J. Phyaiol. (1977), 265, pp. 327-340 327With 4 text-figure.Printed in Great Britain

EXCITATORY RESPONSES OF NEURONESIN RAT BULBAR RETICULAR FORMATION TO BULBARRAPHE STIMULATION AND TO IONTOPHORETICALLY

APPLIED 5-HYDROXYTRYPTAMINE, AND THEIRBLOCKADE BY LSD 25

BY I. BRIGGSFrom the M.R.C. Neuropharmacology Unit, Medical School,

Birmingham, B15 2TJ

(Received 21 April 1976)

SUMMARY

1. The micro-iontophoretic technique has been used to study the re-sponses of single neurones in the bulbar reticular formation to 5-hydroxy-tryptamine and to noradrenaline, ACh or glutamate, and to compare thesewith theresponses to electrical stimulation inor near the bulbar raphe nuclei.

2. In the bulbar reticular formation, most neurones were excitedby 5-hydroxytryptamine; forty-three of fifty-one neurones excited by5-hydroxytryptamine were also excited by stimulation in nucleus raphemagnus, nucleus raphe pallidus or nucleus raphe obscurus. Most of thesestimulation-induced excitations were of long latency: LSD reduced six ofseven of these excitations tested, while 5-hydroxytryptamine excitationswere blocked on all seven.

3. In contrast, stimulation of areas adjacent to the raphe nuclei excitedonly fifteen of forty-six neurones excited by 5-hydroxytryptamine. Most ofthese stimulation effects were of short latency and none of the threetested were reduced by LSD, although 5-hydroxytryptamine excitationswere blocked.

4. The relationship of the long-latency excitatory effects of stimulationwith the position of the stimulating electrode in the raphe nuclei indicatesthat these effects are probably mediated via the raphe neurones and thisis supported by the correlation of the effects of raphe stimulation with theeffects of 5-hydroxytryptamine applied iontophoretically and by theability of LSD to block both effects.

5. The results provide a physiological basis for the excitatory effects ofiontophoretically applied 5-hydroxytryptamine in the bulbar reticularformation.

* Present address: Pharmacology Department, The Wellcome Research Labora-tories, Beckenham, Kent.

I. BRIGGS

INTRODUCTION

Several studies have recently been made concerning the pharmacologyof the responses to iontophoretically applied 5-hydroxytryptamine (5-HT)of neurones in the ponto-bulbar reticular formation (Avanzino, Bradley& Wolstencroft, 1975; Boakes, Bradley, Briggs & Dray, 1970; Bradley &Briggs, 1974a, b; Bradley & Dray, 1973; Bradley & Wolstencroft, 1965;H6sli & Tebecis, 1971) and in the mesencephalic reticular formation(Haigler & Aghajanian, 1974a, b; Straschill & Perwein, 1971). In the ratbulbar reticular formation, the predominant effect of 5-hydroxytrypt-amine is excitation, although inhibition is also seen, whereas in the mesen-cephalic reticular formation, weak inhibitory effects are most common(Haigler & Aghajanian, 1974b).However, the density of 5-hydroxytryptamine containing terminals in

the bulbar reticular formation was found by Fuxe (1965 a, b) to be low, andphysiological evidence was also lacking that there are any serotoninergicsynapses upon the neurones in the bulbar reticular formation which re-spond to micro-iontophoretically applied 5-hydroxytryptamine, althoughprobable serotoninergic synaptic actions of both excitatory and inhibitorytypes have been described in other regions (Bloom, Hoffer, Siggins, Barker& Nicoll, 1972; Couch, 1970; Haigler & Aghajanian, 1974a; Nakamura,1975; Segal, 1975). Doubt has therefore been cast on the validity of studieson the 5-hydroxytryptamine responses, particularly of excitatory type, inthe bulbar reticular formation (Bloom et al. 1972; Haigler & Aghajanian,1974a, b). The present study was made to examine whether the activity ofneurones in the bulbar reticular formation can be affected by stimulationof the bulbar raphe nuclei which are thought to be the probable origin(Fuxe, Hokfelt & Ungerstedt, 1971) of the 5-hydroxytryptamine contain-ing terminals in the bulbar reticular formation and to compare any effectswith the effects of 5-hydroxytryptamine applied by iontophoresis.The excitatory effects of 5-hydroxytryptamine in the bulbar and mesen-

cephalic reticular formation and cerebral cortex are antagonized bylysergic acid diethylamide, (LSD) (Boakes et al. 1970; Haigler& Aghajanian,1974a; Roberts & Straughan, 1967), and in the bulbar reticular formationby certain hallucinogenic tryptamine derivatives also (Bradley & Briggs,1974a). LSD was therefore examined for possible interactions with theeffects of raphe stimulation.

This work was the subject of a communication to the PhysiologicalSociety in December, 1975 (Briggs, 1976).

328

5-HT AND RAPHE-INDUCED EXCITATIONS 329

METHODS

Forty-four male Wistar albino rats weighing 300-400 g were used. Anaesthesiawas induced with halothane (3-5% in oxygen, reduced to 2% after induction) andmaintained either with halothane (10-1-5%) or with urethane (ethyl carbamate,BDH, 1-5-1-8 gfkg i.p.) after operative procedures were completed. Tracheostomywas performed and the floor of the IVth ventricle was exposed by removing the skullcaudal to the lambdoid suture and sucking out the medial parts of the cerebellum.The dura covering the rostral 2-3 mm of spinal cord was reflected. The animal washeld in a Kopf 1404 stereotaxic instrument with a rat adaptor, with the toothplateset at - 10. The stimulating electrode (Kopf SNE-200 bipolar) was inserted into thebrain stem from the rear in the sagittal plane at an angle of 450 to the frame bars, toallow the recording micro-electrode to be placed vertically into the brain stem atabout the same level. The penetration was made from a point 1-2-1-4mm caudal tothe obex, to a depth of 4 7-5-0 mm from the brain surface. Only one penetration ofthe stimulating electrode was made in each rat.

Unit action potentials were recorded extracellularly from neurones in the bulbarreticular formation using five-barrelled glass micro-pipettes with over-all tip dia-meters of 4-10 sm. The recording barrels were filled with 4 M-NaCl and had d.c.resistances of 1-5MO. Two ofthe other barrels contained LSD tartrate (Sandoz, Basle)(0-2- 1-0 x 10-3 Min 0-15M-NaCl) and serotonin bimaleate (Koch-Light, Colnbrook)(0-05-0- 1 M, pH 4 5-5-0) respectively, while the others contained respectively anotherputative transmitter (either ( - )-noradrenaline HCl (Sigma, Kingston-upon-Thames),0-1 M, pH 4-5-5-0; sodium L-glutamate (Koch-Light), 0-2 M, pH 8-0-9-0; oracetylcholine chloride (Sigma), 0-2 M, pH 4.5-5-0) and Pontamine Sky Blue 6BX(G. T. Gurr, Searle Scientific, High Wycombe) for marking recording sites and ascurrent control (Boakes, Bramwell, Briggs, Candy & Tempesta, 1974). lontophoreticcurrents were controlled by automatically timed constant current solid state switches(Blunn & Brown, 1975). Expelling currents of 50 nA or less were used, accordingto the responses produced.

Neuronal activity was amplified, displayed on an oscilloscope and counted by avoltage gate device. Firing rates were printed on paper tape and also plotted on-line in the form of histograms of spike counts in successive 5 s periods. The effectsof stimulation of neurones in the pontomedullary raphe nuclei on the firing ofneurones in the bulbar reticular formation were examined, using a wide range ofstimulus parameters. Rectangular stimulation pulses (0-05-0-1 ms duration) weredelivered from a Devices isolated stimulator controlled by a Devices Digitimer. Theeffects of single pulses were first examined, followed by trains of up to ten pulseswith interpulse intervals of 10 or 100 ms. Occasionally, series of single or short-train stimuli at 3-20 Hz for 5-25 s were used. Stimulation current intensities weremonitored on an oscilloscope and were in the range 50-500 1A. Subsequently theeffects of 5-hydroxytryptamine and other putative transmitters were tested byiontophoretic applications and where possible the interactions of LSD with theseagents and with the stimulation effects were studied. Stimulation effects were moni-tored on a storage oscilloscope and post-stimulus time histograms were formed on-line from gated spike monitor pulses by a Biomac 1010 (Data Laboratories). Storageoscilloscope traces and histograms were photographed.At the end of the experiment, the stimulation site was marked by a lesion pro-

duced by passing 10 #A for 1 min in each polarity, and the rat was then perfused viathe aorta with physiological saline followed by 10% formalin. The brain stem wasremoved for histological examination of stimulation and recording sites after prepara-tion of frozen 50 ,um sections which were stained with Methyl Green Pyronin. Using

a camera lucida, the locations of the stimulation and recording sites were marked onthe nearest of four representative sections of the brain stem at 1 mm intervals.based on sections from the brain stems of several rats.

A

B

Fig. 1. Distribution of stimulating and recording sites in the brain stemof the rat. The four sections are representative sections at 1 mmintervals: A, 2 mm rostral to obex; B, 1 mm rostral to obex; C, sectionthrough obex; D, 1 mm caudal to obex. The locations of Pontamine SkyBlue spots marking recording sites are indicated by filled squares; stimula-tion sites which gave long-latency exictatory effects on neurones in thereticular formation are indicated by filled circles; sites which did not givelong-latency excitations are indicated by open circles. The nomenclature ofPalkcvits et al. (1974) is followed, namely amb, nucleus ambiguus; io, nucleusolivaris inferior; nrp, nucleus reticularis paramedianus; nVII, nucleusorigins nervi facialis; P, tractus corticospinalis; rgi, nucleus reticularisgigantocellularis; rm, nucleus raphe magnus; ro, nucleus raphe obscurus;rp, nucleus raphe pallidus; XII, nucleus originis nervi hypoglossi.

RESULTS

Histological examination of the lesions made at stimulation sites(Fig. 1) showed that in twenty-eight rats, the lesions were in or very nearnucleus raphe magnus, nucleus raphe pallidus or nucleus raphe obscures,corresponding respectively to areas B5, B1 and B2 of Dahlstrom & Fuxe

330 I. BRIGGS

5-HT AND RAPHE-INDUCED EXCITATIONS 331(1964), and in twenty-six of these animals (in which the stimulation sitesare indicated by filled circles in Fig. 1), long-latency excitatory responsesof neurones in the bulbar reticular formation were seen when stimulation

A

.

100

1-0

100.

10

C

;G

100

D* .L -L

A *A

100 10

Fig. 2. Examples of the responses of neurones in the bulbar reticular forma-tion to electrical stimulation. Time scales are given in ms. A, short-latencyexcitatory response typical of those seen when the stimulating electrode wasoutside the raphe nuclei. Single pulse stimulation, ten sweeps. B-F, longer-latency responses to stimulation of the raphe nuclei. B-D; single pulsestimulation; E, five pulses at 100 Hz; F, ten pulses at 100 Hz. B, five sweeps;others one sweep. G and H, effect of increasing stimulation on latency andsize of response; G, single pulse; H two pulses; three sweeps each. Somespikes have been retouched.

was applied. In sixteen other rats, the lesions were outside the raphenuclei and stimulation was unlikely to have been effective; long-latencyexcitatory responses were not seen in these animals.

Extracellular recordings were obtained from 109 units in these forty-four rats. Fig. 1 also shows the locations of recording sites marked withPontamine Sky Blue after the conclusion of testing neuronal responses.

E

332 I. BRIGGS

These marks correspond to neurones which showed long latency excitatoryresponses to raphe stimulation. Most of the spots were in nucleus reticularisgigantocellularis and nucleus reticularis paramedianus. No difference wasapparent between the distributions of neurones affected by stimulation ofthe different raphe nuclei.

TABLE 1. Comparison of electrically evoked responses of rat brain-stem neurones withresponses of the same neurones to iontophoretically applied 5-hydroxytryptamine

5-hydroxytryptamine responses

Excitation No effect Inhibition

Effect of stimulation A, raphe stimulationExcitation 43 1 0No effect 6 3 0Inhibition 2 1 2

B, non-raphe stimulation

Excitation 15 2 1No effect 29 0 1Inhibition 2 0 1

The numbers refer to numbers of neurones in each category. Part A of the tablecorrelates the responses of fifty-eight neurones in twenty-five rats with stimulatingelectrodes placed in nucleus raphe magnus, n.r. pallidus or n.r. obscurus. Part Bcorrelates the responses of fifty-one neurones in eighteen rats with stimulating elec-trodes outside the raphe nuclei.

Examples of the responses to stimulation are shown in Fig. 2. Mostresponses were of an excitatory type. The excitatory responses observedshowed considerable heterogeneity but can be divided broadly into shortlatency and long-latency responses. The short-latency responses (e.g.Fig 2A) were seen mainly in rats with stimulating electrodes placed out-side the raphe; only six responses of thirty-two in these rats had latencieslonger than 5 ms, most being in the range 1-5-3-5 ms. In rats with thestimulating electrode placed in the raphe nuclei, most of the excitatoryresponses were of longer latency (e.g. Fig. 2B-H). Often no precise onset ofexcitation was discernible, because effects were seen only as increasingfiring rates after stimulation with a series of pulses at 5-20 Hz (e.g. Fig. 3).Of forty-four responses in these rats, four had latencies less than 8 ms,four were 8-25 ms, fourteen were 25-70 ms and twenty-two were 'tonic'excitations, appearing as increased firing rates after low frequency stimuli,Thus there appears to be a clear distinction between the latencies of theexcitatory effects of raphe and of non-raphe stimulation.

Occasionally, alternating inhibitory-excitatory effects were seen, similarto those described by Segal (1975); and very rarely, purely inhibitory

5-HT AND RAPHE-INDUCED EXCITATIONS

effects were observed. Both types were seen with both raphe and non-raphestimulation. Neurones excited antidromically were discarded. In Table 1,the effects of electrical stimulation are compared with the effects of ionto-phoretically applied 5-hydroxytryptamine on the same bulbar reticularformation neurones. In rats with the stimulating electrode placed in theraphe nuclei, forty-eight of fifty-eight neurones (83 %) showed similartypes of response to stimulation and 5-hydroxytryptamine application;forty-three of these were excited by both. In contrast, in the rats withstimulating electrodes placed outside the raphe nuclei, of the fifty-oneneurones on which both 5-hydroxytryptamine and stimulation effectswere studied, only sixteen (32 %) showed similar responses to both. As ameasure of correlation between 5-hydroxytryptamine-induced and raphe-stimulation induced effects, the contingency coefficient C (Siegel, 1956) fora two-by-two table of the neurones in Table 1A excited or not excitedby each stimulation mode is 0*426 (P < 0.001) indicating a good correla-tion, whereas C for the neurones in Table 1B (stimulation outside raphe)is 0*028 (P < 0.2). The proportions of the different types of responseto 5-hydroxytryptamine were similar in both groups A and B in Table 1;the dissimilarity between the groups was in the responses to electricalstimulation.

Effects of LSDThe effects of iontophoretically applied LSD on responses to stimulation

and to iontophoretic applications of putative transmitters were studiedon eleven reticular neurones (Figs. 3 and 4; Table 2). Raphe stimulationelicited long-latency excitatory responses from seven of these neurones.LSD blocked or reduced 5-hydroxytryptamine excitations on all seven,and also blocked or reduced the effects of stimulation on six of the seven.Subsequent recovery of the responses was observed on four of these neu-rones. Only one of five excitations by noradrenaline and neither of twoexcitations by acetylcholine was reduced by LSD.LSD was applied to three neurones which showed short-latency excita-

tatory responses to electrical stimulation; on all three, LSD blocked 5-hydroxytryptamine excitations but not stimulation effects or AChexcitations. One neurone showed an inhibitory response to stimulationwhich was not affected by LSD although 5-hydroxytryptamine excitationwas blocked. The excitatory effect of noradrenaline on the neurone was notreduced.

333

I. BRIGGS

_-~ -= I-7 =7 I-m'A t

L u

I-L_ -

.. ........ () o

0 0 0 00 O aoUn f"̂

334

45 0 > 0-4a . a

Z we * X

N as

c

EVE

o o

5-HT AND RAPHE-INDUCED "EXCITATIONS 335

D :.

D~~~-.*I

...A.

B I ! l..... ...............

..... ..............

I+ -hI. _=..... _. . . - .

A I1s100 Ms

20 F

30 50 50S NA Na* S-HT

. I I . I I I. I I

5

LSD 5 nA 5-HT S NA10 3 min IIy D

I INA S 5-HT

0 5I , I *

60 65Time (min)

Fig. 4. A, effects of iontophoretically applied LSD on the responses of a

neurone in the bulbar reticular formation to 5-hydroxytryptamine (5-HT),noradrenaline (NA), current (Na+) and electrical stimulation of nucleusraphe magnus (S; single pulses at 1 Hz, 3-5 V, 1901sA). B and C, unit activityafter raphe stimulation. One sweep per frame; time scale 10 ms intervals.B, before LSD; C, during LSD. D, post-stimulus time histograms fromthe same neurone, each being formed from sixty-four responses, with320 ms sweep duration. The upper two histograms were made before LSDapplication, the third during LSD and the last after recovery of theresponse.

DISCUSSION

For a considerable time, excitatory responses to iontophoretically ap-

plied 5-hydroxytryptamine by neurones in the bulbar reticular formationhave been known to occur (Bradley & Wolstencroft, 1965), and this studyhas now shown that there appears to be a correlation between theseresponses to 5-hydroxytryptamine and the responses of neurones in thebulbar reticular formation to stimulation of the bulbar raphe nuclei. In

0

this part of the reticular formation, excitatory responses were the pre-dominant effects of both 5-hydroxytryptamine and of raphe stimulation.Most previous studies on the effects of stimulation of raphe nuclei on theactivity of single neurones have been confined to areas in which 5-hydroxy-tryptamine has mainly inhibitory effects, and the effects of raphe stimula-tion are also inhibitory (Bloom et al. 1972; Nakamura, 1975; Segal, 1975).

TABLE 2. Effects of LSD on responses of reticular neurones to 5-hydroxytrypt-amine, ACh and noradrenaline applied iontophoretically and on their responses toelectrical stimulation

Effect of electrical stimulation

Long-latency Short-latency Inhibitionexcitation (7) excitation (3) (1)

No No NoBlock block Block block Block block

Stimulation 6 1 0 3 0 15-Hydroxytryptamine 7 0 3 0 1 0(+)

Noradrenaline(+) 1 4 - - 0 1ACh(+) 0 2 0 3 - -

Numbers in parentheses represent the numbers of neurones on which responseswere tested. See text for further explanation. (+) indicates excitation.

However, Couch (1970) produced evidence for an excitatory effect ofserotoninergic synapses upon neurones of the pontine and lower mid-brain raphe nuclei, which was correlated with excitatory effects of 5-hydroxytryptamine applied by iontophoresis; the proposed pathway hasreceived support from autoradiographic and degenerative studies (Bloomet al. 1972). The latter authors also reported excitatory effects of 5-hydroxy-tryptamine on cerebellar granule cells which appear to be contacted by5-hydroxytryptamine containing mossy fibre terminals.

In the present study, the responses to raphe stimulation were of ratherwidely varying latency and duration, possibly depending on the stimula-tion parameters used and on the effectiveness of stimulation of the rapheneurones. The majority of the responses were of 25 ms latency or longer, incontrast with the responses to stimulation outside the raphe nuclei. Con-sidering the short distance (1-4 mm) between stimulating and recordingpoints, the long latencies of these effects are unusual but not without pre-cedent. For example, supposedly monosynaptic effects having latencies inexcess of 100 ms have been reported for serotoninergic neurones (Segal,1975) and for noradrenergic neurones (Segal & Bloom, 1974; Hoffer,Siggins, Oliver & Bloom, 1973). The fineness of serotonin-containing termi-

336 I. BRIGGS

5-HT AND RAPHE-INDUCED EXCITATIONS 337

nals (Descarries, Beaudet & Watkins, 1975) probably results in a signifi-cant presynaptic delay due to slow conduction velocities, but an importantfraction of the latency may be due to a delayed post-synaptic action of thetransmitter. In relation to this, it has been observed that during excita-tions of a reticular neurone induced by 5-hydroxytryptamine or nor-adrenaline, the latencies of the raphe-induced excitations were shortened.Thus it appears that when the membrane potential is initially closer tospike threshold, a shorter period is required for the raphe induced effects toreach threshold level. This implies that the depolarization evoked byraphe stimulation develops slowly; the long latency and time-to-peak ofthe excitatory effects of 5-hydroxytryptamine applied iontophoreticallysupport this idea but intracellular recordings will be necessary to confirmit. It is thus possible that the conduction velocities of the raphe neuronesare greater than the latencies of the post-synaptic effects suggest. Manyneurones showed excitatory effects only as increases in firing rate aftertrains of stimuli to the raphe nuclei; it may be that these neurones receiveonly a weak input from that part of the raphe which was stimulated, orthat the 5-hydroxytryptamine input has a modulatory or tonic function,appearing as a relatively slight but long lasting facilitatory influence ontheir activity.The good correlation between these long-latency excitatory effects and

the excitatory effects of 5-hydroxytryptamine applied by iontophoresis(Table 1) indicates that these effects of raphe stimulation are probablymediated by 5-hydroxytryptamine terminals although it cannot be ex-cluded that the effects are mediated via polysynaptic pathways endingwith a serotoninergic input to the bulbar reticular formation. Furtherstudies involving antidromic activation of raphe neurones from stimula-tion sites in the reticular formation will be needed in connexion with thispoint.

Only the responses of long latency could be reduced by LSD. AlthoughLSD blocks glutamate as well as 5-hydroxytryptamine effects (Boakes et al.1970), it is tempting to suppose that in this case LSD was blocking theactions of 5-hydroxytryptamine released from raphe terminals, in view ofthe correlation of the long latency excitatory effects with the position ofthe stimulating electrode in the raphe nuclei and with the excitatoryeffects of 5-hydroxytryptamine. The short latency effects which LSD didnot reduce were probably due to the stimulation of non-serotoninergicstructures.The ability of LSD to reduce or abolish excitatory responses to 5-

hydroxytryptamine (Boakes et al. 1970; Haigler & Aghajanian, 1974a;Roberts & Straughan, 1967) has been confirmed in the rat bulbar reticularformation. Longer applications of LSD were needed than in previous

experiments, as was to be expected, in view of the lower expelling currentsand much lower concentrations of LSD used, and the addition of sodiumchloride to the LSD solution, reducing the transport number of the LSD(Haigler & Aghajanian, 1974a). The present results from bulbar reticularneurones support the preliminary finding by Couch (1970) that LSD canblock both 5-hydroxytryptamine-induced and stimulation-induced excita-tions of raphe neurones.The antagonism of the excitatory post-synaptic actions of 5-hydroxy-

tryptamine by LSD would be complementary to the potent inhibitoryaction ofLSD on dorsal raphe neurones described by Haigler & Aghajanian(1974 a): both actions would lead to an impairment of serotoninergic trans-mission, each reinforcing the other.

It is possible that the fineness of 5-hydroxytryptamine terminals, andthe weakness and instability of the fluorophore produced from 5-hydroxy-tryptamine may have resulted in an underestimation of the true densityof the terminals in the bulbar reticular formation (Fuxe, 1965a, b).In recent experiments, Candy has found that after treatments designedto enhance fluorescence specifically in 5-hydroxytryptamine terminalsa much higher density of very fine yellow-fluorescing terminals can beobserved in rat nucleus reticularis gigantocellularis than is apparent afterthe usual formaldehyde-induced fluorescence procedure (J. M. Candy,personal communication). Further evidence that the conventional pro-cedure does not provide complete information on the distribution of5-hydroxytryptamine terminals was obtained by Palkovits, Brownstein& Saavedra (1974), who showed by enzymic isotopic assays of 5-hydroxy-tryptamine in very small samples from brain-stem nuclei that 5-hydroxy-tryptamine is present in areas which had previously been reported to haveno 5-hydroxytryptamine terminals. The nuclei of the reticular formationcontained moderate amounts of 5-hydroxytryptamine.

The author is grateful to Professor P. B. Bradley for his direction and encourage-ment during the course of this work.

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