Lesions of rostral ventrolateral medulla abolish some cardio- and cerebrovascular components of the cerebellar fastigial pressor and depressor responses

Brain Research, 508 (1990) 93-104

93

Elsevier BRES 15160

Lesions of rostral ventrolateral medulla abolish some cardio- and cerebrovascular components of the cerebellar fastigial pressor and depressor responses K. Chida*, C. Iadecola and D.J. Reis Division of Neurobiology, Department of Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021 (U.S.A.) (Accepted 3 July 1989)

Key words: Cerebellum; Fastigial nucleus; Rostral ventrolateral medulla; Blood pressure~ Adrenergic neuron

We sought to establish whether the C1 area of the rostral ventrolateral reticular nucleus (RVL) of the medulla oblongata mediates: (1) the elevations in arterial pressure (AP), heart rate (HR) and regional cerebral blood flow (rCBF) elicited by electrical stimulation of the rostral cerebellar fastigial nucleus (FN), the fastigial pressor response (FPR); (2) the reductions in AP and HR elicited by chemical stimulation of intrinsic neurons of FN with excitatory amino acids, the fastigial depressor response (FDR) L~. Studies were conducted on rats anesthetized (chloralose), paralyzed and artificially ventilated. The FN was stimulated electrically through microelectrodes and chemically by microinjection of D,c-homocysteic acid (100 nmol in 100 hi). rCBF was measured in homogenates of 11 brain regions by the ~4C-iodoantipyrine technique. Bilateral electrolytic lesions restricted to the RVL abolished the elevations in AP, HR and rCBF elicited by electrical stimulation as well as the fall of AP and HR elicited by chemical stimulation of the FN. The disappearance of the responses was anatomically selective and could not be attributed to changes in resting AP, HR or rCBF, loss of reactivity of preganglionic sympathetic neurons, or variations in blood gases. Since the FN neither projects to nor receives afferents from the RVL the pathway to RVL is indirect. We conclude that: (1) the FPR results from excitation and the FDR inhibition of reticulospinal sympathocxcitatory axons of RVL; (2) the FPR is a consequence of excitation of axons arising from neurons in an as yet unidentified area of lower brainstem projecting to or through the FN and with collateral branches innervating RVL mono- or polysynaptically; (3) the FDR, in contrast, represents excitation of intrinsic FN neurons with a polysynaptic projection to RVL through unidentified regions of lower brainstem; (4) the RVL is a relay mediating the increase in rCBF associated with the FPR; and (5) the RVL plays a critical role in integrating actions on the systemic and cerebral circulation represented in cerebellum.

INTRODUCTION

e x c i t a t i o n of fibers in the FN. uncovered

E l e c t r i c a l s t i m u l a t i o n of the rostral and v e n t r o m e d i a l p o l e of the c e r e b e l l a r fastigial nucleus ( F N ) elicits a

an

unrecognized

Moreover,

inhibitory

they have

action

of FN

n e u r o n s u p o n s y m p a t h e t i c n e r v e d i s c h a r g e and h e n c e AP, the fastigial d e p r e s s o r r e s p o n s e ( F D R ) ~.

s t i m u l u s - l o c k e d e l e v a t i o n in arterial p r e s s u r e ( A P ) and

The

h e a r t rate ( H R ) , the fastigial p r e s s o r r e s p o n s e ( F P R ) l' 47,48 A s s o c i a t e d is a m a r k e d and global increase in r e g i o n a l c e r e b r a l b l o o d flow ( r C B F ) 27'45"57-59 which, in

output

m a n y r e g i o n s , is u n a s s o c i a t e d with c h a n g e s in regional

is not k n o w n . T h u s while the F P R persists after d e c e r e -

c e r e b r a l glucose utilization 5~.

b r a t i o n , and h e n c e a p p e a r s o r g a n i z e d e n t i r e l y within the l o w e r brainstem 22"4~'49, the e f f e r e n t p a t h w a y f r o m cere-

For m a n y years it has b e e n u n c e r t a i n w h e t h e r the F P R r e p r e s e n t e d a r e s p o n s e to s t i m u l a t i o n of intrinsic n e u r o n s

pathways

through

which

m e d i a t i n g the F P R and the F D R of p r e g a n g l i o n i c

cerebellar

networks

act to influence the

sympathetic

neurons

in the

i n t e r m e d i o l a t e r a l nuclei of the t h o r a c i c spinal c o r d ( I M L )

of the F N o r to e x c i t a t i o n of axons p r o j e c t i n g into or

b e l l u m to I M L is p r o b a b l y not direct, for t h e r e are no p r o j e c t i o n s f r o m c e r e b e l l u m into the I M L 3')'42'75. O n e

through

R e c e n t l y , h o w e v e r , it has b e e n

possible relay might i n v o l v e n e u r o n s within the cardio-

d e m o n s t r a t e d in rat ~3 that e x c i t a t i o n of intrinsic n e u r o n s

t h e nucleus.

vascularly active z o n e of the rostral v e n t r o l a t e r a l me-

of the F N by e x c i t a t o r y a m i n o acids lowers A P and H R

dulla, n a m e l y the C1 a r e a of the rostral v e n t r o l a t e r a l r e t i c u l a r nucleus ( R V L ) ~'473, and its h o m o l o g u e in cat,

by s y m p a t h o i n h i b i t i o n , while after selective d e s t r u c t i o n of F N n e u r o n s by e x c i t o t o x i n s the F P R persists. T h e s e findings

indicate

that

the

FPR

is a c o n s e q u e n c e

of

the s u b r e t r o f a c i a l nucleus 2~. N e u r o n s of the R V L m a k e m o n o s y n a p t i c c o n n e c t i o n s with n e u r o n s of the I M L 46 and

* Present address: Department of Neurobiology, Nihon University School of Medicine, 30-10yaguchi-Kamimachi, Itabashi-ku, lokyo, Japan. Correspondence: D.J. Reis, Division of Neurobiology, Corncll University Medical College, 411 East 69th Street, New York, NY 10021. U.S.A. (XJ06-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

94 play a critical r o l e in t h e r e s t i n g a n d reflex c o n t r o l o f A P 64. M o r e o v e r ,

in

cat

bilateral

electrolytic lesions

p l a c e d j u s t b e n e a t h t h e s o - c a l l e d glycine s e n s i t i v e r e g i o n of the

ventral

surface

o f the

m e d u l l a 3° a b o l i s h t h e

e l e v a t i o n s o f A P a n d H R e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n o f t h e F N '~4. In t h e p r e s e n t s t u d y w e t h e r e f o r e e x a m i n e d w h e t h e r l e s i o n s c o n f i n e d to t h e C1 a r e a o f R V L in rat w o u l d : (1) i m p a i r t h e e l e v a t i o n s in A P , H R a n d also r C B F associa t e d w i t h t h e F P R ; a n d (2) also e f f e c t t h e s y m p a t h e t i c inhibition p r o d u c e d by chemical stimulation of intrinsic FN n e u r o n s . A preliminary p r e s e n t a t i o n of s o m e of the m a t e r i a l h a s b e e n m a d e ~2. MATERIALS AND METHODS

Animals, surgery, and electrical and chemical stimulation The detailed method for surgical preparation of rats, electrical stimulation of FN, production of lesions of RVL, histological procedures, and measurement of rCBF in regional brain homogenates are described elsewhere 14'15'32'36'37"58'59'68 and will be summarized. Experiments were performed on 36 male Sprague-Dawley rats weighing 300-400 g, anesthetized with a-chloralose (40 mg/kg s.c.) after induction with 2% halothane in 100% 0 2. For studies in which rCBF was not analyzed catheters were inserted into the left femoral artery and vein. In rats in which rCBF was measured cannulas were inserted into both femoral arteries and veins. The trachea was cannulated and the animals paralyzed with D-tubocurarine (0.5 mg/kg i.m.) and ventilated on 100% 02 by a respirator. Body temperature was maintaind at 37-38 °C. The animals were mounted in a stereotaxic apparatus (Kopf) with the bite bar adjusted t o - 1 1 mm below the interaural line. The lower brainstem and caudal half of the cerebellum were exposed by an occipital craniotomy. AP and HR were continuously recorded on a chart recorder. The brain was stimulated electrically with negative square wave pulses delivered through a stimulation isolation unit from a constant pulse generator (Grass $88). The anode (ground) was a metal clip attached to a scalp muscle. Electrodes were fabricated from Teflon-coated stainless steel wire with only the cut surface exposed (tip diameter 150/~m). The stimulation current was measured by passing the stimulus through a 10 I2 resistor and displaying it on a cathode ray oscilloscope. The lower brainstem and caudal half of the cerebellum were exposed by an occipital craniotomy. Electrolytic lesions were produced by passage of an anodal (DC) current of 500-750 ~uA for 30 s from a lesion maker (Grass LM5A) through electrodes fabricated from stainless steel needles and insulated by Epoxylite, except for a bare tip of 200 ,am. At the end of each study electrode tips were marked by a small lesion made by passage of an anodal current for 10 s. The FN was chemically stimulated with the excitatory amino acid D.L-homocysteate (DLH) microinjected (100 nmol in 100 nil by a positive pressure system2 through capillary micropipettes with a tip diameter of 50 ,am. This dose of DLH elicits a potent FDR ~5. Microinjections were made over a period of 5-15 s. All agents were dissolved in phosphate-buffered saline, pH 7.4. Fast green dye was added to the injectate for localization of cannula tips. Measurement o f rCBF The procedures for measurement of rCBF by dissection of brain regions in association with brain stimulation has been published in earlier reports 36'37 and will only be summarized, rCBF was measured using the equation developed by Kety4° using [tac]iodoantipyrine (IAP) as a diffusible, inert indicatorTM. Tissue concentration of IAP was determined by tissue sampling and liquid scintillation counting6°.

4-N-methyt-[t4C]IAP (New England Nucic~r (NEN), spot. acl. 40-60 mCi/mmol) was dried under an N~ slrcam and dissolved in about 1 ml of physiological saline. Prio~ to the start of the infusion. animals received 500 1U/100 g b. wt. of heparin i.~. (A-H Robbins). The tracer was infused (5/~Ci/100 g b. wt.) at a constant rate over about 30 s by an infusion pump (Harvard Model 940). Simultaneously, about 50/zl of blood were withdrawn every 2-5 s from the right femoral artery to determine the arterial concentration-time curve of IAP. The sampling catheter was made as short as possible (total length 5 cm), to minimize the 'smearing effcct' o~l the concentration-time curve ~. Aliquots of blood (20-40 ktl) were transferred into scintillation vials containing I ml of tissuc solubilizer (protosol-ethanol, NEN), incubated for 61! min at 55 °C and decolorized with 30% hydrogen peroxide. Alter adding ReadySolve (Beckman), the radioactivity of samples (uCi) was measured in a liquid scintillation spectrophotometer. At about 30 s following the start of the infusion, the animal was killed with an i.v. bolus of saturated KCI. The brain was rapidly removed from the skull, placed on a cooled glass plate, hemisected and samples taken from each side of 11 brain regions: medulla, inferior and superior colliculi, hypothalamus, thalamus, hippocampus, frontal, parietal and occipital cortices, caudate nucleus and corpus callosum. Tissue samples were transferred into preweighed scintillation vials and the vials reweighed. After solubitization of the tissue with Protosol (NEN), 15 ml of scintillation solution (ReadySolve, Beckman) were added and the samples counted. rCBF was calculated from the concentration-time curve of IAP and regional brain concentration of the tracer by using a computerized approximation of the equation developed by Kety4° (see also ref. 58). The blood-brain partition coefficient for IAP was set at 0.8 TM.

Conduct of the experiments Effects of R VL lesions on A P and HR. One hour after completion of surgery, a stimulating microelectrode was lowered into the cerebellum to localize the most sensitive locus (active site) in FN from which electrical stimulation could elicit an elevation of AP and HR, the FPR. Using the obex as stereotaxic zero (0), the electrode was inserted into the cerebellum with a posterior inclination t0 °, at a site +4.6 + 5.4 mm anterior and 0.8 mm lateral to the obex. The stimulating electrode was lowered in 0.5 mm steps. At each step the brain was stimulated with 8 s trains of pulses delivered at 50 Hz, with a pulse duration of 0.5 ms, and stimulation current of 20-100 I~A. Once a pressor response was evoked, the electrode was lowered in 0.2 mm steps and the stimulus current reduced to 5-20/tA. The site from which an elevation of AP of 10 mm Hg was elicited with the lowest current (the threshold current) was determined and designated the active site for that track. Usually the active site was located +0.5 _+ 1.5 mm from dorsoventral 0. In studies comparing the effects of electrical and chemical stimulation on the AP and HR responses of the FDR and FPR a stimulating electrode was inserted into the active site and the stimulus current adjusted to 5-times the threshold current eliciting a minimal rise in AP, usually around 5-21) ~A. A capillary micropipette (50/~m) was then lowered to the same coordinates on the other side of the cerebellum for chemical stimulation of FN. The RVL was then localized by electrical stimulation (8 s train, 100 Hz, 10-20 ~A) with a microelectrode lowered through the floor of the IVth ventricle32'68. This electrode was withdrawn and replaced with an electrode for placement of a lesion. The magnitude of the responses to electrical and chemical stimulation of FN were measured and the RVL lesioned on one and then the other side in rapid succession. AP, which fell to levels below 60 mm Hg after the lesion326~, was maintained within physiological range by continuous infusion of phenylephrine (6-8/~g/kg/min). At least 45 rain elapsed before retesting the effects of RVL lesions on the FPR and FDR. In 3 rats the dorsal medulla was electrically stimulated within the trajectory of the principal tegmental tractS'72, corresponding to the dorsal medullary reticular formation~7. In 4 animals bilateral electrolytic lesions were placed in the cuneate nuclei to control for

95 T A B L E IA

Effect of bilateral electrolytic lesions o f rostral ventrolateral medulla on changes in mean arterial pressure (MAP) and heart rate (HR) elicited by electrical or chemical stimulation of the cerebellar fastigial nucleus in the same animals (n = 5) Values are means ± S.E.M.

Treatment

Resting Electrical stimulation (50Hz, 100,uA,8s) Chemical stimulation (100 nmol/100 nl DLH)

M A P (mm Hg)

Lesioned Intact

132 ± 2

127 ± 4"

417 ± 14 208 ± 17"*

+48±4*

+3±1

+32_+5*

-8 ± 3

-68 _+_8*

o 20o

Lesioned

arterial Mean I ~ pressure 100 (mmHg)

+9±5

-7 ± 4

*P < 0.05 from resting (paired t-test). **P < 0.05 from intact (t-test). a After bilateral lesions A P fell to 60 ± 4 mm Hg and H R 235 ± 17 bpm, values significantly different from resting values (P < 0.005). A P was maintained at resting values by continuous i.v. infusion of phenylephrine (see text for details).

non-specific effects of brain lesions. Effects o f R V L lesions on rCBE In these studies arterial blood gases (pO 2, p C O 2, pH) were measured by a gas analyzer (Instrumentation Laboratories) after completion of the craniotomy and placement of the animal in the stereotaxic frame. The most sensitive pressor region of FN was identified and the electrode left in place. Each R V L was localized by microstimulation and lesioned as described. During placement of the R V L lesion 1-2% halothane was readministered along with transient removal of blood to reduce the pronounced transient rise of A P associated with passage of a lesioning current. Care was taken that A P never rose above 140 mm Hg, the upper limit of the autoregulatory range for rCBF in rat 35. After lesions of the RVL A P was maintained at resting values by infusion of phenylephrine. Blood gases were adjusted with p C O 2 maintained in normocapnic range (34-38 mm Hg) by adjusting the volume of the ventilator. Arterial p O 2 was maintained at a high level by ventilating the rat with 100% 0 2. This results in elevated values of arterial p C O 2 which, however, do not influence rCBF 65. One hour after placement of the R V L lesion and stabilization of blood gases the FN was stimulated. When blood gases were in normal range, the FN was electrically stimulated with intermittent

Chemicalstimulation Intact RVLlesion

Arterial [ pressure I00 (ramHg)

HR (beats/rain)

Intact

-55 ± 4*

Electricalstimulation Intact RVLlesion

0

q

lmin

i

Heartrate 400t (beats/mini 300

f

200 FNstimulation

tlO0tJA,50Hz)

DLH(100nmol/10onil

Fig. 1. Abolition, by bilateral lesions of RVL, of the FRP elicited by electrical stimulation of FN (left panel) and the F D R elicited by chemical stimulation of the FN with D L H (right panel).

stimulus trains (1 s on/1 s off, 50 Hz). The stimulus intensity was gradually increased to reach 5 x threshold current while, at the same time, the evoked rise of A P was gradually reduced by slow controlled removal of blood. In this manner A P never rose during stimulation above 150 mm Hg. We have demonstrated that the controlled hemorrhage has no effect on rCBF under these conditions 3T.

Arterial pressure (mmHg)

& i00

,°°I 0

Mea n

T A B L E IB

Effect o f bilateral electrical lesions of the cuneate nucleus on the changes in mean arterial pressure (MAP) and heart rate (HR) elicited by electrical or chemical stimulation of the fastigial nucleus

Resting Electrical stimulation (50Hz, 100HA,8s) Chemical stimulation (100 nmol/100 nl DLH)

M A P (mm Hg)

HR (beats/min)

Intact

Lesioned Intact

Lesioned

133 ± 4

133 _+ 2

453 + 2

461 + 13

+49+7*

+48+9*

+39+10"

+25+2*

-43±4'

-40±7"

-45±4"

-43±7"

*P < 0.05 from resting (paired t-test).

1min

500I Heart rate I~ats/minI

Values are means ± S.E.M. ; n = 4.

Treatment

arterial pressure 100 (mmHg) 0 40O

2 t rtRVL lesion

t ItRVL lesion

lhr 4____ DMRFstimLllation Phenylephrine 150pA[OOHzl infLlsionm~- - - - - 3

Fig. 2. Effects of bilateral lesions of R V L on resting AP and H R and the effects after lesions of electrical stimulation of the DMRF. Note progressive fall of A P with lesion first of right and then left RVL, recovery of A P with infusion of phenylephrine, and, 1 h later, the preservation of a brisk pressor response to electrical stimulation of the DMRF.

96

Electrical stimulation Intact CuN lesion

Arterial pressure lmmHg)

100

Chemical stimulation Intact CuN lesion

Intact 200 Arterial pressure (mmHgl

I

o 20o

0 200 arterial pressure (mmHg)

Mean arterial pressure (ramHg)

100 f

~

loo

o

0 Heart rate (beats/min)

During pherytephrine infusion

I

5®I

50o

-,/'-"

4OO

lmin

I

lmin

400 Heart rate (beats/min) 300

FN stimulation

~ - [ _ _ __J~-(751JA,50Hz)

DLH (100nmolllO0nl)

Fig. 3. Failure of bilateral lesions of the cuneate nuclei to modify the FPR (left panel) on the FPR or FDR (right panel).

The FN was stimulated for 10 min and blood gases again sampled. IAP was infused, FN stimulation continued and the animals killed 30 s-later. At the end of each experiment the sites of electrical stimulation were marked by passing a 200 ,uA anodal current for 10 s through the stimulating electrode. Dye spots were deposited from micropipettes. Rats were sacrificed by injection of t ml of saturated KCI. Brains were removed, frozen in Freon at -25 °C, and sectioned at 25 #m on a cryostat for reconstruction of stimulation and microinjection sites and lesions. The remainder of the brain was processed for measurement of rCBF as described above.

Statistical procedures Variance in each experimental group was analyzed by Fischer's test. Two group comparisons were analyzed by Student's unpaired t-test. Side-to-side comparisons were analyzed by Student's paired t-test. Multiple comparisons were evaluated by one-way analysis of variance and the Newman-Keuls test. Differences were assessed to be significant for P-values less than 0.05.

Fig. 4. Representative lesions of RVL in rat. Electrolytic lesions were placed bilaterally as described in Materials and Methods.

2o0 FN stimulation

_2_

1'

(IOOpA,50Hz} IIOONA,50HzJ DLH(100nmol/100nl) DLH(I00 nmol/100nl) Fig. 5. Preservation of FDR during infusion of phenylephrine. Left panel: control FPR and FDR. Right panel: during infusion of phenylephrine which elevates AP and reduces HR electrical and chemical stimulation of FN elevate and depress, respectively, AP and HR.

RESULTS

Effects of lesions of RVL on changes in AP and HR associated with the FPR and FDR Electrical stimulation of the FN in 5 rats with an 8 s stimulus train (50 Hz) at a stimulus current 5 x threshold, elicited the well recognized elevation in AP and H R of the FPR 48 (Table IA, Fig. 1). In the same animals chemical stimulation of the contralateral FN with 100 nm of D L H in 100 nl elicited a fall of AP and HR, a typical F D R (Table IA, Fig. 1) 13"14. In all cases, the electrode sites and cannula tips were confined to the rostral one-third of the FN. Bilateral lesions were then placed in RVL resulting in an immediate and significant (P < 0.005) fall of AP and H R to 60 + 4 mm Hg and 235 + 17 bpm, respectively (Fig. 2). AP was rapidly returned to control levels by infusion of phenylephrine (Fig. 2). Such treatment did not elevate the depressed H R (Table IA). Bilateral lesions of RVL, resulted in virtually complete abolition of both the FPR and F D R (Table IA; Fig. 1). In contrast, in 4 rats bilateral lesions were placed in the cuneate nuclei (Table IB). Such lesions had no effect on either the FPR or FDR (Table IB; Fig. 3). Histologically lesions of the RVL consisted of bilateral destruction of the C1 area of RVL as defined in detail

97 T A B L E IIA

Effects of continuous i. v. infusion of phenylephrine (PE) or vehicle (0.9% NaCI) on changes in mean arterial pressure (MAP) and heart rate (HR) elicited by electrical and chemical stimulation of the fastigial nucleus Values are m e a n s _+ S.E.M. ; n = 4. PE infusion: 1-2,ug/kg/min.

Treatment

MAP (mm Hg)

Resting Electrical stimulation (50 Hz, 1 0 0 g A , 8 s) Chemical stimulation (100 nmol/100 nl D L H )

HR (beats/rain)

Vehicle

PE

Vehicle

PE

130_+ 2 +53 _+_6* - 3 9 _+ 9*

156 ± 3** +33 + 3*** -25 ± 4***

417 ± 9 +47 ± 6* - 4 3 ± 9*

288 +_ 21"* + 144 _+ 5*** -41 ± 6*

*P < 0.05 from resting (paired t-test). **P < 0.05 from vehicle (t-test). T A B L E IIB

Effect of local microinjection of phentolamine (5 nmol in 100 nl) (Ph) into bilateral rostral ventrolateral medulla on changes in mean arterial pressure ( MA P) and heart rate (HR ) elicited by electrical or chemical stimulation of the fastigial nucleus Values are m e a n s _+ S. E.M. ; n = 5. n.s. not significantly different from resting (P > 0.05).

Resting

M A P ( m m Hg) H R (bpm)

Electrical stimulation

Chemical stimulation

Before

After Ph

Before

After Ph

Before

After Ph

129 + 5 403 _+ 33

125 + 5 373 _+ 33**

+ 49 ± 7* +36 _+ 11"

+ 38 ± 7* +43 ± 12"

- 5 7 +_ 11 * -61 ± 6*

-26 + 8.... - 8 + 13 n.s.**

• P < 0.05 from unstimulated (t-test) • *P < 0.05 from before.

elsewhere 73. A typical lesion is seen in Fig. 4. Damage in all instances consisted of regions lying ventral to the ambiguus complex at a site just caudal to the caudal pole of the facial nucleus and at the rostral edge of the

Electrical stimulation Intact RVLinjected Arterial pressure (ramH9}

I tO0 t

superior olivary complex. Variably damaged were portions of the lateral reticular nucleus and ambiguus complex. Lesions of the cuneate nuclei damaged that nucleus and variably subjacent portions of the Vth nerve

Chemicalstimulation Intact RVLinjected

Phentolamine

Glutamale

~

0L l~ean arterial pressure (mmHg)

200I ~ .

,,~

i001

--~

0L

train

,

J

1 rain

500I

{beats/min) 400f

~

300L FN stimulation ---[7- -

-- J-~ O,OOMA,5OHz}

L

~' (lOOnmol/10Onl) "~ DLH

J

tOmin ~ 1' 1" rt RVL It RVL Glutamate,It RVL Phentolamine(5nmol/lO0hi) (50nmol/lO0nil

Fig. 6. Effects of microinjection of phentolamine (5 nmol in 100 nl) bilaterally into R V L on F P R and F D R . Left panel: absence of effect on FPR. Middle panel: attenuation of A P and abolition of H R responses of FDR. Right panel: preservation of pressor response to microinjection of l_-glutamate (50 nmol in 100 hi) into region R V L 15 min after treatment with phentolamine.

98 TABLE I11 Mean arterial blood pressure (MAP), blood gases and hematocrit (Hi) with or without electrical stimulation o[ the cerebella,??~stigial nucleus in rat~" following bilateral electrolytic lesions o f the C1 area of rostral ventrolateral medulla

No differences were found between groups (P > ¢).05; Newman-Keuls multiple comparison test). Unstimulated

Body weight MAP (mm Hg) pCO 2 (mm Hg) pO 2 (ram Hg) pH Ht(%)

Electrical stimulation

R V L intact (n = 5)

R V L lesions 07 = 4)

R V L intact (n = 5)

R V L lesions (n = 4)

346 + 19 123 + 4 35.8 _+0.7 386 _+48 7.444 _+0.024 49__+2

342 + 4 121 + 8 35.8 _+0.8 224 + 27 7.390 + 0.027 49-+-t

347 + 8 128 + 7 35.6 + 0.8 260 + 25 7.336 + 0.092 46+3

358 _+6 128 + 6 35.2 + 0.2 242 + 21 7.410 _+0,027 50__+I

complex and portions of the a d j a c e n t reticular formation. A f t e r R V L lesions the pressor response elicited by electrical stimulation (8 s, 100 Hz, 50 n A ) of the D M R F 37 was p r e s e r v e d (Fig. 2). Stimulation at this site excites axons of the descending reticoluspinal projections from neurons of R V L 5'72. Preservation of the pressor response to D M R F stimulation after bilateral lesions of the C1 a r e a d e m o n s t r a t e s that despite the loss of tonic backg r o u n d excitation from neurons of the R V L 64 the reactivity of preganglionic sympathetic neurons was preserved. To rule out the possibility that the phenylephrine utilized to maintain A P could have m a s k e d the fall of A P

and H R of the F D R , c o m p a r a b l e amounts of phenylephrine were infused for 45 min in 4 anesthetized rats in which the R V L was not lesioned. Such t r e a t m e n t , while producing a significant elevation of A P and a reduction in H R (due to baroreflex excitation) (Table I I A ; Fig. 5), did not modify the fall of A P or H R associated with the F D R . The infusion of p h e n y l e p h r i n e also resulted in a substantial elevation of the magnitude of the H R but not A P response of the F P R (Table I I A ; Fig. 5). To d e t e r m i n e w h e t h e r adrenergic receptors in R V L participated in the expression of either the F D R or F P R p h e n t o l a m i n e , an a l - a d r e n e r g i c antagonist, was injected into the R V L bilaterally (5 nmol in 100 nil, Such t r e a t m e n t had no effect on resting A P or H R , nor on the magnitude of the F P R (Table l i B ; Fig. 6). H o w e v e r , it significantly a t t e n u a t e d the reductions in A P and H R elicited by chemical stimulation of F N with D L H . That the effect of p h e n t o l a m i n e was not non-selective was d e m o n s t r a t e d by preservation of the well established 68 potent pressor and c a r d i o a c c e l e r a t o r y responses elicited by microinjection of 50 nmol of L-glutamate (in 100 nil into R V L bilaterally (Fig. 6). E f f e c t s o f R V L lesions o n the e l e v a t i o n o f r C B F associated with the F P R

Toganglia and adrenal medulla

Fig. 7. Diagrammatic representation of proposed neural substrates of fastigial pressor response (FPR, solid line) and fastigial depressor response (FDR, broken line) in rat. The FPR is elicited by antidromic stimulation of axons from as yet unidentified brainstem neurons which, through collaterals, excite negative neurons of the C1 area of RVL. Those, in turn, excite preganglionic sympathetic neurons resulting in rise of AP and HR. The FDR, in contrast, results from excitation of intrinsic neurons of the fastigial nucleus (FN) which project to as yet unidentified neurons of the brainstem. These project, mono- or polysynaptically, to inhibit neurons of the C1 area, thereby inhibiting preganglionic sympathetic neurons and lowering AP.

Studies of r C B F were m a d e in 4 groups of rats: (1) unstimulated controls (n = 5); (2) unstimulated rats with lesions of the C1 area of R V L (n = 4); (3) rats with F N stimulation alone (n = 5); and (4) rats with F N stimulation and R V L lesions. The A P and b l o o d gases did not differ b e t w e e n groups (Table III). Values of r C B F in rats without and with lesions of R V L did not differ from each o t h e r (Table IV) and were similar to those r e p o r t e d in o t h e r studies from this l a b o r a t o r y (e.g. see refs. 36, 37, 55). The failure of R V L lesions to affect resting r C B F is in a g r e e m e n t with the study of U n d e r w o o d et al. 85 from this laboratory. Electrical stimulation of the FN elicited the well

99 TABLE IV Changes in regional cerebral blood flow (rCBF) elicited by electrical stimulation of the cerebral fatigial nucleus (FN) following bilateral electrolytic lesions of the C1 area of rostral ventrolateral medulla (RVL) Regions

Frontal cortex Parietal cortex Occipital cortex Caudate-putamen Hippocampus Corpus callosum Thalamus Hypothalamus Superior colliculus Inferior colliculus Medulla

rCBF (ml/lO0 g x rain) Unstimulated

FN stimulation

RVL intact (n = 5)

RVL lesion (n = 4)

RVL intact (n - 5)

RVL lesion (n = 4)

78"+4 93 +_5 79 -+ 5 80 _+5 68 _+6 57 _+4 82 _+7 87 "+ 5 96 "+ 6 99 +_4 91 _+6

73"+6 78 _+6 7l +_6 65 _+5 59 _+7 49 "+ 7 67 _+7 69 _+9 83 _+9 87 + 7 68 "+ 7

181"+6" 189 +_9* 158 + 5* 138 _+8* 128 + 7* 97 "+ 3* 180 +_9* 143 "+9* 184 _+6* 194 _+6* 125 _+5*

66_+8 58 -+ 6 66 _+8 59 -+ 6 66 _+8 42 _+5 60 -+ 7 66 _+7 81) +_7 83 _+8 73 _+5

*P < 0.05 from unstimulated and RVL lesion groups (Newman -Keuls multiple comparison test).

established elevations of r C B F (Table IV). Bilateral electrolytic lesions of the R V L completely abolished these responses resulting in flow values similar to those of unstimulated animals. Histologically the lesions were c o m p a r a b l e to those described in the experiments above with lesions of m e d u l l a destroying the bulk of R V L bilaterally. DISCUSSION The role o f R V L in mediating changes in A P elicited f r o m FN

In the present study we have observed that bilateral electrolytic lesions of the C1 area of R V L of rat abolishes the elevations of A P and H R elicited by electrical stimulation of the FN, as well as the fall of A P and H R elicited by chemical stimulation of the nucleus. The effects a p p e a r to reflect interruption of a c o m m o n relay in the m e d u l l a and not to be secondary to non-specific effects of the R V L lesion upon sympathetic activity. The interruption of the vasopressor effects of electrically stimulating F N by lesions anatomically highly restricted to the C1 area confirms and extends the anatomically less-precise findings of M c A l l e n 44 in cat. The d i s a p p e a r a n c e of the F P R cannot be attributed to a loss of reactivity of preganglionic neurons since after R V L lesions electrical stimulation of axons of RVLspinal cardiovascular neurons in dorsal medulla 7°'72 still e v o k e d a p o t e n t elevation in AP. Nor can the abolition of the responses of A P either to electrical or chemical

stimulation of FN be attributed to masking or distortion of the cardiovascular c o m p o n e n t s by phenylephrine: both the F P R and F D R were p r e s e r v e d when rats without brainstem lesions were t r e a t e d with c o m p a r a b l e amounts of the drug. The findings also cannot be attributed to non-selective effects of surgery or p l a c e m e n t of brain lesions since bilateral lesions of the cuneate nuclei had no effects on the autonomic responses to cerebellar stimulation, and both responses are stable and reproducible The discovery that the R V L is critical for expression of autonomic effects from c e r e b e l l u m provides an answer to the question of how autonomic effects from cerebellum reach preganglionic neurons in the spinal cord. In contrast to the cerebellospinal projections which primarily act upon spinal m o t o r neurons 39, neurons of the R V L project exclusively to the i n t e r m e d i o l a t e r a l and intermediomedial columns of the thoracic segments of the spinal cord 6~''67 wherein they monosynaptically innervate preganglionic sympathetic neurons 46. It is also p r o b a b l e that the opposing actions upon A P and H R p r o d u c e d by electrical or chemical stimulation of the FN can be attributed to m o d u l a t i o n of the discharge of the p r e m o t o r autonomic neurons of the CI area. These reticulospinal neurons are tonically active 9'5~s2~1~3, and potently symp a t h o - e x c i t a t o r y 2°'51'68. Increases in their activity ~'33,s2,s3 p r o p o r t i o n a l l y elevate while reductions 9"3-<5~s~ reduce sympathetic nerve discharge and AP. Thus modulations in the firing rate of a single p o p u l a t i o n of R V L neurons p r o b a b l y m e d i a t e both the pressor and vasodepressor responses elicited from FN, serving t h e r e b y as a c o m m o n pathway for autonomic control from the d e e p cerebellar nuclei. Neural organization o f the F P R

It is now recognized that the elevations in A P and H R elicited by electrical stimulation of the FN result from excitation of neuronal processes passing into or through the rostral one-third of the FN 4<57. In support of this conclusion are the facts that: (1) the F P R cannot be r e p r o d u c e d by stimulation of the FN with excitatory amino acids s~3 15,50; (2) the F P R persists after destruction of local neurons with an excitotoxin ~2 14: (3) after t r e a t m e n t with an excitotoxin electrical stimulation still evokes an F P R . The identity of the neurons whose processes, when stimulated, elicit the F P R is not known. H o w e v e r the finding that the R V L is a critical link in the projection permits a m o r e detailed formulation of their identity. First, the candidate neurons must have axons projecting into or through the rostral FN since this is the only place from which the response can be elicited 4~'sv. Second the axons must also project mono- or polysynaptically to the R V L since the response is abolished by

100 RVL lesions. Third, the neurons must be contained within the uncinate fasciculus and possibly superior cerebellar peduncle since the response is abolished by selective lesions of these trunks 4s. Fourth, since the FPR cannot be elicited from the caudal FN "~sand persists after chronic removal of the cerebellar vermis (Chida, ladecola, Reis, unpublished), it is unlikely that they are the axons of intrinsic neurons of cerebellar cortex or caudal FN which project through the rostral FN and superior cerebellar peduncle to the brainstem34:~>-<: rather they are contained in brainstem. Therefore. it is most probable that the FPR is the result of antidromically exciting a brainstem neuron with collateralized axons, one branch of which innervates or passes through the FN and another projecting directly to or through an interposed neuron into RVL (see Fig. 7). The assumption implies that the area containing candidate neurons has autonomic activity. Thus, by exclusion, the possible sources would be autonomic nuclei of the lower brainstem which either innervate FN or pass through the nucleus to innervate the cerebellar cortex and which through collaterals innervate, mono- or polysynaptically, the RVL. By these criteria it seems improbable that cell groups innervating the FN are involved. Other than an important input from cerebellar cortex the principal afferents innervating the FN arise from neurons of the vestibular, perihypoglossal and medial accessory olivary nuclei of brainstem (see refs. 39, 71). These are all areas without evident effects upon autonomic activity nor projections to the RVL ll'ts'~9 On the other hand several brainstem nuclei with autonomic connections and activities project through the white matter adjacent to FN to innervate the cerebellar cortex, possibly also sending branches to the FN. These include projections from nuclei with potent autonomic actions including cardiovascular areas of the nucleus tractus solitarii 6977, the parabrachial nucleus 2aTs, the dorsal motor nucleus of the vagus s6, the periaqueductal gray 23, the nucleus locus ceruleus 2*v6, raphe nuclei 6s2 and several nuclei of the hypothalamus including dorsal, lateral, posterior and to a lesser extent periventricular nuclei e~34. In addition some of these regions, including the lateral and dorsal hypothalamic nuclei ~s, the parabrachial nucleus t~~s, the nucleus solitarii ~76'~ and some nuclei of the raphe 69 project to RVL. Whether neurons of these nuclei which innervate cerebellum also project to the RVL is not known. However neurons in some of these areas are highly collateralized including neurons of hypothalamus projecting both to cerebellum and amygdala and neurons of the parabrachial nucleus projecting to both amygdala and cerebellum 24. (Indeed, it is even conceivable that the critical neurons could reside in

hypothalamus if the branch point oi zt presumed hypothalamo-RVL/cerebellar neuron lies bek~w the midcotlicular plane and would therefore still conduct impulses even after an acute midcollicular decerebration ::2"as.) Recently Miura and Takayama 5° have proposed that in cat the FPR arises from stimulation oI commissural axons joining the left and right parabrachial nuclei of the pons. Conceivably some of these fibers may innervate FN or midline cerebellar cortex 7s. This proposition is interesting since, like the FPR, electrical stimulation of portions of the parabrachial nucleus will simulate the FPR increasing globally AP, HR and, globally, cerebral blood flow sa'ss, Lacking, however, is the critical evidence demonstrating that the FPR is eliminated by excitotoxic lesions restricted to neurons of the parabrachial nucleus and that the critical neurons of the parabrachial projecting to FN also innervate RVL. Difficult to reconcile with this study, however, are reports that damage to two brainstem nuclei, the A5 noradrenergic nucleus of ventral pons :8 and the paramedian reticular nucleus of the medulla 4~'49, like the C1 area, will eliminate the FPR. Dormer et al. 2s placed lesions throughout the brainstem of beagle and discovered that the only site effective in abolishing the FPR was a region of ventral pons which they designated the A5 area (although the neurochemical identity of these neurons was not established by cytochemistry). The most obvious reconciliation between that and the present study is to propose that the FPR either relays to the A5 area or the C1 area with each area then relaying to IML to effect AP. The problem with the assumption that the A5 area is critical is that while A5 neurons innervate the IML 4>75, electrolytic lesions of the A5 area do not affect resting AP 33. Moreover, chemical stimulation of the A5 area will lower AP, but through non-spinal mechanisms 4~. The alternative possibility, namely that the effect is mediated via a projection from the noradrenergic neurons of the A5 area into the C1 area also seems implausible (even though the A5 area is innervated m from nuclei which project to cerebellum including NTS 66'6977, the parabrachial nucleus 24'7s and hypothalamus2634). Thus, there is little, if any, evidence that the C1 area is innervated by A5 neurons 7s° while norepinephrine (which would be expected to be released from A5 terminals in the C1 area by A5 activation) lowers rather than elevates AP 33. More likely explanations for the findings of Dormer et al. 2s are that their electrolytic lesions partially damaged the C1 area (inspection of their figures indicates that the caudal edge of their lesion may have impinged on rostral RVL) and/or fibers passing to RVL from candidate brainstem neurons with presumed collaterals to FN as described above. The critical experiment will involve

101 demonstrating that destruction, by excitotoxins of the A5 group (whose identity is unequivocally established by immunocytochemistry) will also block the FPR. The possibility that the paramedian reticular nucleus (PRN) is involved 4.'4~, is also difficult to reconcile with the present data. While Miura and R e i s 48'49 observed that bilateral electrolytic lesions of PRN abolished the FPR in cat, the finding was not replicated by Dormer et al. in dog 2s. While there is anatomical 3"4'39 and electrophysiological 29'49 evidence that the PRN is innervated from the FN and may project to IML 29 it does not project to the C1 area. Conceivably, the lesions of Miura et al. 48"4~J damaged the descending pathway of C1 fibers in dorsal medulla or interrupted projections from candidate neurons with cerebeilar axons which project into the RVL.

Neural organization of the FDR In contrast to the FPR, the FDR is initiated by stimulation of intrinsic neurons of the FN. In support are the facts that: (1) the FDR is elicited by microinjection of some excitatory amino acids into FNIS; (2) the action of the amino acids is blocked by local pretreatment with antagonists of glutamate receptors~5; and (3) destruction of FN neurons by excitotoxins 13"14abolishes the response. Interestingly, the FDR cannot be elicited by local application of the native transmitters glutamate or asparrate into the FN but is easily elicited by low concentrations of kainic acid or D.L-homocysteic acid ~5. The insensitivity to L-Glu most probably reflects their rapid inactivation of the amino acid by uptake into local glia 31. The insensitivity to glutamate may explain the failure of some 8 to elicit a FDR using this amino acid. Like the FPR, the FDR is abolished by lesions of the RVL, a finding which indicates that it is not mediated over fastigiospinal pathways (see ref. 39). Moreover, the facts that the response persists after midcollicular decerebration and that there are no known direct projections from the FN to the R V L 4'11'18"39"72 indicate that the response relays to RVL through an interposed neuron(s) within the lower brainstem. Neurons of the rostral FN innervate the vestibular complex 3'2~'39, neurons within the medial pontine and medullary reticular formation including the nucleus reticularis ventralis, lateral reticular nuclei, nucleus gigantocellularis, and paramedian reticular nuclei and nucleus locus ceruleus, none of which project to RVL. On the other hand projection areas which might exert control over sympathetic activity include the parasolitary division of the nucleus tractus solitarii 3"4"21"39"6~ and the parabrachial nuclei 21 both of which project into R V L 11'1&69'72. At present no information is available on which of these might be essential in mediating the FDR.

Role of the RVL in the cerebrovascular vasodilation from FN Neurons of the ventral medulla also appear to mediate the global increase in rCBF elicited electrical stimulation of the FN 57 59 since the cerebrovascular vasodilation is abolished by lesions of the C1 area. The effects of RVL lesions on rCBF cannot be secondary to alterations in ventilation or AP since these variables were controlled and blood gases and AP did not vary between groups. Likewise the impairment of the cerebrovascular response is unlikely to be a consequence of interruption of the descending sympathoexcitatory outflow and collapse of AP. AP was always maintained by phenylephrine to lie within the autoregulated range in rat 3-~. Moreover, transection of the cervical cord which damages the same reticulospinal projection does not affect the elevations in rCBF produced by electrical stimulation of either FN 5s, dorsal medulla 37 or RVL sS. The RVL, therefore, is a relay through which electrical stimulation of FN acts to globally increase rCBF. Consistent with the findings are recent observations that electrical and chemical stimulation of the RVL will, like electrical stimulation of FN, elicit widespread increases in rCBF unassociated with changes in regional glucose utilization s~. These suggest that the effect of FN stimulation is to excite neurons of RVL acting upon the cerebral circulation much as it does upon reticulospinal neurons which excite preganglionic sympathetic neurons. The elevation of rCBF elicited by electrical stimulation of the FN, like the effects upon AP and HR, is a consequence of stimulation of intracerebellar axons since it persists after destruction of local neurons by ibotenic acid and chemical excitation of the FN results in a fall of rCBF associated with a reduction in rCGU 14'1~'. Thus both the systemic and cerebrovascular effects of electrical stimulation of FN are most probably due to antidromic activation of a brainstem neuron presumably via branching collaterals with one branch innervating the cerebellum another the RVL. Whether the same neurons of brainstem mediate the systemic and cerebrovascular effects of RVL stimulation is unknown. Conceivably, the candidate areas could differ particularly in view of the suggestion that the representation of sites in FN eliciting elevations in AP and rCBF may be topographically distinct 57. However both engage the RVL albeit not necessarily the same neuron. The pathways through which RVL activity acts to increase rCBF in rostral areas of brain and most notably the cerebral cortex is not known. Neurons of RVL project rostrally to innervate a wide range of autonomic regions including the parabrachial nuclei, hypothalamus, the nucleus locus ceruleus 62J2's4 and probably intralaminar nuclei of thalamus 72, a region which when electrically

102 stimulated will increase rCBF but not metabolism in widespread regions of forebrain 53"56. On the other hand, the fact that lesions of basal forebrain will abolish the cerebrovascular c o m p o n e n t s of the F P R 3s indicates that other pathways may be involved. We have recently d e m o n s t r a t e d that chemical excitation of FN n e u r o n will produce reductions in rCBF and r C G U 14. Thus both patterns of autonomic excitation evoked from FN engage the cerebral and systemic

the R V L play a critical role in mediating both the sympathoexcitatory and sympathoinhibitory responses represented within the FN of rat. Presumably the opposing cerebeilosympathetic networks act upon a c o m m o n set of output neurons in R V L

governing, in

turn, the activity of preganglionic sympathetic neurons. Since RVL n e u r o n s are essential in integration of a wide

that the reduction in r C B F associated with the F D R are

range of cardiovascular reflexes (see ref. 64), including those elicited from arterial baro- and chemoreceptors and other cardiopulmonary receptors 32, from stimulation of somatic nerves (pain) 44'52'7'~and by cerebral ischemia ~, it

initiated by excitation of intrinsic neurons of the FN, it is not k n o w n whether this effect relays through the RVL.

might be a site wherein the cerebellum may act to modulate reflexes from visceral and somatic receptors

circulations and in opposite directions. While it is clear

The facts that the elevation of r C B F associated with the F P R is not coupled to changes in r C G U 5~ while the

(see ref. 41). The findings are also of interest with respect

response associated with the F D R is, indicate that the elevation or depressions of rCBF associated respectively with the F P R and F D R are not mirror images of the other.

to the integration of the cerebral and systemic circulations. They again demonstrate that within brain there is an intimate relationship between those regions regulating the output of sympathetic nerves and networks intrinsic

Concluding remarks

to brain which may profoundly regulate cerebral blood flow and metabolism 63.

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