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The site of the origin of the so-called fastigial pressor response
352
Brain Research, 473 (1988) 352-358 Elsevier
BRE 23192
Short Communications
The site of the origin of the so-called fastigial pressor response Mitsuhiko Miura and Kiyoshige Takayama Department of Physiology, 1st Division, Gunma University School of Medicine, Showa-machi, Maebashi (Japan)
(Accepted 26 July 1988) Key words: Fastigial nucleus; Parabrachial nucleus; Electrical stimulation; Blood pressure; Heart rate; Respiration; Autonomic nervous system; Cardiovascular system
The cerebellar nuclei and subnuclear areas of anesthetized cats were electrically stimulated with glass microelectrodes to explore the origin of the so-called fastigiai pressor response (FPR). The FPR Was not derived from the fastigial nucleus per se, but from the subfastigial fiber bundle projecting into the pontine lateral parabrachial nucleus (Pbl). The microinjection of kainic acid did not facilitate the pressor response from the subfastigial fiber bundle, but facilitated the pressor response from the Pbl. When WGA-HRP was deposited in the pressor response site of the Pbl, labeling was found in axons of the subfastigial fiber bundle and in the fiber terminals and cells of the contralateral Pbl. This suggests that the FPR originates in the pressor response site in the Pbl which projects into the contralateral Pbl via the subfastigial fiber bundle. The so-called fastigial pressor response (FPR), a pressor-tachycardiac response which is elicited by electrical stimulation of the rostro-ventro-medial part of the fastigial nucleus (FN), was reported by Miura and Reis 17 and Achari and D o w n m a n 1 in 1969. Since then, there have been many investigations to determine the physiological role of the F P R and the efferent connections involved in the F P R 2. Recently, Chida et al. 5 reported conflicting evidence that chemical stimulation of the rat FN elicited a depressor-bradycardiac response (FDR). We examined the reproducibility of the F D R in the cat, but found no evidence to verify the existence of the FDR. T h e s e conflicting results between the previous studies of the FPR and F D R urged us to re-examine the site of the origin of the FPR. A preliminary report has been published e l s e w h e r e is. Experiments were performed with 15 cats of either sex weighing 2.5-4.5 kg. Anesthesia was induced by ketamine hydrochloride (30 mg/kg, i.m.) and maintained by chloralose (initially 10 mg/kg i.v., with an additional 5 mg/kg every hour). The trachea was cannulated with vinyl tubing (length 7 cm, diameter 7
mm) to sample airway gas (Beckman, LB2 and O M l l ) and to measure the respiratory gas flow rate and tidal volume (Nihon Kohden, AQ-601G and AR-601G). The femoral artery was cannulated to measure the arterial blood pressure. The heart rate was counted with a cardiotachometer triggered by the R wave of the E C G . The arterial blood pressure, heart rate, mean arterial blood pressure, CO2 and 02 concentration in the airway gas, respiratory gas flow rate and tidal volume were continuously displayed on a polygraph. The rectal temperature was maintained at 37 °C by means of a heating lamp. Each animal was placed in a stereotaxic frame with the head flexed at 45 °. A line of small holes was drilled at the appropriate site in the parietal bone above the nuchal ridge. The dorsal surface of the cerebellum was penetrated by a glass microelectrode of the double-barrel coaxial type. The tip of the electrode was beveled obliquely to 18-20 # m at the minor axis and 40-60 # m at the major axis. The inner electrode was filled with a 3 M NaCI solution and the outer electrode with a 0.025 M Tris-HCI buffer (pH 8.6) containing 6% W G A - H R P (Toyobo) and/or 5 m M kainic acid
Correspondence." M. Miura, Department of Physiology, 1st Division, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, 371 Japan.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
353 ( N a k a r a i C h e m . ) . The tip resistance ranged from i to 2 M£2. T h e inner electrode was used to stimulate a small part of the brain, and the o u t e r electrode to inject either kainic acid as a stimulant or W G A - H R P as a m a r k e r or a deposit. The effects of the electrical stimulation of a small part of the brain on blood pressure, heart rate and respiration were e x a m i n e d by a train (6 s) of square pulses (0.5 ms, 5/~A, 50 Hz). To assess the effect of kainic acid on the cerebellar response, the electrode was placed at the site of the potent cerebellar response and kainic acid was injected through the electrode by passing a D C cathodal current of 10 # A from a constant current supply (Nihon Kohden) in 20 repetitive cycles of 0.2 s on, 0.3 s off (40 # A . s ) . W G A - H R P was injected for marking by passing a positive electrical current of 4 # A for 2 min,
A
¢'1
,, C,]N-,
and for depositing by passing a 4 # A current for 200 ms on and 300 ms off for a period of 30 min. Each animal was perfused transcardially by a conventional method. The brain was r e m o v e d , fixed, frozen and sectioned serially at 50 #m. The sections were then processed for the histochemical demonstration of W G A - H R P deposits 14'15. First, we a t t e m p t e d to m a p the precise loci of the potent F P R assessed by electrical stimulation of the brain. Since the potent F P R , i.e., elevation of mean arterial pressure over 15 m m H g , was always associated with a hyperpneic TM and/or tachypneic response 13, we a d o p t e d a potent pressor-hyperpneic response as an indicator. The electrode was inserted from the dorsal surface of lobule V I I A or V I I B and the subcortical area was stimulated at 200 # m steps.
Stim ~--J_l~l ~stl i~ I I
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Fig. 1. Map of sites from which the potent so-called fastigial pressor response (FPR) was elicited by electrical microstimulation of the brain. A - H : frontal sections from the level of the rostral fastigial nucleus to that of the lateral parabrachial nucleus. Small dots indicate sites of the 15-20 mmHg rise in the mean arterial pressure and large dots those of the 20-50 mmHg rise. I: enlarged original microphotograph of D (left) and responses of blood pressure (BP), heart rate (HR) and tidal volume (VT) from the site indicated by an arrow in the subfastigial fiber bundle (right). J: enlarged original microphotograph of H (left) and responses from the site indicated by an arrow in the lateral parabrachial nucleus (right). Scale bar in I: 1 mm. Abbreviations: BC, brachium conjunctivum; DN, dentate nucleus; FN, fastigial nucleus; IN, interposed nucleus; Pbl, lateral parabrachial nucleus; RB, restiform body.
354 In total, 133 tracks from 15 cats, sites of the potent response, were marked by depositing WGA-HRP. Fig. 1 shows the distribution of sites of the potent response, as indicated by black dots mapped on a series of the frontal sections of the brain. Caudomedially, as shown in Fig. 1 A - D , the potent responses were derived from the white matter between the FN and
the lingula cerebelti. Fig. 11, an enlarged original photomicrograph of Fig. 1D, shows the sites of the maximal response in each track marked by a small deposit of WGA-HRP. The responses recorded at the site indicated by an arrow shows potent pressor, tachycardiac and hyperpneic responses. Rostrolaterally, as shown in Fig. 1 E - H , the potent responses
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Fig. 2. Effects of kainic acid on the so-called fastigial pressor response elicited by barely suprathreshold stimulation (5 # A , 50 Hz and 6 s). Left: sites of the tested response as indicated by the arrow. Right: responses before and 8 min after an injection of kainic acid. A: non-facilitation of the responses from the subfastigial fiber bundle. B: facilitation of the responses from the lateral parabrachial nucleus. Scale bar in A: 1 ram.
355 were derived from a subfastigial fiber bundle projecting into the dorsal part to the pontine brachium conjunctivum, i.e., the central part of the lateral parabrachial nucleus (Pbl). Fig. 1J, an enlarged original photomicrograph of Fig. 1H, shows 4 marks which are lined in the central part of the Pbl. The responses recorded at the site indicated by an arrow shows responses similar to those elicited in the subfastigial fiber bundle. No response was recorded in the FN per se.
Second, we attempted to determine whether such a potent response may originate from cells or passing fibers. Since a few cells are distributed in the subfastigial fiber bundle, it is probable that these cells originate in the potent response. Because it is expected that cells in subliminal fringe may be activated to dis-
charge by a combination of conditioning-testing stimulations, a small amount of kainic acid was injected as a conditioning chemical stimulation into the site of the potent response, and a change in excitability of the response was serially assessed by a testing electrical stimulation. Fig. 2A shows that no facilitation of the response was elicited (right) at the cell-rich site in this bundle, as indicated by an arrow in the original microphotograph (left), suggesting that the response does not originate from cells but from passing fibers in the subfastigial fiber bundle. On the other hand, as shown in Fig. 2B, the response to the testing electrical stimulation of the central part of the Pbl was maximally facilitated 8 min after a conditioning kainic acid stimulation. This suggests that the response originates from cells in the Pbl.
Fig. 3. Dark-field microphotographs of frontal sections of rostral pons and cerebellum. A: sites of WGA-HRP injection marked by
brown reaction products of DAB, located in the central part of the lateral parabrachial nucleus. B and D: WGA-HRP labeled fibers traversing white matter under the fastigiai nucleus at the level of rostral pole of the fastigial nucleus (B) and 0.4 mm caudal to it (D). C and E: enlarged microphotographs of the part indicated by white frames in B and D, respectively. Scale bars in A, B and D: 500pm; in C and E: 100/zm.
356 Third, we attempted to determine whether fibers projecting to and from the Pbl may pass the subfastigial white matter. Fig. 3A shows the site of the response in the central part of the Pbl in which a small amount of W G A - H R P was deposited. A part of labeled fibers traversing within the subfastigial fiber bundle was observed both in the area ventrolateral to the rostral pole of the FN (Fig. 3B,C) and the area adjoining the caudal end of the lingula cerebelli (Fig. 3D,E). The course of labeled fibers appeared to accord with the trajectory of sites of the potent responses (see Fig. 1). Both labeled cells and fiber ter-
minals were also observed in the contralateral Pbl (Fig. 4). These results indicate that some neurons in the Pbl give off an efferent pathway projecting to the other side of the Pbl and some of the other neurons receive an efferent pathway coming from the other side of the Pbl via the subfastigial fiber bundle. Therefore, the subfastigial fiber bundle contains countercurrent efferent pathways from both sides of the Pbl. Thus, we have obtained the evidence that the socalled FPR does not originate in the FN, but in the Pbl. This evidence, however, contrasts with the re-
Fig. 4. Dark-field microphotographs of frontal sections of the rostral pons, A: WGA-HRP labeled cells and fiber terminals in the lateral parabrachial nucleus of the contralateral side to the WGA-HRP deposit. B and C: enlarged microphotographs of the part indicated by white arrows in A. Scale bars in A: 200 am; in B and C: 100 pm.
357 cent report by Chida et al. 5. They showed that in rats injection of kainic acid into the FN elicited a depressor and a bradycardiac response. In our preliminary experiment, we could not confirm such responses except when kainic acid leaked into the underlying fourth ventricle. It is highly probable that such an accidental episode may influence the dorsal surface of the medulla oblongata, and excite inhibitory neurons acting on the cardiovascular modulatory pathways. Concerning the sites of the FPR, this study disclosed uncertain results as reported in previous studies. So far, the F1N has been stimulated mostly by an iron wire electrode, and the tip of the electrode was marked by procian blue reactions or small electrolytic lesions. Since the tip of the electrode was large and dull, it always pressed and injured the neural tissues. Such metal electrode caused an inaccurate marking. But use of a glass microelectrode with a sharp tip minimized injury and distortion of the neural tissues and increased the accuracy of marking with a small amount of WGA-HRP. A few anatomical studies have suggested the presence of the parabrachial projection to the cerebellum. Somana and Walberg 26, in the HRP study of cats, found that the cerebellar vermal region receives the bulk of afferent fibers from some neurons in the Pbl. Saper and Loewy25, in the autoradiography of rats, showed that some fibers from the Phi cross the midline of the pons and contribute to a terminal field in the contralateral Pbl, and that some fibers project into the interposed nucleus and the cerebellar white matter but they gave no definite evidence for the presence of the terminal distribution of these fibers. And they did not show any evidence for the presence of the mutual innervation between both sides of the Pbl via the subfastigial fiber bundle. There have been several lines of evidence that properties of the FPR are essentially similar to those of the pressor response derived from the Pbl: (1) the cardiovascular effect of FPR 1'17 and that of the parabrachial pressor response 2°, (2) augmented vasopressin secretion during FPR 7 and the parabrachial pressot response 27, and (3) a hyperpneic and/or tachypneic response associated with the FPR and the parabrachial pressor response, which was verified in this study. The other responses associated with the FPR,
like a predatory attack behavior 24 and an increase in the cerebral blood flow11'22, may be reproduced during the parabrachial pressor response, although a recent study ~9opposed the latter possibility. Recently, there have been many investigations to seek afferent and efferent connections of the parabrachial area including the lateral and medial parabrachial nuclei and the Koelliker-Fuse nucleus. The parabrachial area receives not only ascending visceral information from the nucleus tractus solitarii 1°,12, the buffer nerves 3 and the lateral tegmental field a°, but also descending visceral projections from the amygdaloid nuclei 9'11'28. On the other hand, the parabrachial area sends not only ascending projections to the amygdaloid nuclei 4'28 and the paraventricular nucleus 6, but also descending projections to the nucleus tractus solitarii s,23.25, the ventrolateral medulla 25 and the intermediolateral nucleus of the spinal cord 16. In addition to these longitudinal connections which may have a key role in relaying and integrating information from both ascending and descending pathways, another connection between the two sides of the Pbl has been discovered and it may have an important role in balancing the two sides of neural activities subserving cardiovascular and respiratory control. In conclusion: (1) The site of the so-called FPR to electrical stimulation does not originate in the FN, but in the subfastigial fiber bundle. (2) Facilitation by conditioning kainic acid stimulation did not appear in the response elicited from the subfastigial fiber bundle, but in the response elicited from the Pbl. (3) When WGA-HRP was deposited at the site of the parabrachial pressor response, labeling was positive in the subfastigial fiber bundle and in the contralateral Phi. (4) Both sides of the Pbl are innervated mutually via commissure fibers traversing the subfastigial white matter. The mutual innervation of the two sides of the Pbl is a favorable architecture for balancing information, because an inflow to one side of the Pbl can be distributed to the other side. (5) Since the Pbl is a relay station of the countercurrent pathway of the neural information, the outflow of the so-called FPR may pass on to the suprapontine visceral integrating center and/or medullary and spinal visceral efferent center.
358 1 Achari, N.K. and Downman, C.B.B., Autonomic responses evoked by stimulation of fastigial nuclei in the anaesthetized cat, J. Physiol. (Lond.), 204 (1969) 130-131. 2 Batton, R.R., Jayaraman, A., Ruggiero, D. and Carpenter, M.B., Fastigial efferent projections in the monkey: an autoradiographic study, J. Comp. Neurol., 174 (1979) 281-306. 3 Cechetto, D.F. and Calaresu, F.R., Parabrachial units responding to stimulation of buffer nerve and forebrain in the cat, Am. J. Physiol., 245 (1983) R811-R819. 4 Cechetto, D.F., Ciriello, J. and Calaresu, F.R., Afferent connections to cardiovascular site in the amygdala: a horseradish peroxidase study in the cat, J. Auton. Nerv. Syst., 8 (I983) 97-110. 5 Chida, K., Iadecola, C., Underwood, M.D. and Reis, D J . , A novel vasodepressor response elicited from the rat cerebellar fastigial nucleus: the fastigial depressor response, Brain Research, 370 (1986) 378-382. 6 Ciriello, J., Lawrence, D. and Pittman, Q., Electrophysiological identification of neurons in the parabrachial nucleus projecting directly to the hypothalamus in the rat, Brain Research, 322 (1984) 388-392. 7 Del-Bo, A., Sved, A.F. and Reis, D.J., Fastigial stimulation releases vasopressin in amounts that elevate arterial pressure, Am. J. Physiol., 244 (1983) H687-H694. 8 Hamilton, R.B., Ellenberger, H., Liskowsky, D. and Schneiderman, N., Parabrachial area as mediator of bradycardia in rabbits, J. Auton. Nerv. Syst., 4 (1981) 261-281. 9 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 10 King, G.W., Topology of ascending brainstem projections to nucleus parabrachialis in the cat, J. Comp. Neurol., 191 (1980) 615-638. 11 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol., 178 (1978) 225-254. 12 Loewy, A.D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat, J. Comp. Neurol., 181 (1978) 421-450. 13 Lutherer, L.O. and Williams, J.L., Stimulating fastigial nucleus pressor region elicits patterned respiratory responses, Am. J. Physiol.. 250 (1986) R418-R426. 14 Malmgren, L. and Olsson, Y., A sensitive method for histochemical demonstration of horseradish peroxidase in neurons following retrograde axonal transport, Brain Research, 148 (1978) 279-294. 15 Mesulam, M.-M. and Rosene, K.L., Sensitivity in horse-
radish peroxidase neurohistochemistry: a comparative and quantitative study of nine methods, J. Histochem. Cvtochem., 27 (1979) 763-773. 16 Miura, M., Onai, T. and Takayama, K., Projections of upper structure to the spinal cardioacceleratory center in cats: an HRP study using a new microinjection method, J. Auton. Nerv. Syst., 7 (1983) 119-139. 17 Miura, M. and Reis, D.J., Cerebellum: a pressor response elicited from the fastigial nucleus and its efferent pathways in brainstem, Brain Research, 13 (1969) 595-599. 18 Miura, M. and Takayama, K., Origin of so-called fastigial pressor response, Neurosci. Res., Suppl. 7 (1988) $34. 19 Mraovitch, S., Iadecola, C., Ruggiero, D.A. and Reis, D.3., Widespread reductions in cerebral blood flow and metabolism elicited by electrical stimulation of the parabrachial nucleus in rat, Brain Reseach, 341 (1985) 283-296. 20 Mraovitch, S., Kumada, M. and Reis, D.J., Role of the nucleus parabrachialis in cardiovascular regulation in cat, Brain Research, 232 (1982) 57-75. 21 Nakai, M., Iadecola, C. and Reis, D.J., Global cerebral vasodilation by stimulation of rat fastigial cerebellar nucleus, Am. J. Physiol., 243 (1982) H226-H235. 22 Nakai, M., Iadecola, C., Ruggiero, D.A., Tucker, L.W. and Reis, D.J., Electrical stimulation of cerebellar fastigial nucleus increases cerebral cortical blood flow without change in local metabolism: evidence for an intrinsic system in brain for primary vasodilation, Brain Research, 260 (1983) 35-5/). 23 Onai, T., Takayama, K. and Miura, M., Projections to areas of the nucleus tractus solitarii related to circulatory and respiratory responses in cats, J. Auton. Nerv. Syst., 18 (1987) 163-175. 24 Reis, D.J., Doba, N. and Nathan, M.A., Predatory attack, grooming, and consummatory behaviours evoked by electrical stimulation of cat cerebellar nuclei, Science, 182 (1973) 845-847. 25 Saper, C.B. and Loewy, A.D., Efferent connections of the parabrachial nucleus in the rat, Brain Research, I97 (1980) 291-317. 26 Somana, R. and Walberg, F., The cerebellar projection from the parabrachial nucleus in the cat, Brain Research. 172 (1979) 144-149. 27 Sved, A.F., Pontine pressor sites which release vasopres: sin, Brain Research, 369 (1986) 143-150. 28 Takeuchi, Y., McLean, J.H. and Hopkins, D.A., Reciprocal connections between the amygdala and parabrachial nuclei: ultrastructural demonstration by degeneration and axonal transport of horseradish peroxidase in the cat, Brain Research, 239 (1982) 583-588.
Brain Research, 473 (1988) 352-358 Elsevier
BRE 23192
Short Communications
The site of the origin of the so-called fastigial pressor response Mitsuhiko Miura and Kiyoshige Takayama Department of Physiology, 1st Division, Gunma University School of Medicine, Showa-machi, Maebashi (Japan)
(Accepted 26 July 1988) Key words: Fastigial nucleus; Parabrachial nucleus; Electrical stimulation; Blood pressure; Heart rate; Respiration; Autonomic nervous system; Cardiovascular system
The cerebellar nuclei and subnuclear areas of anesthetized cats were electrically stimulated with glass microelectrodes to explore the origin of the so-called fastigiai pressor response (FPR). The FPR Was not derived from the fastigial nucleus per se, but from the subfastigial fiber bundle projecting into the pontine lateral parabrachial nucleus (Pbl). The microinjection of kainic acid did not facilitate the pressor response from the subfastigial fiber bundle, but facilitated the pressor response from the Pbl. When WGA-HRP was deposited in the pressor response site of the Pbl, labeling was found in axons of the subfastigial fiber bundle and in the fiber terminals and cells of the contralateral Pbl. This suggests that the FPR originates in the pressor response site in the Pbl which projects into the contralateral Pbl via the subfastigial fiber bundle. The so-called fastigial pressor response (FPR), a pressor-tachycardiac response which is elicited by electrical stimulation of the rostro-ventro-medial part of the fastigial nucleus (FN), was reported by Miura and Reis 17 and Achari and D o w n m a n 1 in 1969. Since then, there have been many investigations to determine the physiological role of the F P R and the efferent connections involved in the F P R 2. Recently, Chida et al. 5 reported conflicting evidence that chemical stimulation of the rat FN elicited a depressor-bradycardiac response (FDR). We examined the reproducibility of the F D R in the cat, but found no evidence to verify the existence of the FDR. T h e s e conflicting results between the previous studies of the FPR and F D R urged us to re-examine the site of the origin of the FPR. A preliminary report has been published e l s e w h e r e is. Experiments were performed with 15 cats of either sex weighing 2.5-4.5 kg. Anesthesia was induced by ketamine hydrochloride (30 mg/kg, i.m.) and maintained by chloralose (initially 10 mg/kg i.v., with an additional 5 mg/kg every hour). The trachea was cannulated with vinyl tubing (length 7 cm, diameter 7
mm) to sample airway gas (Beckman, LB2 and O M l l ) and to measure the respiratory gas flow rate and tidal volume (Nihon Kohden, AQ-601G and AR-601G). The femoral artery was cannulated to measure the arterial blood pressure. The heart rate was counted with a cardiotachometer triggered by the R wave of the E C G . The arterial blood pressure, heart rate, mean arterial blood pressure, CO2 and 02 concentration in the airway gas, respiratory gas flow rate and tidal volume were continuously displayed on a polygraph. The rectal temperature was maintained at 37 °C by means of a heating lamp. Each animal was placed in a stereotaxic frame with the head flexed at 45 °. A line of small holes was drilled at the appropriate site in the parietal bone above the nuchal ridge. The dorsal surface of the cerebellum was penetrated by a glass microelectrode of the double-barrel coaxial type. The tip of the electrode was beveled obliquely to 18-20 # m at the minor axis and 40-60 # m at the major axis. The inner electrode was filled with a 3 M NaCI solution and the outer electrode with a 0.025 M Tris-HCI buffer (pH 8.6) containing 6% W G A - H R P (Toyobo) and/or 5 m M kainic acid
Correspondence." M. Miura, Department of Physiology, 1st Division, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, 371 Japan.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
353 ( N a k a r a i C h e m . ) . The tip resistance ranged from i to 2 M£2. T h e inner electrode was used to stimulate a small part of the brain, and the o u t e r electrode to inject either kainic acid as a stimulant or W G A - H R P as a m a r k e r or a deposit. The effects of the electrical stimulation of a small part of the brain on blood pressure, heart rate and respiration were e x a m i n e d by a train (6 s) of square pulses (0.5 ms, 5/~A, 50 Hz). To assess the effect of kainic acid on the cerebellar response, the electrode was placed at the site of the potent cerebellar response and kainic acid was injected through the electrode by passing a D C cathodal current of 10 # A from a constant current supply (Nihon Kohden) in 20 repetitive cycles of 0.2 s on, 0.3 s off (40 # A . s ) . W G A - H R P was injected for marking by passing a positive electrical current of 4 # A for 2 min,
A
¢'1
,, C,]N-,
and for depositing by passing a 4 # A current for 200 ms on and 300 ms off for a period of 30 min. Each animal was perfused transcardially by a conventional method. The brain was r e m o v e d , fixed, frozen and sectioned serially at 50 #m. The sections were then processed for the histochemical demonstration of W G A - H R P deposits 14'15. First, we a t t e m p t e d to m a p the precise loci of the potent F P R assessed by electrical stimulation of the brain. Since the potent F P R , i.e., elevation of mean arterial pressure over 15 m m H g , was always associated with a hyperpneic TM and/or tachypneic response 13, we a d o p t e d a potent pressor-hyperpneic response as an indicator. The electrode was inserted from the dorsal surface of lobule V I I A or V I I B and the subcortical area was stimulated at 200 # m steps.
Stim ~--J_l~l ~stl i~ I I
,
,/F
BP
(mmHg)
B
HR ..,
,,,
;"
F
f
//
w-
]2o
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C
~ i I I I
=
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u
[:~l
I
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:LI !:!i
Vr i i i
i
Fig. 1. Map of sites from which the potent so-called fastigial pressor response (FPR) was elicited by electrical microstimulation of the brain. A - H : frontal sections from the level of the rostral fastigial nucleus to that of the lateral parabrachial nucleus. Small dots indicate sites of the 15-20 mmHg rise in the mean arterial pressure and large dots those of the 20-50 mmHg rise. I: enlarged original microphotograph of D (left) and responses of blood pressure (BP), heart rate (HR) and tidal volume (VT) from the site indicated by an arrow in the subfastigial fiber bundle (right). J: enlarged original microphotograph of H (left) and responses from the site indicated by an arrow in the lateral parabrachial nucleus (right). Scale bar in I: 1 mm. Abbreviations: BC, brachium conjunctivum; DN, dentate nucleus; FN, fastigial nucleus; IN, interposed nucleus; Pbl, lateral parabrachial nucleus; RB, restiform body.
354 In total, 133 tracks from 15 cats, sites of the potent response, were marked by depositing WGA-HRP. Fig. 1 shows the distribution of sites of the potent response, as indicated by black dots mapped on a series of the frontal sections of the brain. Caudomedially, as shown in Fig. 1 A - D , the potent responses were derived from the white matter between the FN and
the lingula cerebelti. Fig. 11, an enlarged original photomicrograph of Fig. 1D, shows the sites of the maximal response in each track marked by a small deposit of WGA-HRP. The responses recorded at the site indicated by an arrow shows potent pressor, tachycardiac and hyperpneic responses. Rostrolaterally, as shown in Fig. 1 E - H , the potent responses
Kainic
before
Gcid inj.
A
Stim.
~
-~,~
offer
6s -- .
~
i ~-Z-
- ~ ~
~
~ ~
200
BP (mmHg)
. . . . . . . . . . . . .
: ~! > ~ (bpm)
-
~
~
B
240
-
w (mO
~ , ; ~
d ~i'i! :]'i -':~: T~!~insp :
:::
!
:~:
:
i
i
.
BP i,j:
HR
'
i
~ - ~
VT i
;
'
Fig. 2. Effects of kainic acid on the so-called fastigial pressor response elicited by barely suprathreshold stimulation (5 # A , 50 Hz and 6 s). Left: sites of the tested response as indicated by the arrow. Right: responses before and 8 min after an injection of kainic acid. A: non-facilitation of the responses from the subfastigial fiber bundle. B: facilitation of the responses from the lateral parabrachial nucleus. Scale bar in A: 1 ram.
355 were derived from a subfastigial fiber bundle projecting into the dorsal part to the pontine brachium conjunctivum, i.e., the central part of the lateral parabrachial nucleus (Pbl). Fig. 1J, an enlarged original photomicrograph of Fig. 1H, shows 4 marks which are lined in the central part of the Pbl. The responses recorded at the site indicated by an arrow shows responses similar to those elicited in the subfastigial fiber bundle. No response was recorded in the FN per se.
Second, we attempted to determine whether such a potent response may originate from cells or passing fibers. Since a few cells are distributed in the subfastigial fiber bundle, it is probable that these cells originate in the potent response. Because it is expected that cells in subliminal fringe may be activated to dis-
charge by a combination of conditioning-testing stimulations, a small amount of kainic acid was injected as a conditioning chemical stimulation into the site of the potent response, and a change in excitability of the response was serially assessed by a testing electrical stimulation. Fig. 2A shows that no facilitation of the response was elicited (right) at the cell-rich site in this bundle, as indicated by an arrow in the original microphotograph (left), suggesting that the response does not originate from cells but from passing fibers in the subfastigial fiber bundle. On the other hand, as shown in Fig. 2B, the response to the testing electrical stimulation of the central part of the Pbl was maximally facilitated 8 min after a conditioning kainic acid stimulation. This suggests that the response originates from cells in the Pbl.
Fig. 3. Dark-field microphotographs of frontal sections of rostral pons and cerebellum. A: sites of WGA-HRP injection marked by
brown reaction products of DAB, located in the central part of the lateral parabrachial nucleus. B and D: WGA-HRP labeled fibers traversing white matter under the fastigiai nucleus at the level of rostral pole of the fastigial nucleus (B) and 0.4 mm caudal to it (D). C and E: enlarged microphotographs of the part indicated by white frames in B and D, respectively. Scale bars in A, B and D: 500pm; in C and E: 100/zm.
356 Third, we attempted to determine whether fibers projecting to and from the Pbl may pass the subfastigial white matter. Fig. 3A shows the site of the response in the central part of the Pbl in which a small amount of W G A - H R P was deposited. A part of labeled fibers traversing within the subfastigial fiber bundle was observed both in the area ventrolateral to the rostral pole of the FN (Fig. 3B,C) and the area adjoining the caudal end of the lingula cerebelli (Fig. 3D,E). The course of labeled fibers appeared to accord with the trajectory of sites of the potent responses (see Fig. 1). Both labeled cells and fiber ter-
minals were also observed in the contralateral Pbl (Fig. 4). These results indicate that some neurons in the Pbl give off an efferent pathway projecting to the other side of the Pbl and some of the other neurons receive an efferent pathway coming from the other side of the Pbl via the subfastigial fiber bundle. Therefore, the subfastigial fiber bundle contains countercurrent efferent pathways from both sides of the Pbl. Thus, we have obtained the evidence that the socalled FPR does not originate in the FN, but in the Pbl. This evidence, however, contrasts with the re-
Fig. 4. Dark-field microphotographs of frontal sections of the rostral pons, A: WGA-HRP labeled cells and fiber terminals in the lateral parabrachial nucleus of the contralateral side to the WGA-HRP deposit. B and C: enlarged microphotographs of the part indicated by white arrows in A. Scale bars in A: 200 am; in B and C: 100 pm.
357 cent report by Chida et al. 5. They showed that in rats injection of kainic acid into the FN elicited a depressor and a bradycardiac response. In our preliminary experiment, we could not confirm such responses except when kainic acid leaked into the underlying fourth ventricle. It is highly probable that such an accidental episode may influence the dorsal surface of the medulla oblongata, and excite inhibitory neurons acting on the cardiovascular modulatory pathways. Concerning the sites of the FPR, this study disclosed uncertain results as reported in previous studies. So far, the F1N has been stimulated mostly by an iron wire electrode, and the tip of the electrode was marked by procian blue reactions or small electrolytic lesions. Since the tip of the electrode was large and dull, it always pressed and injured the neural tissues. Such metal electrode caused an inaccurate marking. But use of a glass microelectrode with a sharp tip minimized injury and distortion of the neural tissues and increased the accuracy of marking with a small amount of WGA-HRP. A few anatomical studies have suggested the presence of the parabrachial projection to the cerebellum. Somana and Walberg 26, in the HRP study of cats, found that the cerebellar vermal region receives the bulk of afferent fibers from some neurons in the Pbl. Saper and Loewy25, in the autoradiography of rats, showed that some fibers from the Phi cross the midline of the pons and contribute to a terminal field in the contralateral Pbl, and that some fibers project into the interposed nucleus and the cerebellar white matter but they gave no definite evidence for the presence of the terminal distribution of these fibers. And they did not show any evidence for the presence of the mutual innervation between both sides of the Pbl via the subfastigial fiber bundle. There have been several lines of evidence that properties of the FPR are essentially similar to those of the pressor response derived from the Pbl: (1) the cardiovascular effect of FPR 1'17 and that of the parabrachial pressor response 2°, (2) augmented vasopressin secretion during FPR 7 and the parabrachial pressot response 27, and (3) a hyperpneic and/or tachypneic response associated with the FPR and the parabrachial pressor response, which was verified in this study. The other responses associated with the FPR,
like a predatory attack behavior 24 and an increase in the cerebral blood flow11'22, may be reproduced during the parabrachial pressor response, although a recent study ~9opposed the latter possibility. Recently, there have been many investigations to seek afferent and efferent connections of the parabrachial area including the lateral and medial parabrachial nuclei and the Koelliker-Fuse nucleus. The parabrachial area receives not only ascending visceral information from the nucleus tractus solitarii 1°,12, the buffer nerves 3 and the lateral tegmental field a°, but also descending visceral projections from the amygdaloid nuclei 9'11'28. On the other hand, the parabrachial area sends not only ascending projections to the amygdaloid nuclei 4'28 and the paraventricular nucleus 6, but also descending projections to the nucleus tractus solitarii s,23.25, the ventrolateral medulla 25 and the intermediolateral nucleus of the spinal cord 16. In addition to these longitudinal connections which may have a key role in relaying and integrating information from both ascending and descending pathways, another connection between the two sides of the Pbl has been discovered and it may have an important role in balancing the two sides of neural activities subserving cardiovascular and respiratory control. In conclusion: (1) The site of the so-called FPR to electrical stimulation does not originate in the FN, but in the subfastigial fiber bundle. (2) Facilitation by conditioning kainic acid stimulation did not appear in the response elicited from the subfastigial fiber bundle, but in the response elicited from the Pbl. (3) When WGA-HRP was deposited at the site of the parabrachial pressor response, labeling was positive in the subfastigial fiber bundle and in the contralateral Phi. (4) Both sides of the Pbl are innervated mutually via commissure fibers traversing the subfastigial white matter. The mutual innervation of the two sides of the Pbl is a favorable architecture for balancing information, because an inflow to one side of the Pbl can be distributed to the other side. (5) Since the Pbl is a relay station of the countercurrent pathway of the neural information, the outflow of the so-called FPR may pass on to the suprapontine visceral integrating center and/or medullary and spinal visceral efferent center.
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