5. Acetylcholine (ACh), catecholamines, histamine, 5-hydroxytrypt

19 Pages·2006·2.95 MB·English

J. Physiol. (1967), 192,pp. 359-377 359
With 10text-figures
Printed in Great Britain
MICRO-IONTOPHORETIC STUDIES ON NEURONES
IN THE CUNEATE NUCLEUS
BY A. GALINDO, K. KRNJEVIO AND SUSAN SCHWARTZ*
From the Wellcome Department of Research in Anaesthesia,
McGill University, Montreal, Canada
(Received 8 March 1967)
SUMMIARY
1. Cuneate cells in anaesthetized cats were strongly excited by
L-glutamate, and somewhat less by D-glutamate; cells which receive
afferents from hair receptors were particularly sensitive.
2. Glutamate could be used to demonstrate post-synaptic inhibitory
inputs from the dorsal column, the medial lemniscus and the frontal
cortex.
3. Many cuneate cells were also strongly excited by adenosinetriphos-
phate (ATP); this was probably due to the chelating action of ATP, as
citric acid was also quite effective.
4. y-Aminobutyric acid (GABA)readilyblockedallformsofspontaneous
and evoked activity, except antidromic invasion of cuneothalamic
neurones; cells which receive proprioceptive afferents were particularly
sensitive to GABA. Glycine had a comparable effect.
5. Acetylcholine (ACh), catecholamines, histamine, 5-hydroxytrypt-
amine (5-HT) and an extract containing substance P mostly had only
weak depressant actions. Cholinergic and mono-aminergic mechanisms
are probably not very significant in the cuneate.
6. These results are consistent with the possibility that glutamate and
GABA (or glycine), or some closely related compounds, are the main
excitatory and inhibitory transmitters in the cuneate nucleus.
7. If ATP is released from afferent nerve endings, it could also play
a significant role in excitation.
INTRODUCTION
Certain amino acids when applied by extracellular micro-iontophoresis
have potent excitatory or inhibitory effects on spinal (Curtis & Watkins,
1960), cortical (Krnjevic6 & Phillis, 1963a) and other central neurones
* NATO Post-doctoral Fellow. Present address: Department ofPsychiatry and Neuro-
logy, NewYorkUniversity School ofMedicine, NewYork City, N.Y. 10016.
360
A. GALINDO AND OTHERS
(Curtis & Watkins, 1965). L-Glutamic acid and GABA, which are found
in the brain in high concentrations (see Tallan, 1962), are particularly
effective. We have recently shown that the action of GABA on cortical
neuronesfaithfullyimitatespost-synapticinhibition(Krnjevic&Schwartz,
1966, 1967). Believing that GABA and L-glutamate may be transmitters
in the cortex, we wondered if they might have a similar function in the
primary somato-sensory pathway.
Someactionsofglutamate,AChanddopamineondorsalcolumnneurones
have been described recently in a briefarticle by Steiner & Meyer (1966).
A preliminary report of our experiments has already appeared (Galindo,
Krnjevic6 & Schwartz, 1966).
METHODS
Experiments were performed on sixteen cats and two monkeys (Macaca mulatta),
mostly anaesthetized with sodium pentobarbitone. Three cats were anaesthetized with
chloralose (Hopkin & Williams) and another cat was decerebrated underether. The rectal
temperature was kept within the range of 35-380C. The femoral blood pressure was
monitoredcontinuously.
Surgical preparation. The foramen magnum was exposed by mid line separation ofthe
cervical musculature and removal of the atlanto-occipital membrane. The posterior arch
ofthe atlas was resected and the occipital bone was chipped away to reveal the inferior
portion ofthe cerebellar vermis. The dura and arachnoid were then removed.
Forcorticalstimulation andthe insertion ofthalamic stereotacticelectrodes, the contra-
lateralcerebralhemispherewasexposedwidely.Allthevisiblesurfacesofthebrainwerekept
moist witha continuous drip ofwarm Lockesolutionandbyathinlayerofpolyethylene.
Recarding. The recordingelectrode was the centre channel ofa 5-barrelled, glass pipette
(tipouterdiameter 5-10j#); this channelwasfilledwith2-7m-NaCl. The outerbarrelswere
used for iontophoresis. The signal was led through a conventional cathode follower to an
amplifying circuit (time constant 1 msec) and displayed on an oscilloscope from which
photographic recordsweretaken. Insomeexperiments therate ofdischarge was computed
by anintegrating circuit and recordedcontinuously onpaperby apenwriter.
Stimulation. In several experiments, an electrode consisting of a hypodermic needle
threaded with insulated copper wire was placed stereotactically into the medial lemniscus
close to the thalamus, near the point corresponding to the coordinates, F 6, L 4-5, and
H -1,asgivenbytheatlasofJasper&Ajmone-Marsan (1960). Theelectrodewasadjusted
to the positionwhich, duringstimulationwith 0-1 msecpulses, gave the largestantidromic
field response inthe cuneate.
Thesurfaceofthecontralateralpericruciatecortexwasstimulatedwith0-1mseccathodal
pulses delivered through a silver ball (1 mm in diameter). Flexible bipolar silver wire
electrodeswere used to stimulate the ipsilateral dorsal columns, atthe lower border ofthe
first cervical segment, with 0-01 msec pulses.
Iontophore8i. The outer barrels ofmultibarrelled pipettes were filled with concentrated
solutionsofvarioussubstances, made upasfollows (whenevernecessarytheywereacidified
or neutralized with 1m-HCI or 1 M-NaOH): GABA (California Biochemicals): 1-2 M,
pH4-5;glycine (Fisher): 1 M,pH4; NaL-glutamate (BritishDrugHouses): 1-2 mr,pH6-7;
D-glutamic acid (Mann): 1-2 M, pH 6-7; noradrenaline bitartrate (British Drug Houses):
0-5M, pH 3-4; noradrenaline HCl (British Drug Houses): 0-5M, pH 3-4; 5-hydroxy-
tryptamine (5-HT) creatinine sulphate (May & Baker): 0-2M, pH3-7; 5-HT bimaleinate
(Light): 0-5M,pH 4; dopamineHCI (Light): 0-6 M,pH4; gallaminetriethiodide (Poulenc):
CUNEATE NEURONES 361
2M, pH 7*5; histamine 2HCl (Hoffmann-La Roche): 08M, pH6; di-sodium adenosine
triphosphate (ATP) (Nutritional Biochemicals): 04M, pH2-3 and 5-4; sodium adenosine
diphosphate (ADP) (Nutritional Biochemicals): 0-5M, pH6-2; citric acid (J.T. Baker
ChemicalCo.):0 5M,pH3-0;a-ketoglutaricacid(NutritionalBiochemicals): 05M,pH3-7;
ethylenediamine tetra-aceticacid(EDTA,Nasalt) (Fisher): 05M,pH5-3.The 'Substance
P' was an extract ofcattle intestine obtainedfrom Hoffmann-La-Roche, Inc., Nutley, 10,
New Jersey (Rol-9256/8), with an original activity of 300u./mg; it was made up in a
concentration of 20mg/ml. of distilled water (6000u./ml.), and acidified to pH4-5 with
1 N-HCI.
Solutionsofpossiblyunstablecompoundssuchascatecholamines, 5-HTandSubstanceP
were prepared and inserted into the pipettes by centrifuging, within about an hour of
recording.
RESULTS
We recorded discharges from about 160 cells in cats, mainly in the
middle third ofthe cuneate nucleus, within the region extending caudally
from the obex for about 3 mm. The cells were identified by their pre-
dominantly negative spikes, which could be recorded for several minutes
without difficulty. In contrast, positive spikes generated by afferent fibres
in the cuneate fascicle were only recorded transiently. Cells were further
identified by antidromic invasion from the medial lemniscus, by synaptic
activation or inhibition from the dorsal column, the medial lemniscus or
the contralateral cortex, and by excitation by L-glutamate.
Classification ofneurones according to effective input
from periphery
An initial group of about 100 cells, recorded at random, consisted of
the following. 42% were hair cells, which gave a fast adapting discharge
in response to movements of hairs; nearly all were found within 0-2-
0*8 mm from the dorsal surface of the medulla (mean 0 40mm). About
16% were touch cells, which responded to prodding ofthe skin and gave
a maintained discharge when a weight was placed on the limb. A few of
them could be excited by moving hairs as well as by touching the skin.
Touch cells were somewhat less superficial than hair cells, being within
the range 04-0 9 mm (mean 0-65 mm). Another 21% were proprioceptive
cells, whose slow adapting discharge dependedontheposition ofthelimb;
they were not related to cutaneous receptors, and were found relatively
deep, all but one within 0-6-1-3 mm (mean 0-91 mm). The remaining
21% were 'insensitive' cells, which could not regularly be excited by
moderate stimulation of the periphery; they were found over a wide
range ofdepths, from 0 35 to 1-5 mm (mean 1-0 mm). The proportions of
different types ofcuneate neurones inthis population agree with previous
findings on dorsal column nuclei (Kruger, Siminoff & Witkowski, 1961;
Perl, Whitlock & Gentry, 1962; Gordon & Jukes, 1964a; Winter, 1965).
362 A. GALINDO AND OTHERS
Their distribution in depth within the nucleus confirms the observations
of Schwartz (1965). Except for touch cells, the different kinds of cells
seem to be arranged in the same way as the corresponding fibres in the
dorsal column (Uddenberg, 1966).
Cuneothalamic neurones
Cuneateneuroneswere alsotestedforantidromicinvasionafterstimula-
tionofthemediallemniscusnearthe contralateralthalamus. The response
was considered to be antidromic ifit occurred regularly, after a short and
constant latency (0.2-2-0 msec, cf. Gordon & Seed, 1961 and Andersen,
Eccles, Schmidt & Yokota, 1964). Using this criterion, we found the
following proportions of cuneothalamic cells in the various groups.
Hair cells: 25 out of35 tested or 72%; touch cells: 8 out of 10, or 80%;
proprioceptive cells: 3 out of 13, or 23%, andinsensitive cells: 1 out of8,
or 13%. Considering the relative inefficiency ofour method ofstimulating
the medial lemniscus (cf. Gordon & Seed, 1961) and the small numbers
ofcellstested, thepercentagesofcellsexcitedantidromicallyinthevarious
groups agree reasonably well with previous findings in the middle region
ofthe gracile nucleus (Gordon & Seed, 1961; Gordon & Jukes, 1964a).
Excitatory substances
Excitation byL-glutamate. Apart from some superficial units, which gave
positive spikes, andwere clearlyafferent fibres (cf. Steiner & Meyer, 1966),
all the neurones tested could be excited by L-glutamate. As in the cortex
(Krnjevic6 & Phillis, 1963a), the action ofL-glutamatewas strikingly quick
in onset and termination, and excitation was easily controlled by varying
the iontophoretic current. There was no sign of desensitization during
prolonged applications, but the cells were readily inactivated by an
excess of glutamate. Strong excitation by L-glutamate can be seen in
Figs. 1, 3 and 4.
Ontheaverage,touch,proprioceptiveand'insensitive' cellswereapprox-
imately equally sensitive to glutamate. The mean iontophoretic currents
neededto exciteeleven touch cells, tenproprioceptive cells and seventeen
insensitive cellswererespectively 63.6 (S.E. 12-0), 51-5 (S.E. 9-7) and 56*8
(s.E. 8.7) nA. Thethirty-one hair cells tested were especiallyeasily excited
by glutamate, the mean current required being 39 9 nA (S.E. 5.1). This is
significantly less than the mean for touch cells, or all the other cells taken
as a group (P < 0.05). Some hair cells could be excited with minute
amounts of glutamate (< 10nA). There was no significant difference
between the currents of glutamate needed to excite twenty-nine cuneo-
thalamic cells (42.7 nA, S.E. 5-5) andtwelve other cells (57.9 nA, S.E. 10.7).
CUNEATE NEURONES 363
The easily controlled excitation by glutamate is very useful for demon-
strating inhibitory or sub-threshold excitatory synaptic actions. For
instance, we could show that the hair cell illustrated in Fig. 2 received
several inhibitory inputs. This cell was initially quiescent (Fig. 2A), but
Fig. 1. Hair cell in cuneate nucleus showing: continuous spontaneous discharge
(large spikes) and strongexcitation when a jet ofairwas blown on to the corre-
sponding hairs on the ipsilateral forepaw (A) or during micro-iontophoresis of
L-glutamate (B); short latencyantidromic invasionfromthe contralateralmedial
lemniscus-severaltracesrecordedwhilestraddlingthreshold (D);clearinhibition
ofspontaneous activityduringiontophoresis ofGABA (C) andaweakdepressant
action of ACh (E), with Na+ current control in F. White lines below traces in
all the figures indicate duration of iontophoretic currents; the numbers above
traces give strength of currents in nA. The traces in E and F show only the
startandend ofrelease ofACh+andNa+, eachlastingabout 1minaltogether.
itwas made to fire steadily byreleasing glutamate (B). Stimulation ofthe
medial lemniscus (which did not excite the cell antidromically) caused a
progressively stronger and longer inhibition of the glutamate-evoked
discharge (C-E). Potent inhibition was also obtained by stimulating the
dorsal column (G-I), andthe contralateral sensorimotor cortex (K). Many
other cells showed similar inhibitory effects, which have previously been
observed by several authors (Gordon & Paine, 1960; Towe & Jabbur,
1961; Gordon & Jukes, 1964a, b; Levitt, Carreras, Liu & Chambers, 1964;
Andersen et al. 1964).
364 A. GALINDO AND OTHERS
The particular significance of the demonstration of these inhibitory
effects against a background of glutamate-evoked firing is that only
post-synaptic inhibition ofthe observed cell would be evident under these
conditions.
Fig. 2. Some inhibitory inputs on a hair cell, which was not invaded anti-
dromicallyfrommediallemnniscus.Eachrecordshowsseveraltracessuperimnposed.
Therewasnospontaneousactivity (A) butiontophoresis ofglutamate excitedthe
cell readily (B-L). The glutamate evoked discharge wasinhibitedbystimulating:
(1)mediallemniscuswithstereotacticneedle (C-E), (2) dorsalcolumnwithsurface
electrodes (H-I) and (3) contralateral somatosensory cortex, also with a surface
electrode (K).
Using small doses of glutamate, we could also detect weak excitatory
inputs from the dorsal column (Fig. 7E), or from the medial lemniscus
(Fig. 8B), which by themselves were insufficient to cause firing.
Specificity of glutamate action. Equal doses Of D-glutamate usually had
a substantial, though somewhat weaker, excitatory action (Fig. 3) and
in a few cases there was no clear dlifference in potency between L- and
D-glutamate (Fig. 3F). (cf. cortical cells (Krnjevi6' & Phillis, 1963a;
Crawford & Curtis, 1964)).
Adenosine triph&osphate (ATP). Holton has suggested that ATP may
be released by the central endings of primary sensor neurones (Harris &
Holton, 1953; Holton & Holton, 1954; Holton, 1959). ATP was therefore
tested, by iontophoretic release with inward currents, on thirty-five
CUNEATE NEURONES 365
cuneate cells in five cats. With a few exceptions (cf. Fig. 4B), most
neurones were clearly excited by iontophoretic currents of ATP similar
to those which excited when releasing glutamate (Figs. 3C, E, F, 4A).
Whentestingwithequalcurrents,ATPwasusuallysomewhatlesseffective
than glutamate but some cells were more strongly excited by ATP (cf.
Fig. 3F).
A L-Glut 28 LG 28 56
_ Yl~~~~2~~84~2t~~1~DG
t t-L Li Li Li
84
160/sec 56
B D-GIut 28 AP28
L LLiLi Li
it t l l l ll
5 sec 25 sec ATP
LG 28 DG
C ATP 28
t t t-i t-
Fig. 3. Excitation oftwo hair cells by L- and D-glutamate, and by ATP. Each
trace records frequency of firing, and arrows indicate time of iontophoretic
applications (allbyinwardcurrents).Thefirstcell (A-E)wasmuchmoresensitive
to L-glutamate than to D-glutamate or ATP-inD and E, various iontophoretic
currents were used to release D-glutamate and ATP respectively. The second cell
(F) gaveasimilarresponsetoequaliontophoretic currentsofL-andD-glutamate,
and an even larger response to an equal current ofATP. Note single frequency
calibrationforalltraces(nearE)anddifferenttimecalibrationsforA-CandD-F.
Neurones receiving inputs from cutaneous afferents tended to be more
sensitive than proprioceptive cells. About 4 of the first group gave a
response to ATP which could be compared with the response to similar
currents of glutamate, whereas only about I of the proprioceptive cells
showed really comparable effects.
TheactionofATPusuallyhadarapidonsetanditstoppedveryquickly;
but in some experiments (particularly when using very acid solutions of
ATP, at pH 2-3) its action was strikingly prolonged, so that the response
366 A. GALINDO AND OTHERS
outlasted the end of the application by several seconds (Fig. 4A). This
kind ofafter-discharge was never seen with glutamate.
Other chelating agents. EDTA excited several cells very powerfully, and
tended to evoke high frequency bursts offiring (Galindo et al. 1967). But
its action, like that ofATP, was less predictable than that ofglutamate.
Fig. 4. Comparing effects produced by equal inward iontophoretic currents of
L-glutamate, ATP or citrate, on a proprioceptive cell (A), a touch cell (B) and
twohaircells (C, D).
Citric acid was somewhat less effective than ATP; nevertheless, about
halfofthe twelve cellstestedgave aresponse comparable totheglutamate
response (Fig. 4). The effect ofcitrate tended to have a slower time course
with a slight after-discharge, in contrast to the depression usually seen
immediately afteraglutamateresponse. ADPwastested onfive cells, with
very little effect.
Otherpossible excitatory agents tested
Substance P. Lembeck (1953) has suggested that substance P (Euler &
Gaddum, 1931; Amin, Crawford & Gaddum, 1954) may be released by
the endings of primary sensory neurones. A tissue extract containing-
substance Pwastestedby 'iontophoresis' on about twenty cuneate cells,
including cells responding to the three types of peripheral stimulation,
using large and prolonged outward and inward currents through the
CUNEATE NEURONES 367
micropipettes. We could not detect a direct excitatory or inhibitory effect
and there was no evidence of any facilitation ofthe action of glutamate;
but the extract sometimes depressed neuronal firing.
The potency of our sample of substance P was tested on two preparations of guinea-
pig ileum, comparing its action with that ofhistamine. In both cases, strong contractions
(about i maximal) were obtained withsolutions ofsubstance P containing 0-03-03u./ml.,
according to the original description ofthe sample. It is evident that the extract had not
lost most ofits initial potency. We are grateful to Dr B. A. Kovacs, ofthe Pharmacology
Department, for performing theseassays.
Fig. 5.Proprioceptivecellexcitedaftershortlatencybydorsalcolumnstimulation
(A). Discharge evoked by extending wrist was very effectively blocked by small
doseofGABA (C), andbylargerdosesof5-HT (B) andnoradrenaline (D). ANa+
currentcontrolisalso in (C).
Some other possible transmitters. ACh was tested on thirty cells, nor-
adrenaline on forty-three cells and 5-HT on twenty-two cells of every
type. Adrenaline, dopamine and histamine were also tested in a few
cases. None of these compounds had a strong or regular excitatory
action. Three cells may have been excited slightly by ACh (cf. Steiner &
Meyer, 1966) and one cell by 5-HT.
Flaxedil (gallamine) had striking excitatory effects, which are being
described separately (Schwartz, Galindo & Krnjevic, 1967; Galindo et at.
1967).
368 A. GALINDO AND OTHERS
Inhibitory substances
Inhibition by GABA. Apart from discharges in afferent fibres and
antidromic invasion of cuneothalamic cells, all kinds ofneuronal activity
were greatly reduced or abolished by GABA. This depressant action is
thus very similar to the effect ofGABA on other central neurones (Curtis
& Watkins, 1960, 1965; Krnjevic6 & Phillis, 1963a). It has asharp onset
and is quickly reversible, and the cells are not obviously desensitized by
repeated applications. Several examples of the blocking action of GABA
are illustrated in Figs. 1C, 5, 9 and 10.
Ondifferenttypesofcells. Proprioceptive cellswere particularly sensitive
to GABA. The mean iontophoretic currents needed to abolish comparable
discharges were: for thirty-two hair cells, 55-1 nA (S.E. 7-8); for eighteen
touch cells, 53-3 nA (S.E. 9.8); for eighteen proprioceptive cells 30-6 nA
(S.E. 5.2), and for seventeen 'insensitive' cells 37 8 nA (S.E. 7.1). The
mean for proprioceptive cells was significantly less (P < 0.05) than the
means for hair and touch cells.
When thirty-one cuneothalamic neurones were compared with twenty-
oneothercells, themeaneffective currentsofGABAwere 50*5 nA (S.E. 7.6)
and 413 nA (S.E. 8.8) respectively: this difference was not significant
(P 0.05).
>
On activity evoked by different inputs. Several kinds ofactivity couldbe
studied: (1) spontaneous firing; (2) excitation from peripheral receptors;
(3) excitation by electrical stimulation of the dorsal column; (4) cortico-
fugal excitation; (5) synaptic activation from the medial lemniscus; (6)
antidromic invasion from the medial lemniscus.
There is some overlap between these categories. Several inputs may contribute to the
spontaneous activity, but an intranuclear drive can be demonstrated by isolating the
nucleus(Schwartz,Giblin&Amassian, 1964).Thedorsalcolumnscontainpropriospinalfibres
aswell asthe dorsalroot afferents (Tower, Bodian & Howe, 1941). Theexcitatoryeffect of
lemniscalstimulationmaybepartlymediatedviathe cortex (Gordon&Jukes, 1964b), but
a more direct pathway is probably present (Amassian & De Vito, 1957; Andersen et al.
1964; Gordon & Jukes, 1964b).
The depression ofspontaneous activity is illustrated in Figs. 1, 5, 9 and
10. Orthodromic excitationwasblockedrelativelyeasily, asintheexample
ofatouch cell in Fig. 6 (E-H), especiallywhen comparedwith antidromic
invasionwhich couldnotbe abolished even byverylarge doses (Figs. 6A-
D). (GABA caused a marked increase in spike amplitude, visible in B:
cf. hyperpolarizing action of GABA on cortical neurones (Krnjevic &
Schwartz, 1966, 1967).) The differential block of orthodromic spikes was
also shown as follows. Antidromic invasion was stopped by a preceding
orthodromic response (Fig. 6I, J). A moderate dose of GABA abolished
this response, and restored antidromic invasion (Fig. 6K).