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Impairment of baroreceptor reflexes following kainic acid lesions of the lateral tegmental field
328
Brain Research, 618 (1993) 328-332 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25752
Impairment of baroreceptor reflexes following kainic acid lesions of the lateral tegmental field Mark E. Clement and Robert B. McCall CardioL,ascular Diseases Research, The Upjohn Co., Kalamazoo, M1 49001 (USA) (Accepted 20 April 1993)
Key words: Baroreceptor reflex; Lateral tegmental field; Kainic acid; Sympathetic activity; Cat
We examined the effects of kainic acid lesions of the lateral tegmental field on baroreceptor function in the anesthetized cat. Kainic acid lesions prevented the reflex inhibition of inferior cardiac sympathetic nerve activity observed during an increase in blood pressure. The temporal locking of sympathetic slow waves to the cardiac cycle was also abolished following lateral tegmental field lesions. Finally, the periodicity of sympathetic nerve discharge shifted to a higher frequency range following kainic acid lesions. These observations are consistent with the conclusion that lesions of the lateral tegmental field impair baroreceptor reflexes.
A large number of studies indicate that neurons in the rostral ventrolateral medulla are critical to the maintenance of sympathetic nerve activity. These sympathetic neurons integrate inputs from many 'autonomic nuclei' including the paraventricular nucleus, lateral hypothalamic area, parabrachial nucleus and the nucleus of the solitary tract (NTS) ~1. In turn, neurons of the rostral ventrolateral medulla project to sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord 5. Sympathetic neurons of the rostral ventrolateral medulla are contained within the sympathetic baroreceptor reflex pathway ~°. Anatomically, baroreceptor input arises from a direct pathway from the NTS to the rostral ventrolateral medulla 14 and an indirect pathway involving the caudal ventrolateral medulla. More recently the lateral tegmental field (LTF) has been proposed as a site for the generation of basal sympathetic nerve activity 1'6. Both chemical and electrical stimulation of the L T F increases nerve activity 4'6. The L T F contains both sympathoexcitatory and sympathoinhibitory neurons. Sympathoexcitatory neurons project to the rostral ventrolateral medulla and evidence exists to suggest that these L T F neurons may
provide a tonic excitatory input to rostral ventrolateral sympathoexcitatory neurons ~'6. Although L T F sympathetic neurons are under baroreflex control, there is little evidence to suggest that the L T F is an important relay in the baroreflex arc. Recently, we demonstrated that the sympathoinhibitory effects of 5-HT1A agonists are mediated, at least in part, in the L T F 3'4. During the course of this investigation we noted that kainic acid lesions of the LTF blocked the baroreceptormediated inhibition of sympathetic activity. The present study details this investigation. Twenty-nine cats (2.5-4.0 kg) received a pre-operative dose of ketamine-HC1 (11 m g / k g ) and were anesthetized by an intravenous injection of chloralose (80 m g / k g ) . Animals were placed in a David Kopf Instruments stereotaxic apparatus and spinal investigation unit, and a glass tracheal tube was inserted. A femoral artery and vein were cannulated to measure arterial blood pressure and to permit intravenous drug administration, respectively. A Fogarty embolectomy catheter was inserted in the opposite femoral artery to permit occlusion of the descending aorta. H e a r t rate was recorded continuously with a Grass 7P4 tachograph triggered by the electrocardiogram. Rectal temperature
Correspondence: R.B. McCall, The Upjohn Co. 7243-209-3, Kalamazoo, MI 49001, USA.
329 was maintained between 36 and 38°C with a heating pad a n d / o r lamp. The dorsal aspect of the medulla was exposed by removal of portions of the overlying occipital bone and cerebellum. Following surgery, animals were immobilized with gallamine triethiodide (4 mg/kg, i.v.) and artificially respired. No experiment lasted longer than the duration of the anesthetic. The obex was used as a surface landmark for placement of the microinjection pipette. Injections were made in an area in which we have previously found sympathetically related neurons in the LTF (i.e. 2.753.0 mm anterior to the obex, 2.8-3.0 mm lateral to the midline and 3.0 mm from the dorsal surface of the medulla). Pipette tip diameters were approximately 40 tzm. Microinjections of 12.5-25 nmol (50-100 nl) of saline, glutamic acid, or kainic acid were made by advancing the plunger of a 1 /zl Hamilton syringe connected to a David Kopf Microdrive. Pontamine sky blue was incorporated into the drug solutions for later identification injection sites. Injection times lasted approximately 5 min per site. Evoked potentials in cardiac nerve discharge produced by electrical (0.5 ms square wave, 5 V) stimulation of the RVLM were used to determine that the descending fibers from the RVLM remained intact. Peripheral sympathetic nerve activity was recorded from the central end of the sectioned left postganglionic inferior cardiac nerve. Potentials were recorded monophasically under mineral oil with a bipolar platinum electrode. A band pass of 1-1000 Hz was used to display the synchronized discharges of the whole sympathetic nerve in the form of slow waves on a Grass polygraph and quantitated using a Grass 7P10B cumulative integrator. Inferior cardiac sympathetic nerve activity, arterial blood pressure, EKG and pulses derived from the electrocardiogram were recorded on magnetic tape. Analog signals were subject to digital conversion and analyzed using in-house software. Methods of analysis included post R-wave average of sympathetic nerve activity, stimulus-triggered average of sympathetic activity and power spectral analysis of sympathetic nerve activity. The brainstem was removed at the end of each experiment. Frozen sagittal sections of 20 /xm thickness were cut with a cryostat microtome and stained with Nuclear fast red. Lesion and stimulation sites were identified in these sections according to the stereotaxic atlas of Berman. In two experiments the extent of the lesion was determined using the 2-deoxyglucose technique to visualize brain glucose metabolism 15. Bilateral microinjection of 50-100 nl kainic acid (12.5-25 nmol) into the LTF was generally followed by
an immediate slight transient decrease in SND (mean = 4.2% of control), although occasionally an immediate increase in sympathetic activity was observed. Pronounced increases in blood pressure were more consistently seen (mean = 25.4 + 7.3 mmHg). There were negligible changes in heart rate. Sympathetic activity rebounded to 120.5 + 16% of control values within 30 min of the microinjections, while blood pressure dropped to within 106 + 5.5% of baseline values. These parameters, along with heart rate, remained stable over the following 30 min. In contrast, microinjection of saline into the LTF failed to affect blood pressure, heart rate or sympathetic nerve activity, and glutamate microinjections had negligible effects on these parameters. Baroreceptor function was assessed by determining (1) sympathoinhibition during increases in blood pressure, (2) R-wave locking of sympathetic slow waves and (3) power spectra of sympathetic slow waves. Sympathetic activity was inhibited during the pressor response elicited by occlusion of the descending aorta in animals prior to microinjection of kainic acid and in animals receiving LTF microinjections of glutamic acid (Fig. 1A). In contrast, increases in blood pressure failed
A
!
°1 z
B
°1 Z
!
W
Fig. 1. LTF lesion blocks the baroreceptor-mediated inhibition of sympathetic nerve discharge (SND) during increases in arterial blood pressure (BP, mmHg). A: baroreflex sympathoinhibition prior to microinjection of kainic acid. B: same animal one hour following kainic acid microinjection into the LTF. Vertical calibration is 100 mV. Horizontal calibration is 1 min.
330 to inhibit sympathetic nerve activity in kainic acid induced LTF lesioned animals (Fig. 1B, n = 7). In untreated and sham animals, sympathetic nerve activity took the form of slow waves which were temporally locked to the cardiac cycle. This is evident by the R-wave triggered average of inferior cardiac sympathetic activity illustrated in Fig. 2A. Following kainic acid microinjection, sympathetic slow waves were no longer temporally related to the cardiac cycle (Fig. 2B). Fig. 3 illustrates the shift in the power spectrum of sympathetic activity following kainic acid microinjection into the LTF. Lesion of the LTF consistently resulted in an increase in power at higher frequencies of sympathetic activity. Histological examination of the microinjection sites revealed the extent of the diffusion of kainic acid to range from 300-500 /zm from the point of injection, with diffusion being slightly greater along the dorsoventral axis. Injection sites were typically 3 mm rostral to the obex, corresponding to the 10.8-10.0 frontal plane coordinates of Berman 2. The extent of the injection as indicated by the dispersion of pontamine sky blue dye was described by a roughly circular area approximately 400 ~ m in diameter (Fig. 4). The symmetrical nature of the injection site was reflected along the rostro-caudal axis. Similar results were seen in sham and glutamate injected animals. The extent of the kainic acid lesion was determined in two animals using the 2-deoxyglucose technique. Examination of injection sites in these animal revealed an area of increased glucose metabolism which was approximately 50% greater than the size indicated by dye diffusion. The affected area, however, remained confined to the A
B
-500
0 +500 msec Fig. 2. LTF lesion blocks the baroreceptor-mediated locking of sympathetic slow wave to the cardiac cycle. A: R-wave triggered average of sympathetic nerve activity before kainic acid microinjection. B: R-wave average of sympathetic activity in same animal taken one hour following kainic acid microinjection into the LTF. Vertical calibration is 150 mV.
1.0
no ,=
0
10
20
i
i
30
40
[Hz] Fig. 3. Power spectral analysis of the frequency components of sympathetic nerve activity (Hz) before (solid line) and following (dashed line) kainic acid lesion of the LTF. L T F lesions shifted the power spectra towards higher frequency components of sympathetic nerve discharge.
LTF. The integrity of the RVLM was supported by demonstration of discharges of the sympathetic nerve and associated pressor response during microstimulation of the RVLM. The present study investigated the importance of the LTF in the baroreceptor reflex. Microinjections of kainic acid were used to lesion the LTF. Kainic acid has been reported to destroy cell bodies while leaving the fibers of passage intact 12. Lesions in the current series of experiments were restricted to the LTF corresponding to the dorsomedial pressor area and including the region in which sympathetically related neurons have been identified by our laboratory and others. The integrity of the RVLM and the associated descending sympathoexcitatory pathway remained intact as evidenced by the evoked sympathetic nerve potentials which could be evoked by microstimulation of the RVLM. Histological examination indicated that the nucleus tractus solitarius was spared by the LTF kainic acid lesions. In this regard glucose metabolism was not altered in the NTS following kainic acid microinjections. Baroreflex function was determined by evaluating the reflex sympathoinhibition during pressor responses, the temporal locking of sympathetic slow waves to the cardiac cycle and the power spectra of sympathetic slow waves. We found that kainic acid lesions of the LTF mimicked baroreceptor denervation in that it abolished the baroreceptor-mediated inhibition of sympathetic nerve activity in response to an increase in blood pressure (Fig. 1). Baroreceptor denervation has also been demonstrated to temporally uncouple sympathetic slow waves and the cardiac cycle7. In the present study, R-wave triggered averages from untreated ani-
331 mals or animals receiving control microinjections of glutamate revealed that sympathetic slow waves were locked in a 1 : 1 fashion. Following kainic acid microinjection, however, sympathetic activity was no longer temporally related to the cardiac cycle (Fig. 2). The results of these experiments are similar to studies in which investigators found that lesion of the caudal ventrolateral medulla (CVL) in rats produced a transient decrease and then an increase in SND, HR, and MAP with a concomitant elimination of pulse synchronous fluctuations of sympathetic nerve activity 9. The injections sites in the present study however, are removed from the CVL and failed to produce the profound increase in sympathetic activity seen in the rodent model. Vayssettes-Courchay et al. have shown that microinjections of kainic acid into the CVL of the cat also abolishes the baroreceptor response. These lesions however, resulted in large increases in SND, HR, and MAP. Furthermore, these animals remained sensitive to the sympatholytic effects of 8 - O H - D P A T t6. These data indicate that in the cat both the CVL and
the L T F are important relays in the baroreceptor reflex arc. However, the LTF, but not the CVL, plays an important role in mediating the sympatholytic effect of 8-OH DPAT. Baroreceptor denervation has also been shown to increase the frequency of sympathetic nerve activity. We used power spectral analysis routines to determine the frequency components contained within sympathetic nerve activity. Prior to kainic acid lesion of the L T F sympathetic activity took the form of primarily 2 - 4 Hz slow waves. As previously shown with baroreceptor denervation 13, the power spectra of sympathetic activity shifted to higher frequencies following L T F lesioning (Fig. 3). The fact that the power spectra retained a large 2 - 6 Hz component suggests that the integrity of the L T F is not critical for the generation of this rhythm. In this regard, neurons with spontaneous discharges temporally related to the 2 - 6 Hz rhythm have been identified in at least two other brainstem regions 8. At any rate, the shift in the periodicity of nerve discharge strongly indicates that kainic acid mi-
Fig. 4. Photograph of a sagittal section of cat medulla 3 mm rostral to obex depicting typical 100 nl microinjectionof a kainic acid/pontamine blue solution. PH = nucleus praepositus hypoglossi;IOD = dorsal accessorynucleus of the inferior olive; IOP = principle nucleus of the inferior olive; 5SP = parvocellulardivision of the alaminar spinal trigeminal nucleus; arrow points to injection site.
332
croinjections into the LTF impair baroreceptor reflexes. Our data suggest that the impairment was severe. However, no attempt to quantitate the degree of baroreceptor loss was made in the present study. Acute baroreceptor denervation is typically associated with increases in arterial blood pressure and sympathetic nerve activity. In contrast, LTF lesions produced minimal cardiovascular effects. This is most easily explained by the heterogenous nature of the LTF. Previous studies indicate that the LTF contains both sympathoexcitatory and sympathoinhibitory neurons 1'3'6. Stimulation of the LTF produces either pressor or depressor responses. Thus kainic acid lesions of the LTF will evoke a complex cardiovascular response. Under these circumstances it is not surprising that blood pressure and sympathetic activity did not consistently increase following microinjections of kainic acid. It is difficult to rule out the possibility that kainic acid microinjection selectively destroyed the baroreceptor fibers arising from the NTS and projecting to the ventrolateral medulla. However, we have demonstrated that fibers projecting through the LTF from the rostral ventrolateral medulla to the spinal cord remain intact following kainic acid microinjection. By analogy, it is unlikely that baroreceptor fibers passing through the LTF were destroyed by the kainic acid microinjections. In conclusion, our data indicates that the LTF is an integral part of the baroreflex and lesions of this area produce an impairment of the baroreceptor effects on sympathetic activity. Moreover, our data implicates the LTF as a critical area in central autonomic regulation.
1 Barman, S.M. and Gebber, G.L., Lateral tegmental field neurons of cat medulla: a source of basal activity of ventral medullospinal sympathoexcitatory neurons, J. Neurophysiol., 57 (1987) 14101424. 2 Berman, A.L., The Brain Stern of the Cat, Univ. of Wisconsin Press, Madison, 1968.
3 Clement, M.E. and McCall, R.B., Evidence that the lateral tegmental field plays an important role in the central sympathoinhibitory action of 8-OH DPAT, Brain Res., 587 (1992) 115-122. 4 Clement, M.E. and McCall, R.B., Lateral tegmental field involvement in the central sympathoinhibitory action of 8-OH DPAT, Brain Res., in press. 5 Farlowe, D.M., Goodchild, A.K. and Dampney, R.A.L., Evidence that vasomotor neurons in the rostral ventrolateral medulla project to the spinal sympathetic outflow via the dorsomedial pressor area, Brain Res., 298 (1984) 313-320. 6 Gebber, G.L. and Barman, S.M., Lateral tegmental field neurons of cat medulla: a potential source of basal sympathetic discharge, J. Neurophysiol., 54 (1985) 1498-1512. 7 Gebber, G.L., Barman, S.M. and Kocsis, B., Coherence of medullary unit activity and sympathetic nerve discharge, Am. J. Physiol. (Regulatory Integrative Comp. Physiol. 28), 259 (1985) R561-R571. 8 Gebber, G.L., Taylor, D.G. and McCall, R.B., Organization of central vasomotor system. In Symposium on Mechanisms of Drug Action on Cardiovascular Control, Proc. Int. Congr. Pharmacol. 6th, 4: 49-58, 1975. 9 Granata, A.R., Numao, Y., Kumada, M. and Reis, D.J., A1 Noradrenergic neurons tonically inhibit neuron of the C1 area in rat, Brain Res., 377 (1986) 127-146. 10 Guyenet, P.G., Sun, M.K. and Brown, D.L., Role of GABA and excitatory amino acids in medullary baroreflex pathways. In J. Ciriello, F.R. Calaresu, L.P. Renaud and C. Polosa (Eds.), Organization of the autonomic nervous system: Central and peripheral mechanisms, Neurology and Neurobiology, Vol. 31, Alan R. Liss, New York, 1987, pp. 215-225. 11 McAllen, R.M., Dampney, R.A.L. and Goodchild, A.K., The sub-retrofacial nucleus and cardiovascular control. In J. Ciriello, F.R. Calaresu, L.P. Renaud and C. Polosa (Eds.), Organization of the autonomic nervous system: Central and peripheral mechanisms, Neurology and Neurobiology, Vol. 31, Alan R. Liss, New York, 1987, pp. 251-263. 12 McGeer, P.L. and McGeer, E.G., Kainic Acid: The neurotoxic breakthrough, CRC Crit. Rev. ToxicoL, 10 (1982) 1-26. 13 McCall, R.B. and Gebber, G.L., Differential effect of baroreceptor reflexes a n d clonidine on frequency components of sympathetic discharge, Eur. J. Pharmacol., 35 (1976) 69-78. 14 Ross, C.A., Ruggiero, D.A and Reis, D.J., Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla, J. Comp. Neurol., 242 (1985) 511-534. 15 Sokolff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O. and Shinohara, M., The 14-C deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure and normal values in the conscious and anesthetized albino rat, J. Neurochem., 28 (1977) 897-916. 16 Vayssettes-Courchay, C., Bouysset, F., Verbeuren, T.J. and Laubie, M., role of the nucleus tractus solitarii and the rostral depressive area in the sympatholytic effect of 8-OH-DPAT in the cat (in press).
Brain Research, 618 (1993) 328-332 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25752
Impairment of baroreceptor reflexes following kainic acid lesions of the lateral tegmental field Mark E. Clement and Robert B. McCall CardioL,ascular Diseases Research, The Upjohn Co., Kalamazoo, M1 49001 (USA) (Accepted 20 April 1993)
Key words: Baroreceptor reflex; Lateral tegmental field; Kainic acid; Sympathetic activity; Cat
We examined the effects of kainic acid lesions of the lateral tegmental field on baroreceptor function in the anesthetized cat. Kainic acid lesions prevented the reflex inhibition of inferior cardiac sympathetic nerve activity observed during an increase in blood pressure. The temporal locking of sympathetic slow waves to the cardiac cycle was also abolished following lateral tegmental field lesions. Finally, the periodicity of sympathetic nerve discharge shifted to a higher frequency range following kainic acid lesions. These observations are consistent with the conclusion that lesions of the lateral tegmental field impair baroreceptor reflexes.
A large number of studies indicate that neurons in the rostral ventrolateral medulla are critical to the maintenance of sympathetic nerve activity. These sympathetic neurons integrate inputs from many 'autonomic nuclei' including the paraventricular nucleus, lateral hypothalamic area, parabrachial nucleus and the nucleus of the solitary tract (NTS) ~1. In turn, neurons of the rostral ventrolateral medulla project to sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord 5. Sympathetic neurons of the rostral ventrolateral medulla are contained within the sympathetic baroreceptor reflex pathway ~°. Anatomically, baroreceptor input arises from a direct pathway from the NTS to the rostral ventrolateral medulla 14 and an indirect pathway involving the caudal ventrolateral medulla. More recently the lateral tegmental field (LTF) has been proposed as a site for the generation of basal sympathetic nerve activity 1'6. Both chemical and electrical stimulation of the L T F increases nerve activity 4'6. The L T F contains both sympathoexcitatory and sympathoinhibitory neurons. Sympathoexcitatory neurons project to the rostral ventrolateral medulla and evidence exists to suggest that these L T F neurons may
provide a tonic excitatory input to rostral ventrolateral sympathoexcitatory neurons ~'6. Although L T F sympathetic neurons are under baroreflex control, there is little evidence to suggest that the L T F is an important relay in the baroreflex arc. Recently, we demonstrated that the sympathoinhibitory effects of 5-HT1A agonists are mediated, at least in part, in the L T F 3'4. During the course of this investigation we noted that kainic acid lesions of the LTF blocked the baroreceptormediated inhibition of sympathetic activity. The present study details this investigation. Twenty-nine cats (2.5-4.0 kg) received a pre-operative dose of ketamine-HC1 (11 m g / k g ) and were anesthetized by an intravenous injection of chloralose (80 m g / k g ) . Animals were placed in a David Kopf Instruments stereotaxic apparatus and spinal investigation unit, and a glass tracheal tube was inserted. A femoral artery and vein were cannulated to measure arterial blood pressure and to permit intravenous drug administration, respectively. A Fogarty embolectomy catheter was inserted in the opposite femoral artery to permit occlusion of the descending aorta. H e a r t rate was recorded continuously with a Grass 7P4 tachograph triggered by the electrocardiogram. Rectal temperature
Correspondence: R.B. McCall, The Upjohn Co. 7243-209-3, Kalamazoo, MI 49001, USA.
329 was maintained between 36 and 38°C with a heating pad a n d / o r lamp. The dorsal aspect of the medulla was exposed by removal of portions of the overlying occipital bone and cerebellum. Following surgery, animals were immobilized with gallamine triethiodide (4 mg/kg, i.v.) and artificially respired. No experiment lasted longer than the duration of the anesthetic. The obex was used as a surface landmark for placement of the microinjection pipette. Injections were made in an area in which we have previously found sympathetically related neurons in the LTF (i.e. 2.753.0 mm anterior to the obex, 2.8-3.0 mm lateral to the midline and 3.0 mm from the dorsal surface of the medulla). Pipette tip diameters were approximately 40 tzm. Microinjections of 12.5-25 nmol (50-100 nl) of saline, glutamic acid, or kainic acid were made by advancing the plunger of a 1 /zl Hamilton syringe connected to a David Kopf Microdrive. Pontamine sky blue was incorporated into the drug solutions for later identification injection sites. Injection times lasted approximately 5 min per site. Evoked potentials in cardiac nerve discharge produced by electrical (0.5 ms square wave, 5 V) stimulation of the RVLM were used to determine that the descending fibers from the RVLM remained intact. Peripheral sympathetic nerve activity was recorded from the central end of the sectioned left postganglionic inferior cardiac nerve. Potentials were recorded monophasically under mineral oil with a bipolar platinum electrode. A band pass of 1-1000 Hz was used to display the synchronized discharges of the whole sympathetic nerve in the form of slow waves on a Grass polygraph and quantitated using a Grass 7P10B cumulative integrator. Inferior cardiac sympathetic nerve activity, arterial blood pressure, EKG and pulses derived from the electrocardiogram were recorded on magnetic tape. Analog signals were subject to digital conversion and analyzed using in-house software. Methods of analysis included post R-wave average of sympathetic nerve activity, stimulus-triggered average of sympathetic activity and power spectral analysis of sympathetic nerve activity. The brainstem was removed at the end of each experiment. Frozen sagittal sections of 20 /xm thickness were cut with a cryostat microtome and stained with Nuclear fast red. Lesion and stimulation sites were identified in these sections according to the stereotaxic atlas of Berman. In two experiments the extent of the lesion was determined using the 2-deoxyglucose technique to visualize brain glucose metabolism 15. Bilateral microinjection of 50-100 nl kainic acid (12.5-25 nmol) into the LTF was generally followed by
an immediate slight transient decrease in SND (mean = 4.2% of control), although occasionally an immediate increase in sympathetic activity was observed. Pronounced increases in blood pressure were more consistently seen (mean = 25.4 + 7.3 mmHg). There were negligible changes in heart rate. Sympathetic activity rebounded to 120.5 + 16% of control values within 30 min of the microinjections, while blood pressure dropped to within 106 + 5.5% of baseline values. These parameters, along with heart rate, remained stable over the following 30 min. In contrast, microinjection of saline into the LTF failed to affect blood pressure, heart rate or sympathetic nerve activity, and glutamate microinjections had negligible effects on these parameters. Baroreceptor function was assessed by determining (1) sympathoinhibition during increases in blood pressure, (2) R-wave locking of sympathetic slow waves and (3) power spectra of sympathetic slow waves. Sympathetic activity was inhibited during the pressor response elicited by occlusion of the descending aorta in animals prior to microinjection of kainic acid and in animals receiving LTF microinjections of glutamic acid (Fig. 1A). In contrast, increases in blood pressure failed
A
!
°1 z
B
°1 Z
!
W
Fig. 1. LTF lesion blocks the baroreceptor-mediated inhibition of sympathetic nerve discharge (SND) during increases in arterial blood pressure (BP, mmHg). A: baroreflex sympathoinhibition prior to microinjection of kainic acid. B: same animal one hour following kainic acid microinjection into the LTF. Vertical calibration is 100 mV. Horizontal calibration is 1 min.
330 to inhibit sympathetic nerve activity in kainic acid induced LTF lesioned animals (Fig. 1B, n = 7). In untreated and sham animals, sympathetic nerve activity took the form of slow waves which were temporally locked to the cardiac cycle. This is evident by the R-wave triggered average of inferior cardiac sympathetic activity illustrated in Fig. 2A. Following kainic acid microinjection, sympathetic slow waves were no longer temporally related to the cardiac cycle (Fig. 2B). Fig. 3 illustrates the shift in the power spectrum of sympathetic activity following kainic acid microinjection into the LTF. Lesion of the LTF consistently resulted in an increase in power at higher frequencies of sympathetic activity. Histological examination of the microinjection sites revealed the extent of the diffusion of kainic acid to range from 300-500 /zm from the point of injection, with diffusion being slightly greater along the dorsoventral axis. Injection sites were typically 3 mm rostral to the obex, corresponding to the 10.8-10.0 frontal plane coordinates of Berman 2. The extent of the injection as indicated by the dispersion of pontamine sky blue dye was described by a roughly circular area approximately 400 ~ m in diameter (Fig. 4). The symmetrical nature of the injection site was reflected along the rostro-caudal axis. Similar results were seen in sham and glutamate injected animals. The extent of the kainic acid lesion was determined in two animals using the 2-deoxyglucose technique. Examination of injection sites in these animal revealed an area of increased glucose metabolism which was approximately 50% greater than the size indicated by dye diffusion. The affected area, however, remained confined to the A
B
-500
0 +500 msec Fig. 2. LTF lesion blocks the baroreceptor-mediated locking of sympathetic slow wave to the cardiac cycle. A: R-wave triggered average of sympathetic nerve activity before kainic acid microinjection. B: R-wave average of sympathetic activity in same animal taken one hour following kainic acid microinjection into the LTF. Vertical calibration is 150 mV.
1.0
no ,=
0
10
20
i
i
30
40
[Hz] Fig. 3. Power spectral analysis of the frequency components of sympathetic nerve activity (Hz) before (solid line) and following (dashed line) kainic acid lesion of the LTF. L T F lesions shifted the power spectra towards higher frequency components of sympathetic nerve discharge.
LTF. The integrity of the RVLM was supported by demonstration of discharges of the sympathetic nerve and associated pressor response during microstimulation of the RVLM. The present study investigated the importance of the LTF in the baroreceptor reflex. Microinjections of kainic acid were used to lesion the LTF. Kainic acid has been reported to destroy cell bodies while leaving the fibers of passage intact 12. Lesions in the current series of experiments were restricted to the LTF corresponding to the dorsomedial pressor area and including the region in which sympathetically related neurons have been identified by our laboratory and others. The integrity of the RVLM and the associated descending sympathoexcitatory pathway remained intact as evidenced by the evoked sympathetic nerve potentials which could be evoked by microstimulation of the RVLM. Histological examination indicated that the nucleus tractus solitarius was spared by the LTF kainic acid lesions. In this regard glucose metabolism was not altered in the NTS following kainic acid microinjections. Baroreflex function was determined by evaluating the reflex sympathoinhibition during pressor responses, the temporal locking of sympathetic slow waves to the cardiac cycle and the power spectra of sympathetic slow waves. We found that kainic acid lesions of the LTF mimicked baroreceptor denervation in that it abolished the baroreceptor-mediated inhibition of sympathetic nerve activity in response to an increase in blood pressure (Fig. 1). Baroreceptor denervation has also been demonstrated to temporally uncouple sympathetic slow waves and the cardiac cycle7. In the present study, R-wave triggered averages from untreated ani-
331 mals or animals receiving control microinjections of glutamate revealed that sympathetic slow waves were locked in a 1 : 1 fashion. Following kainic acid microinjection, however, sympathetic activity was no longer temporally related to the cardiac cycle (Fig. 2). The results of these experiments are similar to studies in which investigators found that lesion of the caudal ventrolateral medulla (CVL) in rats produced a transient decrease and then an increase in SND, HR, and MAP with a concomitant elimination of pulse synchronous fluctuations of sympathetic nerve activity 9. The injections sites in the present study however, are removed from the CVL and failed to produce the profound increase in sympathetic activity seen in the rodent model. Vayssettes-Courchay et al. have shown that microinjections of kainic acid into the CVL of the cat also abolishes the baroreceptor response. These lesions however, resulted in large increases in SND, HR, and MAP. Furthermore, these animals remained sensitive to the sympatholytic effects of 8 - O H - D P A T t6. These data indicate that in the cat both the CVL and
the L T F are important relays in the baroreceptor reflex arc. However, the LTF, but not the CVL, plays an important role in mediating the sympatholytic effect of 8-OH DPAT. Baroreceptor denervation has also been shown to increase the frequency of sympathetic nerve activity. We used power spectral analysis routines to determine the frequency components contained within sympathetic nerve activity. Prior to kainic acid lesion of the L T F sympathetic activity took the form of primarily 2 - 4 Hz slow waves. As previously shown with baroreceptor denervation 13, the power spectra of sympathetic activity shifted to higher frequencies following L T F lesioning (Fig. 3). The fact that the power spectra retained a large 2 - 6 Hz component suggests that the integrity of the L T F is not critical for the generation of this rhythm. In this regard, neurons with spontaneous discharges temporally related to the 2 - 6 Hz rhythm have been identified in at least two other brainstem regions 8. At any rate, the shift in the periodicity of nerve discharge strongly indicates that kainic acid mi-
Fig. 4. Photograph of a sagittal section of cat medulla 3 mm rostral to obex depicting typical 100 nl microinjectionof a kainic acid/pontamine blue solution. PH = nucleus praepositus hypoglossi;IOD = dorsal accessorynucleus of the inferior olive; IOP = principle nucleus of the inferior olive; 5SP = parvocellulardivision of the alaminar spinal trigeminal nucleus; arrow points to injection site.
332
croinjections into the LTF impair baroreceptor reflexes. Our data suggest that the impairment was severe. However, no attempt to quantitate the degree of baroreceptor loss was made in the present study. Acute baroreceptor denervation is typically associated with increases in arterial blood pressure and sympathetic nerve activity. In contrast, LTF lesions produced minimal cardiovascular effects. This is most easily explained by the heterogenous nature of the LTF. Previous studies indicate that the LTF contains both sympathoexcitatory and sympathoinhibitory neurons 1'3'6. Stimulation of the LTF produces either pressor or depressor responses. Thus kainic acid lesions of the LTF will evoke a complex cardiovascular response. Under these circumstances it is not surprising that blood pressure and sympathetic activity did not consistently increase following microinjections of kainic acid. It is difficult to rule out the possibility that kainic acid microinjection selectively destroyed the baroreceptor fibers arising from the NTS and projecting to the ventrolateral medulla. However, we have demonstrated that fibers projecting through the LTF from the rostral ventrolateral medulla to the spinal cord remain intact following kainic acid microinjection. By analogy, it is unlikely that baroreceptor fibers passing through the LTF were destroyed by the kainic acid microinjections. In conclusion, our data indicates that the LTF is an integral part of the baroreflex and lesions of this area produce an impairment of the baroreceptor effects on sympathetic activity. Moreover, our data implicates the LTF as a critical area in central autonomic regulation.
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