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The action of iontophoretically applied acetylcholine and dopamine on single claustrum neurones in the cat
0028-3908
THE ACTION
OF IONTOPHORETICALLY CLAUSTRUM
M.T.
Salerno,
Institutes
M.T.
Zagami,
of Human University
APPLIED
ACETYLCHOLINE
NEURONES
Palermo,
ON SINGLE
and F. Infantellina
and of General
of Palermo. 90134
DOPAMINE
IN THE CAT *
R. Cortimiglia
Physiology
AND
81 090895.05~02.~~0 Perpmon Pres Ltd
Corso
Tukory
Physiology, 129,
Italy
IAceepWf 25 June ?9gI)
The effects of iontophoretically applied acetylY?= c o lne (ACh) and dopamine (DA) on spontaneously active cats, have claustral neurones, in chloralose-anaesthetised
been investigated. Acetylcholine had both facilitatory and inhibitory effects in almost the same proportions. The action of DA was prevalently inhibitory. Two types of inhibitory response to ACh and DA are described: shortlasting inhibition and long-lasting inhibition. The excitatory response always outlasted the period of drug application.
The claustrum is a structure long known to be the seat of multi-modal somato-sensory and somatovisceral convergence (Bonvallet, Dell et Hugelin, 1952; Segundo and Machne, 1956; Urbano, Rapisarda et Infantellina, 1966; Rapisarda, Azzaroni and Infantellina, 1969; Spector, Hassmannova and AlbeFessard, 1974) which has not yet been investigated microiontophoretically in order to identify the neurotransmitters acting at its synapses. With the aim of identifying the chemical transmitters acting on claustral synapses and analysing their effects, the technique of iontophoretic release of drugs was used. Acetylcholine (ACh) and dopamine (DA) were first considered. Acetylcholine, in fact, as is known from the literature, is widely distributed in the central nervous system (Bradley, 1968; Phillis, 1976) and, when applied microiontophoretically, it affects most synapses of the central structures which have been investigated. Also, DA is a putative transmitter in the central nervous system (Bieger and Hockman, 1976) and is known to be the neurotransmitter of the nigrolstriatal pathway controlling motor activity. On the other hand, DA terminals in the claustrum, arising from the pars compacta of the substantia nigra and from the mesencephalic ventromedial tegmentum, have been demonstrated by histofluoresence microscopy in material obtained from aborted foetuses (Nobin and Bjltrklund, 1973). The neurones investigated in this preliminary study were marked with Pontamine Sky Blue and only identified histologically. METHODS The experiments were performed on chloralose-anaesthetised cats (70 mg /kg, i.v.), using the technique of iontophoretic application of drugs from multibarrelled micropipettes. The recording barrel was filled with 4M NaCl, while the other barrels contained: acetylcholine chloride (vials - Sigma), 5% solution in water; 3-hydroxytyramine HCl (Koch-Light), 5% solution in water; L-glutamic acid Na salt (Koch-Light), 2% solution in water at pH 8.0; Pontamine Sky Blue (68x, Gurr). The extracellularly recorded neuronal activity * This work Nazionale
was supported by a grant from delle Ricerche, Rome, Italy.
895
the Consiglio
896
Preliminary
Notes
was counted over successive 5 set epochs by using an electronic counter, connected through an analogue-digital converter to a recording system which traced a histogram of the discharge frequency. A current of 60 nA for 30 set periods was usually employed to expel ACh and DA from the appropriate barrel. Either the ejecting current was increased to 80 nA, or the application period was prolonged to 45 set, to evoke a response to drug application in some neurones. After each application, a current equal to that of the drug ejection current, was applied through the barrel filled with Pontamine Sky Blue, to check the effects of the current. At the end of the experiment the recording sites were marked with Pontamine Sky Blue, using a current of 5uA, for histological examination of serial brain slices, The stereotaxic planes exp'iored were between A12 and A17.5, Lg.5 and L13, Htl and Hi4. according to the atlas of Snider and Niemer (1961). RESULTS Acetylcholine and DA were tested on spontaneously active neurones; because of the known low spontaneous firing rate of claustral neurones (Segundo et al., 1956; Spector et al., 1974), continuous ejection of glutamate was sometimes used to increase the discharge frequency. The effects reported with repeated applications the firing rate'to recover The results
were
were obtained with each neurone more than once of the transmitters at intervals which Dermitted to base line levels.
as follows:
applied ACh affected a large number of the neurones (l) Iontophoretically tested. Of 41 spontaneously active neurones, 36 (88%) were affected by this drug. The effects were both excitatory and inhibitory in almost equal proportions. (2) The inhibitory response was found in 17 neurones (41%). Two types of inhibitory response to ACh could be distinguished on the basis of duration: one, shown by 8 neurones, was limited to the application period (short-lasting response); it began 10 set after the iontophoretic current was switched on and ended in the 5-10 set following the ACh release period (Fig. 1A). The other response, exhibited by 9 neurones, outlasted the end of the ejection current (long-lasting response). The long-lasting inhibitory response was also rapid in onset; the depression in firing rate began within 10 set of the ejection current being switched on and tended to return to its previous level 30-40 set after the end of the release period (Fig. 1s). response, shown by 18 neurones, was invariably of the (3) The excitatory long-lasting type (Fig. 1C). This response was rapid in onset, ie. it began within 10 set of the ACh application; the discharge rate returned gradually towards its previous level over a period of 80 to 120 set after the current was switched off. Occasionally the discharge frequency did not recover to its previous level. (4) Only one neurone showed a biphasic response pattern, inhibitory phase, limited to the ACh release period, excitatory phase lasting for 90 sec.
consisting of an followed by an
(5) Dopamine affected the discharge frequency of 27 (79%) out of the 34 neurones tested. Almost all the neurones tested, ie 23 (68%) showed an inhibitory response and only 4 neurones were excited. (6) The inhibitory responses to DA, like those to ACh, were both short- and long-lasting. Short-lasting inhibition was shown by 16 neurones; it began 5 set after DA ejection and ended within 5 set following drug application (Fig. ZA). The long-lasting inhibition exhibited by 7 neurones was also rapid in onset, beginning 5 set after the ejection current was switched on; the firing rate tended to recover within 40-55 see after the end of the application period (Fig. 26). excited by DA showed a gradually increasing discharge (7) The four neurones during the period of drug application, while the maximum level of excitation was reached after DA ejection (Fig. 2C).
(8) Both ACh and DA were applied to 33 neurones (Table 1) but a comparison of the effects of the two drugs applied to the same neurone did not yield any significant results.
897
Preliminary Notes
c
I
1
I I30
I
I
I
120
Gf-Fi-fO
I
,
1 60
1
0
WC
t
I 120
1 180
1
, ICC
Fi ure 1. Effects of iontophoreticallyapplied acetylcholine (ACh) on r ng rate of three spontaneously active neurones in the claustrum. Abscissa: time in sec. Ordinate: number of spikes in successive 5 set epochs. Horizontal bars indicate the iontophoretic application of ACh or current control; all responses were obtained using 60 nA currents. A: short-lasting inhibition; the inhibitory response was obtained with successive ACh applications. A current control is without effect. B: long-lasting inhibition. C: excitatory response to ACh.
C
OI I
0
I
1 00
I
2% I lz-3
I
I sac
I 0
a
1 *0
&L 1
I 120
I
I I.C
1 0
1
I 60
1
I 120
Figure 2. Effects of iontophoretic application of dopamine (DA) on the 'firingrate of three spontaneously active neurones in the claustrum. A: short-lasting inhibition. B: long-lasting inhibition. C: excitatory response.
I
1
I
‘80
naz
898
Preliminary Notes Table
1.
Comparison of the effects of ACh with DA when applied to the same neurone.
those
of
ACh
0
4
0
0
8
4
1
2
_ -
3
0
2
2
0
3
0
4
0
+ DA
The table represents the number of neurones showing the responses indicated: + = excitation, - = short-lasting inhibition, - - = long-lasting inhibition, o = no effect.
DISCUSSION The data reported here give the first information about the responsiveness of claustral neurones to iontophoretic application of ACh and DA. Although limited to the sample of neurones studied, it is possible to conclude that a large number of units in the claustrum are affected by iontophoretic application of ACh (88%) and DA (79%). The responses to ACh shown by claustral neurones were both excitatory (44%) and inhibitory (41%) in almost the same proportions and resemble those such as the brain stem (Bradley, Dhawan found in other cerebral structures, and Wolstencroft, 1966) and the caudate nucleus (Bloom, Costa and Salmoiraghi, 1965), while in the cerebral cortex there is a predominatly excitatory effect (KrnjeviC and Phillis, 1963). The response to DA exhibited by claustral neurones was predominantly inhibitory (68%) and this is analogous with the stiuation in the caudate nucleus where DA, studied under various experimental conditions, was found to act as an inhibitory neurotransmitter (McLennan and York, 1967; Bloom et al. 1965; York, 1967). According to their time course, two types of inhibitory responses to period was called ACh and DA were found: one, limited to the drug application the iontophoretic ejection the short-lasting type; the other, outlasting type. Both short- and long-lasting period, was called the long-lasting inhibition occurred almost equally following iontophoretic application of ACh, the former being shown by 47% and the latter by 53% of the neurones inhibition appeared to be more prevalent which were inhibited. Short-lasting among the neurones inhibited by DA and was present in 69% of the neurones depressed by DA. The excitatory response to both drugs was invariably of the longlasting type; however, for the neurones excited by DA, the slow onset and gradual time-course of excitation might suggest an indirect effect. In conclusion, from these preliminary results, it appears that these two drugs play an important role in chemical transmission at ClaUStral synapses. and further investigations should reveal the physiological significance of the ACh and DA inputs to the claustrum.
899
Preliminary Notes REFERENCES
On the physiology and pharmacology of Bieger, D. and Hockman, C.H. (1976). Chemical Transmission in the Mammalian cerebral dopamine neurones. In: Central Nervous System, Ed. Hockman, C.H. and Bieger, D., University Park press, Baltimore, pp. 215-325. Anesthesia and the Bloom, F.E., Cosat, E. and Salmoiraghi, G.C. (1965). responsiveness of individual neurons of the caudate nucleus of the cat to norepinephrine and dopamine administered by microelectroacetylcholine, 150: 244-252. phoresis. J.Pharmac.exp.Ther. Projections olfactives, Bonvallet, M., Dell, P. et Hugelin, A. (1952). gustatives, viscerales, vagales, visuelles et auditives au niveau des formations grises du cerveau anterieur du Chat. J.Physiol. (Paris), 44: 222-224. Synaptic transmission in the central nervous Bradley, P.B. (1968). and its relevance for drug action. Int.Rev.Neurobiol., 11: l-56.
system
J.H. (1966).-Pharmacological Bradley, P.B., Dhawan, B.N. and Wolstencroft, properties of cholinoceptive neurones in the medulla and pons of the cat. J.Physiol., 183: 658-674. Acetylcholine-sensitive J.W. (1963). Krnjevic, K. andhillis, cerebral cortex. J.Physiol., 166: 296-327. McLennan, H. and York, D.H. (1967). the caudate nucleus. J.Physiol.,
The action of dopamine 189: 393-402.
cells
in the
on neurones
of
Nobin, A. and BjBrklund, A. (1973). - Topography of the monoamine neurone systems in the human brain as revealed in fetuses. Acta physiol.scand. (Suppl.), 388: l-40. Acetylcholine and synaptic transmission in the central Phillis, J.W. (1976). Chemical Transmission in the Mammalian Nervous nervous system. In: System, Ed. Hockman, C.H. and Bieger, D. , University Park Press, Baltimore, pp. 159-213. An electrophysiolRapisarda, C., Azzaroni, A. and Infantellina, F. (1969). ogical analysis of the visual projections to the claustrum in unanaesthet53: 130-148. ized cats. Arch.Sci.biol., Unitary responses Segundo, J.P. and Machne, X. (Isa). in lenticular nucleus and claustrum. J.Neurophysiol., A stereotaxic Snider, R.S. and Niemer, W.T. (1961). The University of Chicago Press, Chicago.
to afferent volleys -19: 325-339.
atlas
of the cat brain.
Sensory Spector, I., Hassmannova, J. and Albe-Fessard, D. (1974). of single neurones of cat's claustrum. Brain Res. -66: 39-65. Urbano, A., Rapisarda, ique des afferences 50: 41-54. biol., York, D.H. 1967). caudate nucleus.
C. et Infantellina, F. (1966). somatiques au claustrum, chez
The inhibitory action of+dopamine Brain Res., 5_:263-266.
properties
Etude microphysiologle chat. Arch.Sci. on neurones
of the
THE ACTION
OF IONTOPHORETICALLY CLAUSTRUM
M.T.
Salerno,
Institutes
M.T.
Zagami,
of Human University
APPLIED
ACETYLCHOLINE
NEURONES
Palermo,
ON SINGLE
and F. Infantellina
and of General
of Palermo. 90134
DOPAMINE
IN THE CAT *
R. Cortimiglia
Physiology
AND
81 090895.05~02.~~0 Perpmon Pres Ltd
Corso
Tukory
Physiology, 129,
Italy
IAceepWf 25 June ?9gI)
The effects of iontophoretically applied acetylY?= c o lne (ACh) and dopamine (DA) on spontaneously active cats, have claustral neurones, in chloralose-anaesthetised
been investigated. Acetylcholine had both facilitatory and inhibitory effects in almost the same proportions. The action of DA was prevalently inhibitory. Two types of inhibitory response to ACh and DA are described: shortlasting inhibition and long-lasting inhibition. The excitatory response always outlasted the period of drug application.
The claustrum is a structure long known to be the seat of multi-modal somato-sensory and somatovisceral convergence (Bonvallet, Dell et Hugelin, 1952; Segundo and Machne, 1956; Urbano, Rapisarda et Infantellina, 1966; Rapisarda, Azzaroni and Infantellina, 1969; Spector, Hassmannova and AlbeFessard, 1974) which has not yet been investigated microiontophoretically in order to identify the neurotransmitters acting at its synapses. With the aim of identifying the chemical transmitters acting on claustral synapses and analysing their effects, the technique of iontophoretic release of drugs was used. Acetylcholine (ACh) and dopamine (DA) were first considered. Acetylcholine, in fact, as is known from the literature, is widely distributed in the central nervous system (Bradley, 1968; Phillis, 1976) and, when applied microiontophoretically, it affects most synapses of the central structures which have been investigated. Also, DA is a putative transmitter in the central nervous system (Bieger and Hockman, 1976) and is known to be the neurotransmitter of the nigrolstriatal pathway controlling motor activity. On the other hand, DA terminals in the claustrum, arising from the pars compacta of the substantia nigra and from the mesencephalic ventromedial tegmentum, have been demonstrated by histofluoresence microscopy in material obtained from aborted foetuses (Nobin and Bjltrklund, 1973). The neurones investigated in this preliminary study were marked with Pontamine Sky Blue and only identified histologically. METHODS The experiments were performed on chloralose-anaesthetised cats (70 mg /kg, i.v.), using the technique of iontophoretic application of drugs from multibarrelled micropipettes. The recording barrel was filled with 4M NaCl, while the other barrels contained: acetylcholine chloride (vials - Sigma), 5% solution in water; 3-hydroxytyramine HCl (Koch-Light), 5% solution in water; L-glutamic acid Na salt (Koch-Light), 2% solution in water at pH 8.0; Pontamine Sky Blue (68x, Gurr). The extracellularly recorded neuronal activity * This work Nazionale
was supported by a grant from delle Ricerche, Rome, Italy.
895
the Consiglio
896
Preliminary
Notes
was counted over successive 5 set epochs by using an electronic counter, connected through an analogue-digital converter to a recording system which traced a histogram of the discharge frequency. A current of 60 nA for 30 set periods was usually employed to expel ACh and DA from the appropriate barrel. Either the ejecting current was increased to 80 nA, or the application period was prolonged to 45 set, to evoke a response to drug application in some neurones. After each application, a current equal to that of the drug ejection current, was applied through the barrel filled with Pontamine Sky Blue, to check the effects of the current. At the end of the experiment the recording sites were marked with Pontamine Sky Blue, using a current of 5uA, for histological examination of serial brain slices, The stereotaxic planes exp'iored were between A12 and A17.5, Lg.5 and L13, Htl and Hi4. according to the atlas of Snider and Niemer (1961). RESULTS Acetylcholine and DA were tested on spontaneously active neurones; because of the known low spontaneous firing rate of claustral neurones (Segundo et al., 1956; Spector et al., 1974), continuous ejection of glutamate was sometimes used to increase the discharge frequency. The effects reported with repeated applications the firing rate'to recover The results
were
were obtained with each neurone more than once of the transmitters at intervals which Dermitted to base line levels.
as follows:
applied ACh affected a large number of the neurones (l) Iontophoretically tested. Of 41 spontaneously active neurones, 36 (88%) were affected by this drug. The effects were both excitatory and inhibitory in almost equal proportions. (2) The inhibitory response was found in 17 neurones (41%). Two types of inhibitory response to ACh could be distinguished on the basis of duration: one, shown by 8 neurones, was limited to the application period (short-lasting response); it began 10 set after the iontophoretic current was switched on and ended in the 5-10 set following the ACh release period (Fig. 1A). The other response, exhibited by 9 neurones, outlasted the end of the ejection current (long-lasting response). The long-lasting inhibitory response was also rapid in onset; the depression in firing rate began within 10 set of the ejection current being switched on and tended to return to its previous level 30-40 set after the end of the release period (Fig. 1s). response, shown by 18 neurones, was invariably of the (3) The excitatory long-lasting type (Fig. 1C). This response was rapid in onset, ie. it began within 10 set of the ACh application; the discharge rate returned gradually towards its previous level over a period of 80 to 120 set after the current was switched off. Occasionally the discharge frequency did not recover to its previous level. (4) Only one neurone showed a biphasic response pattern, inhibitory phase, limited to the ACh release period, excitatory phase lasting for 90 sec.
consisting of an followed by an
(5) Dopamine affected the discharge frequency of 27 (79%) out of the 34 neurones tested. Almost all the neurones tested, ie 23 (68%) showed an inhibitory response and only 4 neurones were excited. (6) The inhibitory responses to DA, like those to ACh, were both short- and long-lasting. Short-lasting inhibition was shown by 16 neurones; it began 5 set after DA ejection and ended within 5 set following drug application (Fig. ZA). The long-lasting inhibition exhibited by 7 neurones was also rapid in onset, beginning 5 set after the ejection current was switched on; the firing rate tended to recover within 40-55 see after the end of the application period (Fig. 26). excited by DA showed a gradually increasing discharge (7) The four neurones during the period of drug application, while the maximum level of excitation was reached after DA ejection (Fig. 2C).
(8) Both ACh and DA were applied to 33 neurones (Table 1) but a comparison of the effects of the two drugs applied to the same neurone did not yield any significant results.
897
Preliminary Notes
c
I
1
I I30
I
I
I
120
Gf-Fi-fO
I
,
1 60
1
0
WC
t
I 120
1 180
1
, ICC
Fi ure 1. Effects of iontophoreticallyapplied acetylcholine (ACh) on r ng rate of three spontaneously active neurones in the claustrum. Abscissa: time in sec. Ordinate: number of spikes in successive 5 set epochs. Horizontal bars indicate the iontophoretic application of ACh or current control; all responses were obtained using 60 nA currents. A: short-lasting inhibition; the inhibitory response was obtained with successive ACh applications. A current control is without effect. B: long-lasting inhibition. C: excitatory response to ACh.
C
OI I
0
I
1 00
I
2% I lz-3
I
I sac
I 0
a
1 *0
&L 1
I 120
I
I I.C
1 0
1
I 60
1
I 120
Figure 2. Effects of iontophoretic application of dopamine (DA) on the 'firingrate of three spontaneously active neurones in the claustrum. A: short-lasting inhibition. B: long-lasting inhibition. C: excitatory response.
I
1
I
‘80
naz
898
Preliminary Notes Table
1.
Comparison of the effects of ACh with DA when applied to the same neurone.
those
of
ACh
0
4
0
0
8
4
1
2
_ -
3
0
2
2
0
3
0
4
0
+ DA
The table represents the number of neurones showing the responses indicated: + = excitation, - = short-lasting inhibition, - - = long-lasting inhibition, o = no effect.
DISCUSSION The data reported here give the first information about the responsiveness of claustral neurones to iontophoretic application of ACh and DA. Although limited to the sample of neurones studied, it is possible to conclude that a large number of units in the claustrum are affected by iontophoretic application of ACh (88%) and DA (79%). The responses to ACh shown by claustral neurones were both excitatory (44%) and inhibitory (41%) in almost the same proportions and resemble those such as the brain stem (Bradley, Dhawan found in other cerebral structures, and Wolstencroft, 1966) and the caudate nucleus (Bloom, Costa and Salmoiraghi, 1965), while in the cerebral cortex there is a predominatly excitatory effect (KrnjeviC and Phillis, 1963). The response to DA exhibited by claustral neurones was predominantly inhibitory (68%) and this is analogous with the stiuation in the caudate nucleus where DA, studied under various experimental conditions, was found to act as an inhibitory neurotransmitter (McLennan and York, 1967; Bloom et al. 1965; York, 1967). According to their time course, two types of inhibitory responses to period was called ACh and DA were found: one, limited to the drug application the iontophoretic ejection the short-lasting type; the other, outlasting type. Both short- and long-lasting period, was called the long-lasting inhibition occurred almost equally following iontophoretic application of ACh, the former being shown by 47% and the latter by 53% of the neurones inhibition appeared to be more prevalent which were inhibited. Short-lasting among the neurones inhibited by DA and was present in 69% of the neurones depressed by DA. The excitatory response to both drugs was invariably of the longlasting type; however, for the neurones excited by DA, the slow onset and gradual time-course of excitation might suggest an indirect effect. In conclusion, from these preliminary results, it appears that these two drugs play an important role in chemical transmission at ClaUStral synapses. and further investigations should reveal the physiological significance of the ACh and DA inputs to the claustrum.
899
Preliminary Notes REFERENCES
On the physiology and pharmacology of Bieger, D. and Hockman, C.H. (1976). Chemical Transmission in the Mammalian cerebral dopamine neurones. In: Central Nervous System, Ed. Hockman, C.H. and Bieger, D., University Park press, Baltimore, pp. 215-325. Anesthesia and the Bloom, F.E., Cosat, E. and Salmoiraghi, G.C. (1965). responsiveness of individual neurons of the caudate nucleus of the cat to norepinephrine and dopamine administered by microelectroacetylcholine, 150: 244-252. phoresis. J.Pharmac.exp.Ther. Projections olfactives, Bonvallet, M., Dell, P. et Hugelin, A. (1952). gustatives, viscerales, vagales, visuelles et auditives au niveau des formations grises du cerveau anterieur du Chat. J.Physiol. (Paris), 44: 222-224. Synaptic transmission in the central nervous Bradley, P.B. (1968). and its relevance for drug action. Int.Rev.Neurobiol., 11: l-56.
system
J.H. (1966).-Pharmacological Bradley, P.B., Dhawan, B.N. and Wolstencroft, properties of cholinoceptive neurones in the medulla and pons of the cat. J.Physiol., 183: 658-674. Acetylcholine-sensitive J.W. (1963). Krnjevic, K. andhillis, cerebral cortex. J.Physiol., 166: 296-327. McLennan, H. and York, D.H. (1967). the caudate nucleus. J.Physiol.,
The action of dopamine 189: 393-402.
cells
in the
on neurones
of
Nobin, A. and BjBrklund, A. (1973). - Topography of the monoamine neurone systems in the human brain as revealed in fetuses. Acta physiol.scand. (Suppl.), 388: l-40. Acetylcholine and synaptic transmission in the central Phillis, J.W. (1976). Chemical Transmission in the Mammalian Nervous nervous system. In: System, Ed. Hockman, C.H. and Bieger, D. , University Park Press, Baltimore, pp. 159-213. An electrophysiolRapisarda, C., Azzaroni, A. and Infantellina, F. (1969). ogical analysis of the visual projections to the claustrum in unanaesthet53: 130-148. ized cats. Arch.Sci.biol., Unitary responses Segundo, J.P. and Machne, X. (Isa). in lenticular nucleus and claustrum. J.Neurophysiol., A stereotaxic Snider, R.S. and Niemer, W.T. (1961). The University of Chicago Press, Chicago.
to afferent volleys -19: 325-339.
atlas
of the cat brain.
Sensory Spector, I., Hassmannova, J. and Albe-Fessard, D. (1974). of single neurones of cat's claustrum. Brain Res. -66: 39-65. Urbano, A., Rapisarda, ique des afferences 50: 41-54. biol., York, D.H. 1967). caudate nucleus.
C. et Infantellina, F. (1966). somatiques au claustrum, chez
The inhibitory action of+dopamine Brain Res., 5_:263-266.
properties
Etude microphysiologle chat. Arch.Sci. on neurones
of the