Neuroscience Letters, 13 (1979)151--155 ~) E ~ e r / N o r t h - H o ~ d Scientific Publkhem Ltd. ,
PO~E
151
i
~FE~i~
TO THE M E r ~ ~ R ~ y RESPIRATORY
S Y ~.,.......... ... ~A~MOFUNCTIONAL":: I~ORRELATION
D. RICHE, M. DENAVIT-SAUBI]~and J, CHAMPAGNAT Laboratoire de Physiologic ne.~euse, D~partement de NeuropI:~ysiologie appliqu~e, C.N,R.8. 91199, Gif-~u~Yvel~te (France) (Received December 20th, 1'978) (Recked version received Febrmry 10th, 1979) (Accepted Arril 24th, 197~)
SUMMARY
A direct projection frown the parabrachialis medialis, KSlliker-Fuse complex (PMKF) to the bulbar respiratory area is shown using autoradiography. Perikarya of P M K F project to the medulla, labelled fibers and terminals being located radially to the fourth ventricle from the nucleus hypoglossus to the zmbiguus. The functional importance of this projection is demonstrated in pl~macological studiesusing iontophoresis of L-glutamab~.• which enable identification of somatodendritic discharges. A good correlation exists between the localization in the medulla of labelled fibers and terminals and that of an inspiratory background activity related to inspiratory ~.~bersor terminals.
Mechanisms underlying the central production of respiratory rhythm are localized at the medullary level. Here, mapping of respiratory units has generally been undertaken with the aid of extracellular recording [ 2,5,19, 20]. Except with kdz~-ellular recording [ 16] it is difficult to distinguish between potentials J~icm a soma or from an axon. It is useful to combine the electrophysiolol~Lcal and the neuroanatomical approach in order to afferents, perikarya and efferents. avestigate, with these two.different echniquesi.theinvo]ivement of afferent fibers arising from the pons in ulbar respiratory mechanisms. atomical data obtained by retrograde [13.] have shown that the medulla arabrachialis medialis, K6lliker-Fuse Berman [3]). R,~spiratory activity has b e e n recorded fm this complex which corresponds functionally to the
152
pneumotaxic center [4,7]. Lesions made in this structure in bivagotomized cats stron~'ly physiologica] prompted us localize axo~ complex. Anatomical results have been obtained ~from 6 cats. Slow unilateral injections of tritiated prolinewere madein PMKF with a Hamilton syringe. Each anim~ received 5 0 - 6 0 pCi of L-[3H]prolme(spec. ~ t . , 27 Ci/mM) di~olved in 1 ,~1o f n o m a l saline. The b ~ s were ~ e d by peffusion with 2% paraformaldehyde, 0.5% glutaraldehyde in phosphate buffer 7 0 - 7 4 h post-operatively. B~ains were embedded in paraffin and sectioned in a plane parallel to the tentorium cerebelli (45 ° relative to t h e Horsley-Clarke reference plane). Slices 10 #m thick were coated with diluted K5 e~Lulsion (Ilford), stored in light-tight containers at 4°C for 3--9 weeks, developed° and stained with cresyl violet. When the injection site included the whole PMKF region (Fig. 1, inset) involv~lg nucleus parabrachialis medialis and lateralis surrounding the brachium conjuctivum, an important part of the KSlliker-Fuse nucleus and a part of t h e 'nucleus subcoe.-uleus' of Meessen and Olszewski [18], heavily labelled fibers and dense terminals appear around the injection
B
J
5SP
rL
"
lll,r Imm
Fig. 1. Localization of PMKF. projections to...... the ......medulla activity. A: Anatomicaldata: in the inse~i~lo . . . . el of PMKF is indica~d bY hatched area~At ~c the labelled efferent pathways aLt buibar Ii FTL: lateral tegment~l field;LP pyramidalis; SP: spit !al trigemim ~,n the right, half h~toiogiCai se¢ methyl. blue . . dot . . ( ~. o w )!Onth recorded insp~ato~ RBA (bli~C (triangles ...... . . . . . ~!a indi~tes the: lo~lizatio! ~ of*4!heil iif .... i
~¸/ :~ ,
~ ~
i background ilion ~ ~atthe level ~e;ip : tractus onai data :
~ei~trae~ ~ d a
153
site. They form an ipsilateral descending projection through the dorsolateral reticular formation. More caudally labelling was found in a longitudinal bundle extending beneath the spinal l~rigeminal nucleus. A section of this bundle appeared in coronal planes at the level of the obex as an elongated area extending:~radially with respect to the fourth ventricle between the nucleus hypoglossus and thenucleus ambiguus (Fig. 1A). At more caudal levels thesilver grain distribution diminishes in density. Such labelling did not appear t o t h e k:vel of the obex iv 2 cats where the injection did not include the FMKF but was restricted to the dorsal superficial part of the pons. In these cases labelling was found only up to a p~ane 4 mm anterior to the obex. Functional approach. Local application of L-glutamate to ~ o n s is ineffective; on the contrary, it greatly increases the discharge of neurones when applied near somata or dendrites [6,12]. We used this pharmacological method combined with histological localization in order to map in the medulla respiratory activity which was not coming from soma. Results were obtained from 15 cats, anaesthetized with nembutal, bivagotomized, curarised and artificially ventilated. Bipolar recording from one exposed phrenic nerve was used as an index for central respiratory rhythm. Multibarrelled micropipettes were usc~ for extracellular recording and iontophoretic application of L-glutamate and methyl blue. The latter was used to localize the recording sites and to test for possible current artefacts (for further technical details see refs. 9 and 10). At the end of the experiment, the animals were perfused and brains sectioned for determination of electrode tracks. The cat medulla was explored, from antelior 3 to posterior I ram, taking the obex as zero. The micropipette was lowered progressively perpendicular to the dorsal surface. The activity recorded was identified as non-respiratory, inspimtory or expirs~nry according to the po.~dtion of maximal firing in the respiratory cycle. Respiratory activi~os were classified as unit activities when sp~kes with stable shape av.P sufficient amplitude were recorded. Other respiratory activities ~.ppe~ed as large rhythmic changes in the background noise (respiratory background activity (RBA)) in which unitary spikes were not clearly isolated [ 1,8]. L-Glutamate was applied by iontophon~sis and its effect was tested on the recorded unit activity. The minimal dose which readily increased the firing frequency of a neuronal discharge was defined on soma recorded at the beginning of the track (in the dorsal column nuclei, for instance). Then, as soon as activity was correlated with the phrenic nerve discharge its response to L-glutamate applicatio:n was tested. A n inspiratory R B A was constantly found below dorsal column nuclei. It was not affected by dosage of L.glutmnate ten times larger than the one which was effective )n soma: itdid not originate from multiunita~ somato~lendritic recording aud was thus considered to be a multiaxonal discharge, a sum of synaptic
!54:
,°
.
ACKNOWLEDGEMENTS
The authors wish tothauk E.Boudinot andB, Guibert for theirtechnical assistance.This study wassupp0rted by an A.T.P. from the D,G.R.S.T. No. "/8.7.2776 ~nd A.T.P,from the I.N.S.E.R,M. No. 76-61. REFERENCES 1 Achard. D~~and Buche~, V,M., Courants d'action bulbaires i rythme respiratoire. Helv, Physiol. Acta,. 12 (1954) 265-.283.~ 2 Batsel, H.L., Loca]izationofbulbar respiratory centerby microelectrode sounding, Exp. Neuroi,, 9 (1964) 410-'~426. " ...... 3
4 5
1 1 I
155 fibre eonnectionit to the tr~eminal, facial and h y p o g ] o ~ l motor nuclei. II. An at 100 (1977) 265--286. ~f the respiratory centers, Fed. Proe., 36 p,~thways possibly subversing respiration chniques, Proc. Inter~. Union of Physiol. nd Senekowitsch, R., ~orphological and tory neurons of the cat, Pfliiggers Arch., dry centers in ~be cat, J. Phy~oL (Lond.), iktonischer a t l ~ der rautenhho~ des kmfin~ens, Karger, ]Basel, 1949. 19 Merrill, E.G., laindiul[ a respiratory function for the medullary respiratory ~eurons. In R. B e l k ~ and E.G. Gray (Eds.), Essays on the Nervous System, Clarendon Press, Oxford, 1974, pp. 451-486. 20 Vibert, J.F., Bertrand, F., Denavit-Saubi6, M. and HugeUn, A., Three dimensional representation of the bulbo pontine respiratory networks architecture from unit dem/ty maps, Brain Res., 114 (1976) 227--244.
PO~E
151
i
~FE~i~
TO THE M E r ~ ~ R ~ y RESPIRATORY
S Y ~.,.......... ... ~A~MOFUNCTIONAL":: I~ORRELATION
D. RICHE, M. DENAVIT-SAUBI]~and J, CHAMPAGNAT Laboratoire de Physiologic ne.~euse, D~partement de NeuropI:~ysiologie appliqu~e, C.N,R.8. 91199, Gif-~u~Yvel~te (France) (Received December 20th, 1'978) (Recked version received Febrmry 10th, 1979) (Accepted Arril 24th, 197~)
SUMMARY
A direct projection frown the parabrachialis medialis, KSlliker-Fuse complex (PMKF) to the bulbar respiratory area is shown using autoradiography. Perikarya of P M K F project to the medulla, labelled fibers and terminals being located radially to the fourth ventricle from the nucleus hypoglossus to the zmbiguus. The functional importance of this projection is demonstrated in pl~macological studiesusing iontophoresis of L-glutamab~.• which enable identification of somatodendritic discharges. A good correlation exists between the localization in the medulla of labelled fibers and terminals and that of an inspiratory background activity related to inspiratory ~.~bersor terminals.
Mechanisms underlying the central production of respiratory rhythm are localized at the medullary level. Here, mapping of respiratory units has generally been undertaken with the aid of extracellular recording [ 2,5,19, 20]. Except with kdz~-ellular recording [ 16] it is difficult to distinguish between potentials J~icm a soma or from an axon. It is useful to combine the electrophysiolol~Lcal and the neuroanatomical approach in order to afferents, perikarya and efferents. avestigate, with these two.different echniquesi.theinvo]ivement of afferent fibers arising from the pons in ulbar respiratory mechanisms. atomical data obtained by retrograde [13.] have shown that the medulla arabrachialis medialis, K6lliker-Fuse Berman [3]). R,~spiratory activity has b e e n recorded fm this complex which corresponds functionally to the
152
pneumotaxic center [4,7]. Lesions made in this structure in bivagotomized cats stron~'ly physiologica] prompted us localize axo~ complex. Anatomical results have been obtained ~from 6 cats. Slow unilateral injections of tritiated prolinewere madein PMKF with a Hamilton syringe. Each anim~ received 5 0 - 6 0 pCi of L-[3H]prolme(spec. ~ t . , 27 Ci/mM) di~olved in 1 ,~1o f n o m a l saline. The b ~ s were ~ e d by peffusion with 2% paraformaldehyde, 0.5% glutaraldehyde in phosphate buffer 7 0 - 7 4 h post-operatively. B~ains were embedded in paraffin and sectioned in a plane parallel to the tentorium cerebelli (45 ° relative to t h e Horsley-Clarke reference plane). Slices 10 #m thick were coated with diluted K5 e~Lulsion (Ilford), stored in light-tight containers at 4°C for 3--9 weeks, developed° and stained with cresyl violet. When the injection site included the whole PMKF region (Fig. 1, inset) involv~lg nucleus parabrachialis medialis and lateralis surrounding the brachium conjuctivum, an important part of the KSlliker-Fuse nucleus and a part of t h e 'nucleus subcoe.-uleus' of Meessen and Olszewski [18], heavily labelled fibers and dense terminals appear around the injection
B
J
5SP
rL
"
lll,r Imm
Fig. 1. Localization of PMKF. projections to...... the ......medulla activity. A: Anatomicaldata: in the inse~i~lo . . . . el of PMKF is indica~d bY hatched area~At ~c the labelled efferent pathways aLt buibar Ii FTL: lateral tegment~l field;LP pyramidalis; SP: spit !al trigemim ~,n the right, half h~toiogiCai se¢ methyl. blue . . dot . . ( ~. o w )!Onth recorded insp~ato~ RBA (bli~C (triangles ...... . . . . . ~!a indi~tes the: lo~lizatio! ~ of*4!heil iif .... i
~¸/ :~ ,
~ ~
i background ilion ~ ~atthe level ~e;ip : tractus onai data :
~ei~trae~ ~ d a
153
site. They form an ipsilateral descending projection through the dorsolateral reticular formation. More caudally labelling was found in a longitudinal bundle extending beneath the spinal l~rigeminal nucleus. A section of this bundle appeared in coronal planes at the level of the obex as an elongated area extending:~radially with respect to the fourth ventricle between the nucleus hypoglossus and thenucleus ambiguus (Fig. 1A). At more caudal levels thesilver grain distribution diminishes in density. Such labelling did not appear t o t h e k:vel of the obex iv 2 cats where the injection did not include the FMKF but was restricted to the dorsal superficial part of the pons. In these cases labelling was found only up to a p~ane 4 mm anterior to the obex. Functional approach. Local application of L-glutamate to ~ o n s is ineffective; on the contrary, it greatly increases the discharge of neurones when applied near somata or dendrites [6,12]. We used this pharmacological method combined with histological localization in order to map in the medulla respiratory activity which was not coming from soma. Results were obtained from 15 cats, anaesthetized with nembutal, bivagotomized, curarised and artificially ventilated. Bipolar recording from one exposed phrenic nerve was used as an index for central respiratory rhythm. Multibarrelled micropipettes were usc~ for extracellular recording and iontophoretic application of L-glutamate and methyl blue. The latter was used to localize the recording sites and to test for possible current artefacts (for further technical details see refs. 9 and 10). At the end of the experiment, the animals were perfused and brains sectioned for determination of electrode tracks. The cat medulla was explored, from antelior 3 to posterior I ram, taking the obex as zero. The micropipette was lowered progressively perpendicular to the dorsal surface. The activity recorded was identified as non-respiratory, inspimtory or expirs~nry according to the po.~dtion of maximal firing in the respiratory cycle. Respiratory activi~os were classified as unit activities when sp~kes with stable shape av.P sufficient amplitude were recorded. Other respiratory activities ~.ppe~ed as large rhythmic changes in the background noise (respiratory background activity (RBA)) in which unitary spikes were not clearly isolated [ 1,8]. L-Glutamate was applied by iontophon~sis and its effect was tested on the recorded unit activity. The minimal dose which readily increased the firing frequency of a neuronal discharge was defined on soma recorded at the beginning of the track (in the dorsal column nuclei, for instance). Then, as soon as activity was correlated with the phrenic nerve discharge its response to L-glutamate applicatio:n was tested. A n inspiratory R B A was constantly found below dorsal column nuclei. It was not affected by dosage of L.glutmnate ten times larger than the one which was effective )n soma: itdid not originate from multiunita~ somato~lendritic recording aud was thus considered to be a multiaxonal discharge, a sum of synaptic
!54:
,°
.
ACKNOWLEDGEMENTS
The authors wish tothauk E.Boudinot andB, Guibert for theirtechnical assistance.This study wassupp0rted by an A.T.P. from the D,G.R.S.T. No. "/8.7.2776 ~nd A.T.P,from the I.N.S.E.R,M. No. 76-61. REFERENCES 1 Achard. D~~and Buche~, V,M., Courants d'action bulbaires i rythme respiratoire. Helv, Physiol. Acta,. 12 (1954) 265-.283.~ 2 Batsel, H.L., Loca]izationofbulbar respiratory centerby microelectrode sounding, Exp. Neuroi,, 9 (1964) 410-'~426. " ...... 3
4 5
1 1 I
155 fibre eonnectionit to the tr~eminal, facial and h y p o g ] o ~ l motor nuclei. II. An at 100 (1977) 265--286. ~f the respiratory centers, Fed. Proe., 36 p,~thways possibly subversing respiration chniques, Proc. Inter~. Union of Physiol. nd Senekowitsch, R., ~orphological and tory neurons of the cat, Pfliiggers Arch., dry centers in ~be cat, J. Phy~oL (Lond.), iktonischer a t l ~ der rautenhho~ des kmfin~ens, Karger, ]Basel, 1949. 19 Merrill, E.G., laindiul[ a respiratory function for the medullary respiratory ~eurons. In R. B e l k ~ and E.G. Gray (Eds.), Essays on the Nervous System, Clarendon Press, Oxford, 1974, pp. 451-486. 20 Vibert, J.F., Bertrand, F., Denavit-Saubi6, M. and HugeUn, A., Three dimensional representation of the bulbo pontine respiratory networks architecture from unit dem/ty maps, Brain Res., 114 (1976) 227--244.
