucleus submedius, the ventrolateral orbital cortex, and the midbrain in the rat, J. Comp. Neurol. 321 Ž1992. 488–499. w5x S.J. Cooper, Anaesthetisation of prefrontal cortex and response to noxious stimulation, Nature ŽLondon. 254 Ž1975. 439–440. w6x A.D. Craig, S.J. Weigand, J.L. Price, The thalamo-cortical projections of the nucleus submedius in the cat, J. Comp. Neurol. 206 Ž1982. 28–48. w7x J.O. Dostrovsky, G. Guilbaud, Nociceptive responses in medial thalamus of the normal and arthritic rat, Pain 40 Ž1990. 93–104. w8x J.O. Dostrovsky, G. Guilbaud, Noxious stimuli excite neurons in nucleus submedius of the normal and arthritic rat, Brain Res. 460 Ž1988. 269–280. w9x N. El-Yassir, J.O. Dostrovsky, Activation of neurones in the orbital region of rat cortex by noxious stimulation and by stimulation of nucleus submedius, Neurosci. Abstr. 16 Ž1990. 706. w10x K.A. Follett, B. Dirks, Responses of neurons in ventrolateral orbital cortex to noxious visceral stimulation in the rat, Brain Res. 669 Ž1995. 157–162. w11x B. Gernandt, Y. Zotterman, Intestinal pain: an electrophysiological investigation on mesenteric nerves, Acta Physiol. Scand. 12 Ž1946. 56–72.
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w12x K. Kawakita, J.O. Dostrovsky, J.S. Tang, C.Y. Chiang, Responses of neurons in the rat thalamic nucleus submedius to cutaneous, muscle and visceral nociceptive stimuli, Pain 55 Ž1993. 327–338. w13x H. Korn, Donnees sur le type fibres conduisant les afferences splanchniques au cortex orbitaire chez le chat, J. Physiol. ŽParis. 58 Ž1966. 243. w14x J.F. McGinty, D.V.D. Kooy, F.E. Bloom, The distribution and morphology of opioid peptide immunoreactive neurons in the cerebral cortex of rats, J. Neurosci. 4 Ž1984. 1104–1117. w15x V. Miletic, J.A. Coffield, Responses of neurons in the rat nucleus submedius to noxious and innocuous mechanical cutaneous stimulation, Somatosens. Motor Res. 6 Ž1989. 567–587. w16x T.J. Ness, G.F. Gebhart, Visceral pain: a review of experimental studies, Pain 41 Ž1990. 167–234. w17x P.J. Snow, B.M. Lumb, F. Cervero, The representation of prolonged and intense, noxious somatic and visceral stimuli in the ventrolateral orbital cortex of the cat, Pain 48 Ž1992. 89–99. w18x T. Tsubokawa, Y. Katayama, Y. Ueno, N. Moriyasu, Evidence for involvement of the frontal cortex in pain-related cerebral events in cats: increase in local cerebral blood flow by noxious stimuli, Brain Res. 217 Ž1981. 179–185. w19x S.W. Yang, K.A. Follett, J.G. Piper, T.J. Ness, The effect of morphine on responses of mediodorsal thalamic nuclei and nucleus submedius neurons to colorectal distension in the rat, Brain Res. 779 Ž1998. 41–52. w20x A. Yoshida, J.O. Dostrovsky, C.Y. Chiang, The afferent and efferent connections of the nucleus submedius in the rat, J. Comp. Neurol. 324 Ž1992. 115–133.
Short communication
The effect of morphine on responses of ventrolateral orbital cortex ŽVLO . neurons to colorectal distension in the rat Shou-wei Yang, Kenneth A. Follett
)
DiÕision of Neurosurgery, The UniÕersity of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA Accepted 28 July 1998
Abstract In 49 halothane-anesthetized rats, we characterized the responses of single neurons in the ventrolateral orbital cortex ŽVLO. to a noxious visceral stimulus Žcolorectal balloon distension, CRD., and studied the effects of intravenous morphine on these responses using standard extracellular microelectrode recording techniques. One hundred and four neurons were isolated on the basis of spontaneous activity. Fifty-seven Ž55%. responded to CRD, of which 32% had excitatory and 68% had inhibitory responses. Neurons showed tendencies toward graded responses to graded CRD pressures Ž20–100 mmHg., with maximum excitation or inhibition occurring at 80 or 100 mmHg, respectively. Responses to noxious Žpinch, heat. and innocuous Žbrush, tap. cutaneous stimuli were studied in 80 of the VLO neurons isolated. Thirty-three Ž41%. of these neurons Ž21 CRD-responsive and 12 CRD-nonresponsive. had cutaneous receptive fields, of which 79% were large and bilateral, 18% were small and bilateral, 3% were small and ipsilateral. Ninety-four percent of these neurons responded only to noxious cutaneous stimulation, 6% responded to both noxious and innocuous stimulation. No neurons responded solely to innocuous stimulation. Cumulative doses of morphine Ž0.0625, 0.125 and 0.25 mgrkg i.v.. produced statistically significant dose-dependent attenuation of neuronal responses to CRD. Naloxone Ž0.4 mgrkg i.v.. reversed the effects of morphine. Morphine and naloxone had no significant effects on spontaneous activity. These data support the involvement of VLO neurons in visceral nociception. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Visceral; Analgesia; Supraspinal; Opiate
Neurons in ventrolateral orbital cortex ŽVLO. respond to noxious stimuli, suggesting a role in nociception w1,2,9,10,17x. Most experimental studies of nociceptive properties of VLO neurons have been based on superficial or cutaneous pain models w1,2,9,17x. While data derived from studies of cutaneous nociception are important in building our knowledge of pain mechanisms, visceral pain is of greater clinical significance. The role of VLO neurons in visceral nociception is unclear and has received relatively little attention. Orbital cortex receives afferent input from the splanchnic nerve conveyed by A-d, g-fibers w13x. These small diameter fibers can be activated by application of natural noxious stimuli to intraperitoneal structures w11x, suggesting that VLO may play a role in visceral nociception. Direct evidence for the involvement of VLO in visceral nociception is provided by the observations in rats
) Corresponding author. Fax: q1-319-353-6605; E-mail: [email protected]
w10x and cats w17x that neurons respond to the noxious visceral stimulation produced by distension of the colon and gallbladder, respectively. Colorectal balloon distension ŽCRD. is a particularly useful animal model because it mimics acute intestinal obstruction and distension, a clinically significant cause of visceral pain, and has been established as a reliable, quantifiable, reproducible physiologic model of visceral pain, and has been used extensively to study visceral pain mechanisms w16x. In the present study, we have evaluated the responses of VLO neurons to CRD in halothane-anesthetized animals. In addition, we have studied the effects of intravenous morphine on VLO neuron responses evoked by CRD. If VLO responses to CRD are nociceptive, then we anticipate those responses would be attenuated by analgesic administration. Experiments were performed on 49 halothane-anesthetized male Sprague–Dawley rats ŽBiolab, St. Paul, MN. weighing 280–420 g. The procedure has been described previously w10x, with the exception that halothane was used for anesthesia in this study. Rats were administered 1–1.5%
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 0 4 - X
S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105
102
halothane via mask during initial surgical preparation, including placement of a tracheal cannula, and anesthesia was maintained at 0.5–0.7% inhaled halothane via the tracheal cannula during experimental observations. As described previously w10,19x, standard extracellular microelectrode recording techniques were used to isolate single neurons. We characterized the responses of single neurons to inflation of the colorectal balloon to a pressure of 80 mmHg. Three types of units were recognized according to their responses to the noxious visceral stimulation: CRD-excited, CRD-inhibited, and CRD-nonresponsive units. Each unit’s activity was measured for 10 s before, 20 s during, and 20 s after CRD and was evaluated as a peristimulus–time histogram with 1-s bins. A unit was deemed to respond to CRD if its activity changed Žduring CRD. at least 10% from its predistension baseline frequency. Stimulus–response curves were developed through testing 20, 40, 60, and 100 mmHg CRD for those units which responded to 80 mmHg CRD and could be held for a sufficiently long period of time. The location of a cutaneous receptive field for each neuron was determined qualitatively in response to innocuous brush and tap and noxious pinch Žforceps. and heat Žophthalmic cautery probe.. The dose-related effects of morphine Ž0.0625, 0.125, 0.25, 0.5 and 1 mgrkg i.v., cumulative doses. on responses to 80 mmHg CRD were studied in 15 inhibited and 4 excited neurons. For eight inhibited and three excited neurons in which CRD-evoked discharges were suppressed completely by morphine, the effect of subsequent naloxone Ž0.4 mgrkg i.v.. was studied. Data are expressed as the mean " S.E.M. Stimulus–response curves and differences among the effects of morphine doses were assessed by analysis of variance, using post-tests with Bonferroni correction to compare groups. A p-value less than 0.05 was considered statistically significant. At the conclusion of each experiment, animals were sacrificed by overdose of saturated KCl solution, and locations of recorded neurons were determined histologically. Data reported are only for those neurons that were confirmed histologically to be within VLO.
1. Responses of VLO neurons to CRD One hundred and four neurons isolated on the basis of spontaneous activity were studied. Fifty-seven of the 104
neurons Ž55%. responded to CRD. Eighteen of the CRDresponsive neurons Ž32%. had excitatory responses to CRD, 39 Ž68%. had inhibitory responses. Spontaneous neuronal activity remained relatively consistent over time Ž7.3 " 0.4 Hz, n s 15, continuous observation for 40 min.. Spontaneous discharge activity was not related significantly to time ŽANOVA, p ) 0.8.. Responses to CRD were independent of changes in spontaneous activity. For neurons excited by CRD, graded increases in distension pressure from 20 to 100 mmHg produced peak neuronal activity at 80 mmHg Žaverage increase to 40% greater than baseline. ŽTable 1.. For inhibited neurons, graded increases in CRD pressures caused maximal inhibition at 100 mmHg Žactivity attenuated to 60% of baseline. ŽTable 1.. Both excited and inhibited neurons showed tendencies toward graded changes in activity related to graded changes in distension pressures, but ANOVA analyses of the stimulus–response curves demonstrated no statistically significant dependence of responses on distension pressures ŽTable 1.. Eighty VLO neurons Ž39 CRD-responsive and 41 CRD-nonresponsive. were tested for cutaneous receptive fields. Twenty-one CRD-responsive and 12 CRD-nonresponsive neurons had cutaneous receptive fields, of which one Ž3%. was small and ipsilateral, six Ž18%. were small and bilateral, and 26 Ž79%. were large and bilateral Žsometimes whole body. cutaneous receptive fields. Ninety-four percent of the neurons with cutaneous receptive fields responded only to noxious pinch and heat. Two neurons Ž6%. responded to both noxious and non-noxious cutaneous stimulation. No neuron responded exclusively to innocuous cutaneous stimulation.
2. Effects of morphine on CRD-evoked activity of VLO neurons Morphine had statistically significant dose-dependent effects on CRD-evoked activity ŽTable 2.. For inhibited neurons, the change produced by morphine was statistically significant for the 0.25 mgrkg dose Ž p - 0.05.. For excited neurons, a trend toward dose-dependent attenuation by morphine was evident, but the small sample size precludes statistical significance Ž p ) 0.05.. For both inhibited and excited neurons, naloxone restored CRD-evoked responses to a level not statistically different from premorphine. Spontaneous activity was not altered significantly by any dose of morphine or by naloxone ŽTable 2..
Table 1 CRD-evoked activities ŽHz. of VLO neurons Response to CRD
Number of neurons
CRD ŽmmHg. 20
40
60
80
100
ANOVA
Excited Inhibited
6 9
0.4 " 0.9 y1.1 " 0.6
1.8 " 0.8 y1.5 " 0.9
3.6 " 1.5 y2.2 " 0.9
4.3 " 1.3 y3.1 " 0.8
3.8 " 0.8 y3.2 " 0.9
F4,23 s 1.730 F4,35 s 1.110
p s 0.178 p s 0.367
Neuronal activity
Response to CRD
Number of neurons
Baseline
Evoked
Inhibited Excited Inhibited Excited
15 4 15 4
y3.1"0.7 5.1"1.7 9.4"1.7 12.6"6.1
Spontaneous
) p- 0.05, post-test with Bonferroni correction.
Morphine Žmgrkg i.v.. 0.0625 0.125
0.25
y2.2"0.8 4.1"3.1 9.8"2.3 13.1"6.9
y0.01"0.5) 1.9"0.6 9.1"2.6 13.3"5.3
y1.6"0.5 2.2"0.8 8.2"1.8 14.5"5.0
0.5
1
1.6"0.5
y0.2"0.6
14.3"6.6
10.4"3.4
Naloxone Ž0.4 mgrkg.
ANOVA
y2.4"0.7 2.6"0.8 10.5"3.2 7.4"3.4
F4,50 s 2.728 ps 0.039 F6,14 s1.635 ps 0.210 F4,51 s 0.126 p) 0.8 F6,15 s 0.1759 p) 0.8
S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105
Table 2 Effects of morphine on CRD-evoked and spontaneous activities ŽHz. of VLO neurons
103
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S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105
Our present data show that VLO neurons respond to physiologic noxious visceral stimulation, CRD, and these responses can be attenuated significantly by an intravenous analgesic Žmorphine.. These findings support a role of VLO in visceral nociception. Evidence has accumulated suggesting that VLO may be an important site for processing of nociceptive information. Cooper w5x has shown that application of local anesthetics to the VLO in rats decreases aversive responses to noxious stimulation; Tsubokawa et al. w18x have shown that electrical stimulation of C-fibers in the sciatic nerve or perfusion of bradykinin into the extremity in cats increases blood flow in VLO. In addition, it has been demonstrated that single neurons in VLO respond to noxious cutaneous w1,2,9,17x and visceral w10,17x stimulation. Further support for a role of VLO in visceral nociception is provided through noting its relationship to the Nucleus Submedius ŽSm. of medial thalamus. Anatomical studies in rats w4,20x and cats w6x have demonstrated that the Sm projects primarily to the VLO and that this cortical region projects reciprocally back to Sm. Sm has been demonstrated in numerous studies to be involved in nociception w3,7,8,12,15x. In a recent study w19x, we demonstrated that Sm neurons respond to noxious visceral stimulation ŽCRD.. Our present data show that the responses of VLO neurons to CRD have some physiologic properties similar to those we have observed for Sm neurons w19x, suggesting that the anatomical connections between VLO and Sm may have functional significance. In both sites, the CRD-responsive neurons demonstrate tendencies toward graded responses to graded CRD, and the responses can be attenuated by morphine in a dose-dependent fashion. Of the neurons which have cutaneous receptive fields, we have observed that in both Sm and VLO the majority Ž72% and 79%, respectively. demonstrate very large Žsometimes whole body. and usually bilateral cutaneous receptive fields. An additional 14% of Sm and 18% of VLO neurons have small receptive fields that are bilateral. Only 14% of Sm and 3% of VLO neurons have small contralateral or ipsilateral cutaneous receptive fields. These data suggest that both Sm and VLO neurons have little ability to encode stimulus location, and support a role of neurons of both sites in affective–motivational rather than discriminative aspect of nociception. We have observed that the majority of neurons in both Sm and VLO Ž90% and 94%, respectively. respond exclusively to noxious cutaneous stimulation. A few neurons in each site Ž10% in Sm, 6% in VLO. respond to both noxious and non-noxious cutaneous stimulation, but no Sm or VLO neurons respond exclusively to non-noxious stimulation. These characteristics of nociceptive-specific cutaneous responses indicate that Sm and VLO neurons have little ability to encode cutaneous stimulus intensity, and provide further support for the involvement of Sm and VLO neurons in affective–motivational aspects of nociception. Sm neurons demonstrate statistically significant
graded responses to graded CRD, suggesting the ability to encode stimulus intensities. In contrast, VLO neurons show a tendency toward graded responses to graded colorectal distension but the correlation is not statistically significant, suggesting a lesser ability to encode stimulus intensities. If VLO neurons encode the intensity of visceral stimulation, it may be based upon patterns of activity, since the grouped responses ŽTable 1. show a prominent trend toward greater responses at greater distension pressures. Based on the similarities of response properties of Sm and VLO neurons, and in view of data showing that VLO is the main efferent target of Sm in the cortex, it seems reasonable to postulate that Sm and VLO may be components of a pathway for the transmission of noxious visceral signals within brain, and this pathway might be important in subserving affective–motivational functions of nociception. Responses of VLO neurons differ from those of Sm neurons in several aspects. The proportion of neurons responding to CRD is lower in VLO than in Sm Ž55% and 83%, respectively.. Of the CRD-responsive neurons, a much higher proportion of inhibitory neurons has been noted in VLO Ž68%., contrasting with that in Sm Ž11%.. If the CRD-evoked responses of VLO neurons are nociceptive in nature, as we believe, then the responses should be attenuated by morphine. In the present study, responses of VLO neurons to noxious visceral stimulation were inhibited significantly by intravenous morphine in a dose-dependent fashion, providing additional support for the involvement of VLO in nociception. To our knowledge, this is the first report describing such an effect of morphine on the responses of VLO neurons to noxious visceral stimulation. In the VLO of rats, enkephalin-immunoreactive neurons have been demonstrated by McGinty et al. w14x. In the present study, we have observed that the effect of morphine on the evoked activities of VLO neurons can be reversed by intravenous naloxone, suggesting that the antinociceptive effect of morphine may be mediated by opioid receptors. The specific site at which morphine acts to attenuate VLO responses to noxious visceral stimulation is unknown, and could be at spinal andror supraspinal levels. In summary, our present data indicate that VLO neurons may be involved in visceral nociception. The data are most consistent with a role of VLO in affective–motivational aspects of nociception. Acknowledgements The authors thank Ms. Qiao-ling Cui for her assistance. References w1x M. Backonja, V. Miletic, Responses of neurons in the rat ventrolateral orbital cortex to phasic and tonic nociceptive stimulation, Brain Res. 557 Ž1991. 353–355.
S.-w. Yang, K.A. Follettr Brain Research 808 (1998) 101–105 w2x M. Backonja, B. Wang, V. Miletic, Responses of neurons in the ventrolateral orbital cortex to noxious cutaneous stimulation in a rat model of peripheral mononeuropathy, Brain Res. 639 Ž1994. 337– 340. w3x J.A. Coffield, V. Miletic, Responses of rat nucleus submedius neurons to enkephalins applied with micropressure, Brain Res. 630 Ž1993. 252–261. w4x J.A. Coffield, K.K. Bowen, V. Miletic, Retrograde tracing of projections between the nucleus submedius, the ventrolateral orbital cortex, and the midbrain in the rat, J. Comp. Neurol. 321 Ž1992. 488–499. w5x S.J. Cooper, Anaesthetisation of prefrontal cortex and response to noxious stimulation, Nature ŽLondon. 254 Ž1975. 439–440. w6x A.D. Craig, S.J. Weigand, J.L. Price, The thalamo-cortical projections of the nucleus submedius in the cat, J. Comp. Neurol. 206 Ž1982. 28–48. w7x J.O. Dostrovsky, G. Guilbaud, Nociceptive responses in medial thalamus of the normal and arthritic rat, Pain 40 Ž1990. 93–104. w8x J.O. Dostrovsky, G. Guilbaud, Noxious stimuli excite neurons in nucleus submedius of the normal and arthritic rat, Brain Res. 460 Ž1988. 269–280. w9x N. El-Yassir, J.O. Dostrovsky, Activation of neurones in the orbital region of rat cortex by noxious stimulation and by stimulation of nucleus submedius, Neurosci. Abstr. 16 Ž1990. 706. w10x K.A. Follett, B. Dirks, Responses of neurons in ventrolateral orbital cortex to noxious visceral stimulation in the rat, Brain Res. 669 Ž1995. 157–162. w11x B. Gernandt, Y. Zotterman, Intestinal pain: an electrophysiological investigation on mesenteric nerves, Acta Physiol. Scand. 12 Ž1946. 56–72.
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