Facilitation of the lumbar monosynaptic reflexes by locus coeruleus stimulation

FACILITATION OF THE LUMBAR MONOSYNAPTIC REFLEXES BY LOCUS COERULEUS STIMULATION J. C. STKAHLENDOKF,~

H.K.

J. GINTAUTAS,’ ‘Department of Physiology. 2Center for Medical Education.

R. E. KINGSLY~Y.’ and C. D. BARNES’

STKAHLENDORF.'

Texas Tech University School of Medicine. Lubbock. TX 79430 and Indiana University School of Medicine, Notre Dame, IN 46556. U.S.A.

conditioning train of stimuli delivered in the vicinity of the locus coeruleus elicited a marked (average 104”;,) facilitation of the lumbar monosynaptic reflex in decerebrated cats. Placement of the electrode in periaqueductal gray resulted in a biphasic facilitatory action in the MSR. Phenoxybenzamine (2 m&kg) and chlorpromazine (l-2 mg;kg) caused marked reductions in LC-induced augmentations. Haloperidol appeared to potentiate LC-elicited facilitation. These data further verify the noradrenergic nature of fibers originating from the locus coeruleus and assign a physiological role to coeruleo-spinal projection in the regulation of lumbar MSR. Summary--A

Experimental evidence from studies examining the effects of supraspinal structures on the monosynaptic reflex (MSR) in the lumbar cord indicates that there are three classes into which most spinal reflex responses can be categorized: inhibition of flexor and extensor motoneuron pools; inhibition of both pools preceded by facilitation; or reciprocal inhibition and facilitation of flexor and extensor pools (Chan and Barnes, 1972; Engberg, Lundberg and Tyall, 1968: Pompeiano. 1973). However. recent reports suggest that the nigro-striatal complex facilitates both flexor and extensor spinal reflexes in the cat (York, 1972a, 1973). These nigral-induced responses were postulated to be relayed i?u midbrain structures by way of reticula-spinal systems to the lumbo-sacral level. Several studies in rat have revealed the existence of noradrenergic projections coursing in the ventral funiculus and ventral parts of the lateral funiculus of the spinal cord, terminating within alpha motoneuron pools (Commissiong. Hellstrom and Neff. 1978: Dahlstriim and Fuxe. 1965; Nygren and Olson, 1977). These fibers originate in the locus coeruleus (LC), a pontine tegmental structure known to possess a predominance of norepinephrine-containing cell bodies (Chu and Bloom, 1974) and to receive a large number of nigral e&rent terminations (Sakai, Touret. Salvert, Leger and Jouvet, 1977). Similarly, in cat, spinal projections from the locus coeruleus and subcoeruleus have been demonstrated by retrograde transport of horseradish peroxidase (Hancock and Fougerousse. 1976). The present investigation was initiated to examine the influence of LC stimulation on the lumbar MSR in cats and to characterize further the functional relationships and types of transmitters which may be involved between these areas. 225

METHODS

Twenty-three cats of either sex, decerebrated at the precollicular level under ether anesthesia, were subsequently subjected to lumbar laminectomies (L55L7) and prepared for MSR recording. Animals were paralyzed with Flaxedil (2 mg/kg) and artificially ventilated. End tidal CO, was continuously monitored by a Beckman LB2 gas analyzer and maintained at 3.8 k 0.5y,0 by adjusting the rate of respiration. Arterial blood pressure was recorded from a cannulated femoral artery contralateral to the recording site. Experiments were discarded if mean blood pressure fell below 70 mm Hg. Body temperature was maintained at 37°C with a proportional controlled thermoregulator (Kingsley. Barnes and Pompeiano. 1976). After exposure of the spinal cord. L6 and L7 dorsal and ventral roots were cut and positioned on platinum hook electrodes. In some animals the popliteal fossa was opened and the gastrocnemius and common peroneal nerves were prepared for recording instead of the severed ventral root. The MSR was elicited by single shocks (%&IO0 htsec) at a strength of two times threshold for the MSR. Modulation of MSR activity was achieved by a train of four shocks (50 100 lrsec pulses at 1Omsec intervals) delivered through a concentric electrode (Rhodes Medical Instruments, Model NE 100). stereotaxically directed toward the locus coeruleus (PO.~rO~.~. Lr.7r0~.r. Hml.sro -z.o). Placement of the electrode was aided by visual landmarks. All stimulus shocks were optically isolated from ground reference by WPI constant current isolators. Recorded potentials were preamplified by a WPI DAM 6A differential amplifier (band pass of 0.1 to > 30 kHz). A programmable stimulator was used to deliver stimuli every 4 sec. The interval between the oscillo-

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STRAHLF.NDORF t’r ul.

scope trigger pulse and the test MSR shock was automatically incremented 2 msec after each sweep. A constant interval of 25 msec between the oscilloscope trigger and the conditioning train was maintained: MSR activity was displayed on a storage oscilloscope. In this manner. a time versus amplitude histogram of the MSR at conditioning-testing interv,als from -25 to 175 msec was created and stored on Polaroid film. Phenoxybenzamine (PBZ) (2 mg.kg). chlorpromazine (CPZ) (l-2 mg/kg) and haloperidol (OS mg/kg) were dissolved in physiologic saline and injected slowly into the inferior vena cava via a catheter inserted into the femoral vein. At the termination of each experiment. small electrolytic lesions were created at the anode of the conditioning electrode, and animals were perfused with formalin solution saturated with potassium ferrocyanide. Electrode placements were visualized with the aid of the Prussian blue reaction on frozen serial 40 pm sections stained with cresyl violet. RESULTS

Stimulation in the area of the ipsilateral LC produced a marked facilitation of lumbar spinal MSR (Fig. 1A). The average potentiation in all instances observed was 1040;,, with a range of 39.-380”/;,. A differential effect of LC conditioning was seen on MSRs recorded from L6 vs L7. Enhancement of L6 MSR activity averaged 60”/, (range 39-925;); while facilitation of L7 MSR averaged 1540;, (range of 50-380”:,). The onset of induced increases was approximately 30 msec and lasted 135 msec after the beginning of the conditioning train. Peak facilitatory effects were seen approximately 75 msec after conditioning. On several occasions, a transient increase in MSR amplitude occurred as the conditioning electrode was lowered into place. It is interesting to note that contralateral stimulation in the area of the LC also showed similar potentiation of the MSR. In some instances. ventral root discharges were elicited by ipsilateral stimulation A

Some placements of the brainstem electrode were found to lie dorsal to LC, i.e. in periaqueductal gray (PAG). In 753, of these animals, conditioning stimulation resulted in a biphasic facilitatory action on MSR (Fig. IB). The onset of the first facilitatory period was 20 msec after conditioning stimulation. The minimum height between the two peaks coincided in time (72 msec) with the peak facilitatory action in those experiments which demonstrated a single peak. The duration of the biphasic response was similar to that observed in experiments with monophasic changes. Since the LC of the cat is a diffuse structure intertwined with central gray and a wide range of stimuli intensities were necessary to elicit facilitatory activity. a mathematical correlation was derived to further implicate LC as a source of the observed response. A correlation index was derived by dividing the average percent facilitation by the average stimulus strength in /tA between each response and histologically verified electrode placements. Electrode placements less than l.Omm from the center of the LC had an index of 0. I I. For distances greater than 1.0 mm. the index was 0.04. This demonstrates that there exists a maximally effective locus for chciting facilitation in the vicinity of LC with the stimulus strength required to illicit the spinal cord effect higher with electrode placements outside a I mm radius surrounding this pontine tegmental nucleus. Figure 2 depicts the actual electrode placements within the vicinity of LC. Various pharmacological interventions were performed to clarify the nature of the induced facilitation of MSR activity. In 12 animals. PBZ. an alphaadrenergic blocking agent, was administered (2 mgikg. i.v.) resulting in a marked reduction in the degree of LC-induced augmentation of the MSR (Fig. 3). This antagonism was generally expressed in the absence of marked effects on control MSR activity. although a slight increase in MSR amplitude was occasionally observed. The onset of PBZ blockade was npproximately 20 min; peak effects occurred within 45 min following drug administration. In two experiments. amperage of the conditioning train was increased to B

Fig. I. Effect of midbram stimulation on the monosynaptic reflex in a decerebrate cat. Monosynaptic reflex elicited by stimulation of dorsal root of L7 and recorded from ventral root of L7. A: Locus coeruleus conditioning stimulation delivered at marks. B: Periaqueductal gray conditioning stimulation delivered during gate pulse. Conditioning stimulation in both instances consisted of 4 pulses at 100 HI. Calibration: 20 msec, I mV.

Monosynaptic

Fig. 2. The distribution

CONT

of electrode

reflex and locus coerulcus

placements

in the dorsolateral

221

tegmentum

of the pons.

PBZ

Fig. 3. Effect of phenoxybenzamine on locus coeruleus-induced facilitation of the monosynaptic recorded from L7. Locus coeruleus conditioning stimulation delivered at marks. CONT: Control. 20 min post phenoxybenzamine. 2 mg/kg. Calibration: 20 msec. I mV.

reflex PBZ:

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J. C. STRAHLWIIORF or al. CPZ

CONT

_-. --

--

l Fig. 4. Effect of chlorpromazine on locus cocruleus-induced facilitation of the monosynaptic reflex recorded from L6. Locus coeruleus conditioning stimulation delivered at gate pulse. CONT: Control. CPZ: 30 min post chlorpromazine. 1mg/kg. Calibration: 20 msec. 2 mV.

five times initial stimulus strength to assess the efficacy of the blockade. In both instances, it was not possible to surmount PBZ actions. Chlorpromazine was given to 6 animals. Similar to PBZ. intravenous CPZ l-2 mgikg produced a diminution of LC-induced enhancements (Fig. 4). The onset of action was about IO min and maximal antagonism was observed 20 min after drug administration. The CPZ-initiated blockade occurred in the presence of a slight reduction in amplitude of the control MSR. Since CPZ has adrenergic and general aminergic blocking properties. it was advantageous to investigate further the possibility that dopaminergic neurotransmission contributed to LC facilitatory actions by inlccting halopcridol intravenously (OS mg;kg) in 5 animals (Fig. 5). In contrast to PBZ and CPZ, halopcridol appeared to potentiate LC-elicited facilitation. The apparent enhancement may be attributed to a reduction in MSR amplitude without a concomitant decrease in LC-induced facilitation. The onset of haloperidol-elicited potentiation was approximately 10 min; peak effects wcrc manifest within 30min. During administration. haloperidol produced an initial transient enhancement followed by a prolonged decrease in MSR amplitude. In one animal. this agent caused a reduction in LC-initiated facilitation of MSR similar to CPZ and PBZ.

A comparison between blood pressure, MSR, and the LC-elicited facilitation was made since all three drugs involved have cardiovascular effects. No consistent correlation could be made in any one animal or between animals. All three drugs produced a decrease in blood pressure. Although haloperidol and CPZ often resulted in a decreased control MSR. PBZ was attended by no change or an increase. Phenoxybenzamine with an increased MSR. produced a decreased facilitation as did CPZ, although the latter had a decreased control MSR. Furthermore. haloperidol. though showing the same blood pressure and control MSR effects as CPZ. produced an increase rather than a decrease in facilitation.

DISCL’SSION

Brainstem stimulation in the vicinity of LC produced a marked enhancement of the lumbar monosynaptic reflex. Previous studies (York. 1972a. 1973) have revealed similar enhancements of MSR activity after stimulation of the n&al-striatal complex, the effects of which were thought to be relayed [?a the midbrain to the reticula-spinal tract. These changes were abolished by an ipsilateral ventral quadrant cord section (York. 1972b). an area shown to have the majority of descending cocruleus projections in rat.

Fig. 5. Effect of haloperidol on locus coeruleus-induced facilitation of the monosynaptic reflex recorded from L6. Locus coeruleus conditioning stimulation delivered at marks. CONT: Control. HAL: 30min post haloperidol. 0.5 mg./kg. Calibration: 20 msec. 2 mV.

Monosynaptic reflex and locus coerulcus Furthermore, anatomical studies in rat have revealed that noradrenergic fibers originating from LC terminate in ventral regions of the dorsal horn (laminac I-IX) (Nygren and Olson, 1977). Coeruleo- and subcoeruleo-spinal projections have also been described in the cat and monkey (Hancock and Fougerousse. 1976). Sakai et (I/. (1977) have demonstrated a large number of afferent fibers to locus coeruleus from the ipsilateral substantia nigra. Collectively. these data suggest that the nigral-induced increase of MSR. as reported by York (1973) may be relayed at least in part through LC. Fluorescent techniques have revealed that many of the norepinephrine-containing terminals surround large neurons believed to be alpha motoneurons in the ventral column of the spinal cord (Commissiong er u/.. 1978). Ventral root discharges elicited by LC area stimulation. as observed in the present study. may reflect synaptically coupled effects directly on alpha motoneurons. Barnes, Fung and Gintautas (1979) have antidromically activated LC cells from stimulation of the lumbar ventral column and demonstrated their spontaneous activity to coincide with tonic facilitatory effects on the cord from the brainstem. The MSR increase following LC stimulation may be interpreted as a descending system with conduction velocities about 9 mlsec to onset. 4 m;scc to peak effect and I m#sec to end of effect. These values correlate well with the reported values for cat locus coeruleus fibers which had a mean of 3 m set and a range of 1 to 9 misec (McBride and Sutin. 1976). Studies are presently underway in the authors’ laboratory to determine the correlation between the chemical depletion of NE in the spinal cord and the amount of MSR facilitation produced bq LC stimulation. Preliminary data indicate that animals treated with 6-hydroxydopamine have a decreased lumbar MSR facilitation from LC stimulation as well as a decreased spinal cord NE content, Contralateral stimulation in the region of LC also facilitated the MSR. In this regard. it has been demonstrated that some LC fibers decussate in the spinal cord (Dahlstriim and Fuxe. 1965). Furthcrmore, a study (Buda. Roussel. Renaud and Pujol. 1975) in which a lesion placed in one LC caused changes in tyrosinc hqdroxylase activity in the opposite LC suggested the existence of coeruleo-coerular connections. The contralatcral facilitatory response on the MSR therefore. could be due to an axon reflex where the contralateral terminal endings were stimulated with the activit) then crossing antidromically to the origin of the collateral and descending orthodromically without synapsing. Since maximal facilitntory effects were obtained with the more accurate electrode placements. it seems probable that facilitation of MSR activity aas the result of direct activation of descending coeruleospinal fibers. Locus coeruleus stimulation produced a greater facilitatory efTect on the MSR of L7 than of L6. Since L7 contains both flexor and extensor nerves.

229

while L6 is comprised primarily of flexor elements to the ankle but extensors of the hip and flexors and extensors of the knees, it appears that LC may have a preferential effect on extensor muscles or on distal .joints. In this regard. Sinclair and Sastry (1974) have proposed a tonically active supraspinal system which has an overall excitatory influence on extensor motoneurons. This is interesting in light of the work of And&. Engle and Rubenson (1972) who found that late flexor reflex activity in the rat was enhanced by dopamine and norepinephrine, whereas the early activity was depressed. Investigations are currently underway to clarif! this disparity. Conditioning trains delivered to electrodes which were placed dorsall) to LC in the PAG also resulted in a facilitation of MSR activity. although it was biphasic in nature. These data are in accord with the anatomical findings of Russell (1955) which demonstrate afferents to LC originating from the mesencephalic central gray. Consequently. stimulation of PAG ma) have resulted in coactivation of the dorsolateral LC which manifested itself as a complex interaction of excitation and inhibition of the MSR. Although an explanation for the biphasic nature of the response is not clearly evident, it is possible that stimulation of the area encompassing PAG also activates another arca or arcas that elicit(s) inhibition of MSR activity. Periaqueductal gray-elicited facilitation of lumbar MSR is particularly interesting in the light of studies by Liebeskind. Guilhaud. Besson and Olivcras (1973) which ascribe the primarily inhibitory activity of PAG to Group III and IV fibers. That phenoxybenzamine antagonized the facilitatory effect lends further support to the contention that LC-initiated facilitation is mediated by noradrenergic neurotransmission. It is well established that I-DOPA enhances spinal reflexes in cat and rat presumably via release of NE from nerve terminals of descending noradrcnergic axons (And& ut d.. 1972). Phenoxybenzamine is thought to antagonize NE irreversibly at post-junctional sites: ho\ve\,cr. the exact anatomical site of PBZ action is not immediately evident from this stud\. In this regard. Sastry and Sinclair (1976) have shown diminution of flexor and facilitation of extensor recurrent inhibition by PBZ. leading to the speculation that there exists a tonically active descending inhibitory noradrenergic system. Single test shocks delivered every 4 sec. as employed in this study. precluded effects of recurrent inhibition, however. Chlorpromazine effectively antagonized the facilitatory response to midbrain stimulation. A similar action of CPZ against I-DOPA-induced facilitation of the MSR has been demonstrated in cat (Baker and Anderson. 1970). In the present study. chlorpromazine was shown to diminish the amplitude of the unconditioned MSR. which suggested that CPZ may have a spinal site of action, as has been shown in rats (Herman and Barnes. 1967). Alternatively. in the pre-collicular deccrebrate cat. MSR activity may be under the

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J. C. STKAHLFYDOKF et ul.

influence of a tonically active descending facilitatory system which was antagonized by CPZ at the supraspinal Icvel. The dopamine antagonist, haloperidol, appeared to potcntiate the MSR. In contrast. York (1973) demonstrated haloperidol to be almost as effective as CPZ in blocking nigral-induced facilitation of the MSR. If nigral-elicited enhancement of MSR activity were relayed through LC. haloperidol would fail to antagonize facilitation induced by direct LC stimulation. Accordingly. these data suggest the involvement of a facilitator) dopaminergic pathway between the substantia nigra and locus coerulus which appears to activate coeruleo-spinal projections. Although the evidence supports the concept that the present findings are due to coeruleo-spinal projections. no evidence precludes the possibility that the effects from LC stimulation are being relayed through some other neurons in the brainstem before entering the cord. Also. an alternative to NE being facilitatory, which is consistent with all of the reported findings. is that the descending NE system has a modulatory action on Ia synapses such as to potentiate their efficacy. Such an action has been demonstrated at other sites in the neuraxis (Woodward. Moises. Waterhouse, Hoffer

and

Freedman.

1979).

.-l~~~,~o~r/c~/~//,r,~c,,~r.s~ This research was supported in part hq grants from the Tarbox Parkinson’s Disease Institute. NIH Grant HL 7289 and Merck Frosst Laboratories.

REFERENCES AndOn. N. E.. Engle. J.. and Rubenson,

A. (1972). Mode of on central noradrenaline mechanisms. ,~~rlrtt!,rt-S~/I~~li[,~~[,b[,r~/s .4r(hs c’.x,>. Purh. Phcvr~~ac~. 273:

action

of I-DOPA

I IO. Baker. R. G. and Anderson. E. G. (1970). The antagonism of the effects of I-3.4-dihydroxyphenqlalanine on spinal rctlcxes by adrencrgic blocking agents. J. Phurrwc. c’sy. 7’1wr. 173: X4-23 I. Barnes, C. D.. Fung. S. J. and Cintautas. J. (1980). Brainstem noradrenergic system depression by cyclobenzaprine. r~c~~r~opliurrf~~~~~lo~~!~ 19: 22 I 224. Buda. M.. Ronsscl. B.. Rcnaud. B.. and PuJol. J. F. (1975). Inc~wtsc in tqrosinc hJdrox>lasr activity in the locus cocruleus of the rat brain after contralateral lesioning. B,-tlitl Rc.y. 93: 564 569. C’han. S. H. H. and Barnes. C. D. (1972). A prcsynaptic mechanism choked from brain stem reticular formation in the lumbar cord and its temporal sigmficancc. Bwrl Rv\. 45: 101~ 114. Chu. N.-S. and Bloom, F. E. (1974). The catecholaminecontuinlng neurons in the cat dorsolateral pontinc tegmentum: distrihution of the ccl1 bodies and some axonal proJections, Brrri~ Rra. 66: I 21. Commissiong. J. W.. HrllstrGm. S. 0.. and Neff. N. H.

(1978). A new proJection from locus coeruleus to the spinal ventral columns: histochemical and biochemical evidence. Errrir~Rcs. 148: 207-213. Dahlstriim A. and Fuxe. K. (1965). Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of hulhospinal neuron systems, Actu physiol. .scuizd 64: suppl. 247. I 36. Enghcrg, I.. Lundberg. A., and Tyall. R. W. (1968). Reticulospinal inhibition of transmission in reflex pathways. J. PIl~SiOl. 194: ‘01 223. Hancock. M. B. and Fougerousse, C. L. (1976). Spinal projections from the nucleus locus coeruleus and nucleus suhcoerulcus in the cat and monkey as demonstrated by the retrograde transport of horseradish peroxidase. Brclirl Rcs. Bull. 1 : ‘29 ‘34. Herman. E. H. and Barnes. C. D. (1967). Observations of drug effects on a tqpc of spinal motor activity. Arc/u irlf. Phrrrm~~r/~r~. Thu. 165: 425-429. Kingsle], R. E.. Barnes, C. D., and Pompeiano, 0. (1976). A proportional controlled thermoregulator circuit of simple design. E. E. G. Clin. .\‘wwph~sio/. 40: 30&30X. Licheskind J. C.. Guilhaud. G.. Besson. J. M.. and Oliveras. J. L. (1973). Analgesia from electrical stimulation of the perwqueductal gray matter in the cat: behavioral ohservations and inhlbltory effects on spinal cord interneurons. Brclifl Re. 50: 44l- 446. McBride. R. L. and Sutm. J. (1976). Projections of the locus cocruleus and adjacent pontine tegmentum in the cat. J. co,,lp. :\‘c~rr. 165: ‘65-284. Nbgrcn, L.-G. and Olson L. (1977). A new major projection from locus cocrulcus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord. Brclin Rec. 132: 85 -93. Pompeiano. 0. (1973). Reticular formation. In: Hmdhook 01 Set~.sory Physiology. Somutosensory Spstern. (Iggo. A.. Ed.). Vol. II pp. 3X1-4X8. Springer-Verlag, New York. Russell. G. V. (1955). The nucleus locus coeruleus (dorsolateralis tcgmenti). Tcu. Rep. Bid. Med. 13: 939 -988. Sakai. K.. Tourct, M., Salvert. D.. Leger. L., and Jouvct. M. (1977). A&rent projections to the cat locus coeruleus as >isuali/cd h> the horseradish peroxidase technique. Brrrill Rc.\. 119: 71 ill. Sastry. B. S. R. and Sinclair. J. G. (1976). Tonic inhibitory influence of a supraspinal monoaminergic system on recurrent inhibition of an extensor monosynapic reflex. Sraiv Rv\. 117: 69 76. Sinclair, J. G. and Sastrq. B. S. R. (1974). Supraspinai monoaminergw involvement in the blockade of recurrent inhlhition of the monosynaptic reflex. ,VrfrrophLlr,?ltI(.Okx! 13: 741 747. Wooduard. D. J., Molscs. H. C.. Waterhouse, B. D.. Hoffcr, B. J. and Freedman. R. (1979). Modulatory actions of norepmephrine in the central nervous sqstcm. F<,tlr, Proc. Fcth~ AUI. .Soc.\ r.yp. Biol. 38: 2109~ 21 16. York, D. H. (1972a). Potentiation of lumho-sacral monosynaptic reflexes by whstantia nigra. E\-pl .%‘cwol. 36: 437 448. York. D. H. (1972b). Potcntiation of spinal monosynaptic rcflexcs hq the suhstantia nigra. F& Pro<,. Fdn .4rn. Sots