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Brain Research &&tin, Vol. 24, pp. 457-163. @Pergamon Press plc, 1990. Printed in the U.S.A.
Direct Vagal Input to Neurons in the Area Postrema Which Project to the Parabrachial Nucleus: An Electron Microscopic-HRP Study in the Cat’ S. M. STRAIN, D. G. GWYN,’
J. G. RUTHERFORD AND B. J. LOSJER
Received 22 September 1989
STRAIN, S. M., D. G. GWYN, J. G. RUTHERFORD AND B. J. LOSIER. LXrect vagaf input to netcrons in the areupostrema which project to the purabrachiaf nucleus: An electron microscopic-HRP study in the cut. BRAIN RES BULL 24(3) 457-463, 1990. -This study in cat examines the synaptic relationship of vagal afferents to parabrachial projecting neurons in the area postrema (AP) using anterograde and retrograde transport of horseradish peroxidase (HRP). Wheat germ agglutinin-HRP injected into the parabrachial nucleus (PBN) produced retrogradeneuronal labeling in the AP and in the nucleus of the tractus solitarius bilaterally, but with an ipsilateral predominance. Labeled neurons were confined mainly to the caudal s’s of the AP. Following injection of WGA-HRP into the PBN and HRP into the nodose ganglion in the same animal, examination of sections of the AP with the electron microscope revealed anterogradely labeled axon terminals in apposition to retrogradely labeled somata and dendrites. In some instances, labeled terminals were observed to form synaptic contacts with retrogradely labeled neurons. We conclude that in the cat a vagal input to neurons in the AP is monosynaptic~ly relayed to the PBN.
Area postrema
Parabrachial nucleus
Vagus nerve
Cat
THJZarea postrema (AP) is a highly vascular structure lying in the floor of the caudal part of the IV ventricle (30). Several functions have been attributed to the AP including the regulation of cardiovascular function (15,17) and the control of food intake (12,48). In addition there is evidence to suggest a role for the AP as a chemoreceptive trigger zone for emesis (4, 5, 8, 9). Since capillaries with the AP are fenestrated (14, 28, 30) and therefore are permeable to circulating substances, it is possible for chemical stimuli to impinge upon the AP via the blood stream as well as via the cerebrospinal fluid. In addition, afferent nerve fibers terminating in the area convey information to the AP from peripheral nerves and from a variety of regions within the central nervous system. The neural connections of the AP have been extensively studied at the light microscopic level, by ourselves and by others [see (3 1,45)]. The AP has been found to receive sensory fibers from the vagus nerve (3, 11, 18, 19, 24, 32) and the glossopharyngeal nerve (I I) with specifically identified input from the aortic nerve (26) and the carotid sinus nerve (13). Descending projections to the AP have been shown to arise from the dorsomedial hypothalamus in rat (23,45) and from the paraventricular hypothalamic nucleus in cat (22) and rat (45). Neurons in the AP project to the ventrolateral medulla (7,45),
Horseradishperoxidase
Electron microscopy
to the subjacent nucleus of the tractus solitarius (NTS) and to the
dorsal motor nucleus of the vagus (45). The major ascending projection of the AP is to the parabrachial nucleus (PBN) (29, 34, 45, 49). The PBN, in turn, appears to be a focal point for the distribution of information derived from the tongue and from visceral sources. The PBN has been shown to be involved in the regulation of cardiovascular (39) and respiratory activity (6) and gives rise to projections to the cerebral cortex, the amygdala, the thalamus and the hy~~~arnus (1641, 43,44). In the rat electrical stimulation of the cervical vagus nerve has demonstrated a second order vagal input to the PBN (21). The purpose of the present study is to determine in the cat whether vagal afferent Bbres synapse directly upon neurons in the AP which project to the PBN. Such a cotmection would provide a route whereby a variety of visceral sensory impulses could be distributed to such regions as the insular cortex, the amygdala and the thalamus. To achieve this goal horseradish peroxidase (HRP) was injected into the nodose ganglion and lectin-conjugated horseradish peroxidase (WGA-HRP) was injected into the PBN in the same animal. The AP was then examined in the electron microscope for the presence of anterogradely labeled vagal terminals synapsing upon retrogradely labeled PBN-p~jecting neurons.
‘A preliminary report of this study was presented at the Canadian Federation of Biological Societies, Winnipeg, 1987. ‘Requests for reprints should be addressed to Dr. D. G. Gwyn.
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AND LOSIER
METHOD In all of the operative procedures described below, animals were initially anesthetized with an intravenous injection of sodium pentobarbital and then were maintained on a halothane-oxygen mixture delivered by a respirator. In order to determine the distribution of neurons in the AP which project to the PBN, unilateral injections of 0.08 to 0.12 ~1 of a 5.0% aqueous solution of lectin-conjugated horseradish peroxidase (WGA-HRP) containing 0.1% Neutral red and 2.0% dimethyl sulfoxide (DMSO) were made stereotaxically in the PBN of 5 cats. Following survival periods of 3 or 4 days, the animals were anesthetized and perfused transcardially with normal saline followed by a fixative consisting of 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer (pH=7.4), both at room temperature. The fixative was washed out with cold (4°C) 10% phosphate buffered sucrose solution (pH = 7.4). The brain was exposed and blocked in the stereotaxic plane. Transverse, 40 pm frozen serial sections were cut through the injection site and transverse or sagittal, frozen serial sections were cut through the medulla. Sections were processed for the light microscopic demonstration of HRP activity using the tetramethylbenzidine (TMB) technique of Mesulam (36), and were then divided into three series. Two series were lightly stained with Neutral red, while the third series was left unstained. Sections were examined in the light microscope using both bright- and darkfield illumination; the distribution of retrogradely labeled neurons in the AP and the extent of the injection site were plotted with the aid of a camera lucida. In a further 5 cats, unilateral injections of 0.08 to 0.12 (*l of 5.0% WGA-HRP were placed in the PBN, and the animals were allowed to survive for 3 days. The tissue was processed for electron microscopic demonstration of reaction product in retrogradely labeled neurons in the AP, using the protocol described below. In 3 cats, between 0.10 to 0.12 p.1 of a 5.0% aqueous solution of WGA-HRP containing 0.1% Neutral red and 2.0% DMSO was stereotaxically injected into the right PBN. Following this procedure, the animals were removed from the stereotaxic frame, and the right nodose ganglion was exposed in the neck. Between 15 and 25 l.~l of a 30% aqueous solution of unconjugated HRP was injected into the ganglion in each animal. After a survival period of 3 days, animals were anesthetized and perfused transcardially with 1 1of phosphate buffered saline (pH = 7.4) followed by 3 to 4 1 of fixative consisting of 2.0% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer at 4°C. The brain was exposed and blocked in the stereotaxic plane. Blocks containing the injection site and the nodose ganglion were placed in the 10% buffered sucrose solution, while the blocks which included the AP were placed in 0.1 M phosphate buffer. Both groups of tissue blocks were stored overnight at 4°C. Frozen, transverse serial sections (40 pm) were cut through the injection site in the PBN and 40 p,m longitudinal sections were cut through the nodose ganglia. These sections were processed for light microscopic demonstration of HRP activity as described above. Transverse
FIG. 1. (A) A light micrograph of a transverse section through the pons showing the extent of the WGA-HRP injection site in DC 369. PB, = Lateral parabrachial nucleus, PB, = Medial parabrachial nucleus, KF = KGlliker-Fuse nucleus. Bar= 1 .O mm. (B through D). Light micrographs of transverse sections through the medulla, arranged from caudal (B) to rostra1 (D) levels, which illustrate the distribution of retrogradely labeled neurons in the area postrema (AP) and the nucleus of the tractus solitarius (NTS) ipsilateral to the injection shown in (A). Note the paucity of labeled neurons in the rostra1 AP (D). SNG=Subnucleus gelatinosus, TS= Tractus solitarius, X = Dorsal motor nucleus of the vagus. Bar = 100 km.
VAGAL, INPUT TO PBN PROJECTING
NEURONS
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PREVIOUS
PAGE
FIG. 2. (A) A light micrograph of a transverse section through the pons showing the extent of the WGA-HRP injection site in DC 396. Abbreviations as in Fig. 1A. Bar= 1 .O mm. (B) An electron micrograph showing HRP retrogradely labeled neurons in the AP resulting from the injection illustrated in A. The labeled neurons are indicated by an asterisk (*), while the crystals characteristic of the TMB reaction for HRP are indicated by arrowheads. The open block arrows indicate the extensive extracellular spaces typical of the AP. G= Golgi apparatus, N=Nissl substance, Nu -Nucleus. Bar= 5.0 km. (C) The area included within the rectangle in B is shown here at a higher magnification. TMB crystals are indicated by block arrows. Bar= 1 .O pm. (D) An axon terminal (AT) containing round, clear and dense-cored (arrowhead) vesicles is shown contacting (arrow) a labeled soma in the AP of DC 396. The TMB reaction product in the soma is indicated by a solid block arrow. Bar=0.5 pm. (E) An axon terminal (AT) containing round, clear vesicles and forming a synapse (arrow) upon a labeled dendrite (Dn) is shown in the AP of DC 396. The TMB reaction product is indicated by a solid block arrow. Bar=OS pm.
FIG. 3. (A) A labeled axon terminal (AT) containing round, clear vesicles and a single dense-cored vesicle (arrowhead) is shown in close apposition to a labeled soma (S) in the AP of DC 400. The TMB reaction product is indicated by solid block arrows. (B) A labeled axon terminal (AT) containing round, clear and dense-cored vesicles (arrowheads) is shown in close apposition to a labeled dendrite (Dn) in the AP of DC 416. Solid block arrows indicate the TMB reaction product. (C) A heavily labeled axon terminal (AT) is seen in apposition to a labeled dendrite (Dn) in the AP of DC 416. The TMB reaction product is indicated by solid block arrows, while the small arrow indicates the region of an obliquely sectioned synaptic density between the labeled preand postsynaptic elements. (D) A labeled axon terminal (AT) is shown synapsing upon a large labeled dendrite (Dn) in the AP of DC 416. Solid block arrows indicate the TMB reaction product in both the terminal and the dendrite. (E) The axon terminal in D is shown here at a higher magnification. The small arrows indicate the location of the postsynaptic densities, while the TMB reaction product is indicated by the solid block arrows. Bars =O.S pm.
461
VAGAL INPUT TO PBN PROJECTING NEURONS
vibratome sections (100 to 125 pm) were cut through the AP. These sections were processed using a modification of Mesulam’s (36) TMB technique for revealing HRP activity at the electron microscopic level. The region including the AP and the NTS were cut out of each section. The excised tissue was osmicated, stained with saturated aqueous many1 acetate, and embedded in TAAB resin. Thick (1 .O pm) sections were cut and examined in the light microscope to determine the location of the AP. Using camera lucida drawings of these sections as a guide, blocks were retrimmed to include only the AP and NTS. Thin sections were then cut from the blocks, stained with lead citrate, and examined in a Zeiss EM 10 electron microscope for the presence of labeled axon terminals in contact with retrogradely labeled neurons. As a control between 20-40 p1 of 30% HRP was unilaterally injected into the nodose ganglion in three cats. Tissue from these cases was processed as described above for the doubly injected animals. Sections through the AP were examined in the electron microscope for the presence of any transsynaptic labeling of
RESULTS
In five cases the distribution of neurons in the AP retrogradely labeled from the PBN was studied. The results from one cat, DC 369, will be presented in detail because these are representative of our findings in general. The central zone of the injection extended throughout the length of the PBN and included the medial and lateral parabrachial nuclei (PB, and PB, , respectively) and the Kolliker-Fuse nucleus (KF). The peripheral zone of the injection site extended into the region of the locus ceruleus and the dorsal division of the principal sensory nucleus of the trigeminal nerve, while at rostral levels, a small amount of HRP spread into the region of the dorsal nucleus of the lateral lemniscus. In the AP, retrogradely labeled neurons were found bilaterally, but in far greater numbers ipsilateral to the injection site. Labeled neurons were most abundant in the caudal 2/3’sof the area (Fig. 1B and C), while very few labeled cells were found throughout the rostral % of the AP (Fig. 1D). In addition, labeled neurons were distributed bilaterally, though with an ipsilateral predominance, throughout the length of the NTS (Fig. IB-D). While present in most of the subnuclei of the NTS, HRP-positive cells were most numerous in the subnucleus gelatinosus (SNG) and the medial subnucleus. No evidence of anterograde terminal labeling was found in either the APortheNTS. Four of five animals which received unilateral injections of WGA-HRP into the PBN and which were subsequently processed for electron microscopy demonstrated retrograde labeling in the AP. Representative findings from one of these four cases (DC 396) are illustrated in Fig. 2. The injection site in this animal, as in the other three positive cases included both the PB, and PB, , as well as the KF (Fig. 2A). Some spread of the HRP into the locus ceruleus occurred. In the AP, retrogradely labeled neurons could be identified easily by the presence in the cytoplasm of darkly staining crystalline reaction product (Fig. 2C). The somata of labeled neurons were small (7 to 12 pm in diameter), and were scattered, singly or in groups of two or three, throughout the neuropil (Fig. 2B). The nuclear envelope of labeled neurons was frequently infolded and the cytoplasm contained the usual complement of organelles, including rough endoplasmic reticulum, Golgi apparatus and lysosomes. Labeled somata received synapses (Fig. 21)) typically from axon terminals which contained round, clear vesicles or a combination of round, clear and dense-cored vesicles. Terminals containing pleomorphic vesicles were not observed in these preparations. HRP reaction product was also found in dendrites in the AP (Fig. 2E). Axon terminals forming
synapses with labeled dendrites contained round, clear vesicles or both round, clear and dense-cored vesicles, although terminals containing only round, clear vesicles were more commonly encountered than those with a mixed population of vesicles (Fig. 3E). The diameter of round, clear vesicles ranged from 30 to 50 nm, while dense-cored vesicles were 60 to 100 nm in diameter. No labeled axon terminals were encountered in the AP or NTS in any of the four cases comprising this portion of the study. In all three cases in which WGA-HRP was injected into the PBN and HRP was injected into the nodose ganglion, many labeled terminals were seen in the AP in apposition to labeled profiles, most of which were dendritic in nature. Results from two of these cases (DC 400 and DC 416) are illustrated in Fig. 3. The injection sites in both of these animals were similar to those shown in Figs. 1 and 2. The HRP injections were centered on the PBN, with some spread into surrounding structures such as the dorsal principal sensory and motor nuclei of the trigeminal nerve, and the dorsal nucleus of the lateral lemniscus. Labeled terminals contained round, clear vesicles (Fig. 3D) or a combination of round, clear and dense-cored vesicles (Fig. 3A and B). In Fig. 3A the labeled axon terminal is seen in close apposition to a labeled soma while in Fig. 3B the labeled terminal is apposed to a labeled dendrite. No synaptic densities are associated with the membranes which are included in the plane of section in these examples. In Fig. 3C, however, a heavily labeled axon terminal is apposed to a lightly labeled dendrite. An obliquely sectioned synaptic density can be observed at the margin of the terminal. In Fig. 3D and E, a labeled axon terminal is shown which clearly forms an asymmetrical synaptic contact with a large labeled dendrite. In control animals in which HRP was injected into the nodose ganglion alone, anterograde terminal labeling was seen in the AP and NTS at the EM level, but no labeling was seen in any ~stsynaptic structures, DISCUSSION
The existence of a projection from the AP to the PBN has been reported from several laboratories (34, 45, 49). Due to inconsistent findings it was argued in one study in cat (27) that HRP labeling observed in the AP following injections of the enzyme into the PBN could not be construed to be proof of a connection between the two structures. In contrast to that study, we consistently observed HRP neuronal labeling in the AP at the light microscopic level, using both bright- and darkfield illumination. Our electron microscopic studies confirmed that HRP reaction product was located in neurons and their processes in the AP. Moreover, a number of other studies at the light microscopic level have shown a large number of retrogradely labeled neurons in the AP following injection of HRP into the PBN in both cat (34) and rat (45,49). In fact, in rat it has been suggested (49) that the majority of neurons in the AP project to the PBN. The present study has confiied a major efferent pathway from the AP and the NTS to the PBN in cat. Retrogradeiy labeled neurons were observed ~oughout the rostr~audal extent of the NTS in most subnuclei including the commissural, medial, lateral, intermediate and interstitial nuclei and in the SNG. The largest number of retrogradely labeled neurons were observed in the medial subnucleus and the SNG. In the AP retrogradely labeled neurons were mainly confined to the caudal %‘s of this region while the rostra1 % contained few or no retrogradely labeled neurons. The distinct caudo-rostral ~s~bution within the AP observed in this study has not been reported previously in cat, but has been described in rat (45). Retrogradely labeled cell bodies in the AP which project to the PBN were found to be small in size, ranging from 7-12 pm in
STRAIN, GWYN, RUTHERFORD
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diameter, and occurred singly or in groups in the neuropil. These data correspond to a previous description of normal neurons in the AP of the cat (28). Previous light microscopic studies in monkey (3,19), cat (l&24) and rat (32) have reported a vagal projection to the AP. In cat it has been reported that vagal projections to the AP arise from the trachea, lungs, heart and stomach (25). Using a combined retrograde and anterograde electron microscopic HRP labeling technique, the present study has demonstrated that vagal afferent fibers synapse directly upon neurons in the AP which in turn project to the PBN. Such vagal axon terminals contain round, clear vesicles or mixed populations of clear vesicles and dense-cored vesicles. No pleomorphic vesicle containing terminals were observed in the present study, although the existence of terminals containing pleomorphic vesicles has been reported in the AP of the rat (30). Many labeled vagal terminals were seen apposed to labeled neuronal somata or dendrites. By comparison, far fewer labeled vagal terminals were observed forming distinct synaptic contacts with labeled postsynaptic elements, although such synaptic relationships were seen in each animal examined. Thus, while there is clear evidence of a vagal projection to neurons in the AP which project to the PBN in the cat, the magnitude of the projection is uncertain. The ultrastructure of vagal sensory terminals in the AP is similar to that previously described for vagal terminals in the NTS. Following vagotomy or HRP labeling, vagal axon terminals in the
AND LOSIER
subnucleus gelatinosus of the NTS (32,33), the medial subnucleus of the NTS (20) and the commissural subnucleus of the NTS (10) were found to contain mainly round, clear vesicles and a few dense-cored vesicles. In an autoradiographic study in rat, it was reported that vagal sensory terminals in the caudal NTS contained small round, clear vesicles and large dense-cored vesicles (46). That study further showed that vagal terminals formed predominantly axodendritic synapses, a feature which was also observed in the present study. In the rat, a very small number of retrogradely labeled neurons were found in the PBN following an injection of HRP into the AP [see (45), Fig. 4B]. In a study in the cat in which a silver degeneration technique was employed, a projection from the PBN to the NTS dorsolateral to the tractus solitarius was shown; however, no projection to the AP was described or illustrated (47). In the present study, following injections of WGA-HRP into the PBN in a total of ten cats, no anterograde labeling was seen in the AP at either the light or electron microscopic level. We conclude that at least in the cat the AP is not reciprocally connected to the PBN. ACKNOWLEDGEMENTS
The authors thank Ms. Christine Anjowski for typing the manuscript and Mr. Frank Sasinek for assistance with the photography. This work was supported by a grant from the M.R.C. of Canada.
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Brain Research &&tin, Vol. 24, pp. 457-163. @Pergamon Press plc, 1990. Printed in the U.S.A.
Direct Vagal Input to Neurons in the Area Postrema Which Project to the Parabrachial Nucleus: An Electron Microscopic-HRP Study in the Cat’ S. M. STRAIN, D. G. GWYN,’
J. G. RUTHERFORD AND B. J. LOSJER
Received 22 September 1989
STRAIN, S. M., D. G. GWYN, J. G. RUTHERFORD AND B. J. LOSIER. LXrect vagaf input to netcrons in the areupostrema which project to the purabrachiaf nucleus: An electron microscopic-HRP study in the cut. BRAIN RES BULL 24(3) 457-463, 1990. -This study in cat examines the synaptic relationship of vagal afferents to parabrachial projecting neurons in the area postrema (AP) using anterograde and retrograde transport of horseradish peroxidase (HRP). Wheat germ agglutinin-HRP injected into the parabrachial nucleus (PBN) produced retrogradeneuronal labeling in the AP and in the nucleus of the tractus solitarius bilaterally, but with an ipsilateral predominance. Labeled neurons were confined mainly to the caudal s’s of the AP. Following injection of WGA-HRP into the PBN and HRP into the nodose ganglion in the same animal, examination of sections of the AP with the electron microscope revealed anterogradely labeled axon terminals in apposition to retrogradely labeled somata and dendrites. In some instances, labeled terminals were observed to form synaptic contacts with retrogradely labeled neurons. We conclude that in the cat a vagal input to neurons in the AP is monosynaptic~ly relayed to the PBN.
Area postrema
Parabrachial nucleus
Vagus nerve
Cat
THJZarea postrema (AP) is a highly vascular structure lying in the floor of the caudal part of the IV ventricle (30). Several functions have been attributed to the AP including the regulation of cardiovascular function (15,17) and the control of food intake (12,48). In addition there is evidence to suggest a role for the AP as a chemoreceptive trigger zone for emesis (4, 5, 8, 9). Since capillaries with the AP are fenestrated (14, 28, 30) and therefore are permeable to circulating substances, it is possible for chemical stimuli to impinge upon the AP via the blood stream as well as via the cerebrospinal fluid. In addition, afferent nerve fibers terminating in the area convey information to the AP from peripheral nerves and from a variety of regions within the central nervous system. The neural connections of the AP have been extensively studied at the light microscopic level, by ourselves and by others [see (3 1,45)]. The AP has been found to receive sensory fibers from the vagus nerve (3, 11, 18, 19, 24, 32) and the glossopharyngeal nerve (I I) with specifically identified input from the aortic nerve (26) and the carotid sinus nerve (13). Descending projections to the AP have been shown to arise from the dorsomedial hypothalamus in rat (23,45) and from the paraventricular hypothalamic nucleus in cat (22) and rat (45). Neurons in the AP project to the ventrolateral medulla (7,45),
Horseradishperoxidase
Electron microscopy
to the subjacent nucleus of the tractus solitarius (NTS) and to the
dorsal motor nucleus of the vagus (45). The major ascending projection of the AP is to the parabrachial nucleus (PBN) (29, 34, 45, 49). The PBN, in turn, appears to be a focal point for the distribution of information derived from the tongue and from visceral sources. The PBN has been shown to be involved in the regulation of cardiovascular (39) and respiratory activity (6) and gives rise to projections to the cerebral cortex, the amygdala, the thalamus and the hy~~~arnus (1641, 43,44). In the rat electrical stimulation of the cervical vagus nerve has demonstrated a second order vagal input to the PBN (21). The purpose of the present study is to determine in the cat whether vagal afferent Bbres synapse directly upon neurons in the AP which project to the PBN. Such a cotmection would provide a route whereby a variety of visceral sensory impulses could be distributed to such regions as the insular cortex, the amygdala and the thalamus. To achieve this goal horseradish peroxidase (HRP) was injected into the nodose ganglion and lectin-conjugated horseradish peroxidase (WGA-HRP) was injected into the PBN in the same animal. The AP was then examined in the electron microscope for the presence of anterogradely labeled vagal terminals synapsing upon retrogradely labeled PBN-p~jecting neurons.
‘A preliminary report of this study was presented at the Canadian Federation of Biological Societies, Winnipeg, 1987. ‘Requests for reprints should be addressed to Dr. D. G. Gwyn.
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AND LOSIER
METHOD In all of the operative procedures described below, animals were initially anesthetized with an intravenous injection of sodium pentobarbital and then were maintained on a halothane-oxygen mixture delivered by a respirator. In order to determine the distribution of neurons in the AP which project to the PBN, unilateral injections of 0.08 to 0.12 ~1 of a 5.0% aqueous solution of lectin-conjugated horseradish peroxidase (WGA-HRP) containing 0.1% Neutral red and 2.0% dimethyl sulfoxide (DMSO) were made stereotaxically in the PBN of 5 cats. Following survival periods of 3 or 4 days, the animals were anesthetized and perfused transcardially with normal saline followed by a fixative consisting of 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer (pH=7.4), both at room temperature. The fixative was washed out with cold (4°C) 10% phosphate buffered sucrose solution (pH = 7.4). The brain was exposed and blocked in the stereotaxic plane. Transverse, 40 pm frozen serial sections were cut through the injection site and transverse or sagittal, frozen serial sections were cut through the medulla. Sections were processed for the light microscopic demonstration of HRP activity using the tetramethylbenzidine (TMB) technique of Mesulam (36), and were then divided into three series. Two series were lightly stained with Neutral red, while the third series was left unstained. Sections were examined in the light microscope using both bright- and darkfield illumination; the distribution of retrogradely labeled neurons in the AP and the extent of the injection site were plotted with the aid of a camera lucida. In a further 5 cats, unilateral injections of 0.08 to 0.12 (*l of 5.0% WGA-HRP were placed in the PBN, and the animals were allowed to survive for 3 days. The tissue was processed for electron microscopic demonstration of reaction product in retrogradely labeled neurons in the AP, using the protocol described below. In 3 cats, between 0.10 to 0.12 p.1 of a 5.0% aqueous solution of WGA-HRP containing 0.1% Neutral red and 2.0% DMSO was stereotaxically injected into the right PBN. Following this procedure, the animals were removed from the stereotaxic frame, and the right nodose ganglion was exposed in the neck. Between 15 and 25 l.~l of a 30% aqueous solution of unconjugated HRP was injected into the ganglion in each animal. After a survival period of 3 days, animals were anesthetized and perfused transcardially with 1 1of phosphate buffered saline (pH = 7.4) followed by 3 to 4 1 of fixative consisting of 2.0% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer at 4°C. The brain was exposed and blocked in the stereotaxic plane. Blocks containing the injection site and the nodose ganglion were placed in the 10% buffered sucrose solution, while the blocks which included the AP were placed in 0.1 M phosphate buffer. Both groups of tissue blocks were stored overnight at 4°C. Frozen, transverse serial sections (40 pm) were cut through the injection site in the PBN and 40 p,m longitudinal sections were cut through the nodose ganglia. These sections were processed for light microscopic demonstration of HRP activity as described above. Transverse
FIG. 1. (A) A light micrograph of a transverse section through the pons showing the extent of the WGA-HRP injection site in DC 369. PB, = Lateral parabrachial nucleus, PB, = Medial parabrachial nucleus, KF = KGlliker-Fuse nucleus. Bar= 1 .O mm. (B through D). Light micrographs of transverse sections through the medulla, arranged from caudal (B) to rostra1 (D) levels, which illustrate the distribution of retrogradely labeled neurons in the area postrema (AP) and the nucleus of the tractus solitarius (NTS) ipsilateral to the injection shown in (A). Note the paucity of labeled neurons in the rostra1 AP (D). SNG=Subnucleus gelatinosus, TS= Tractus solitarius, X = Dorsal motor nucleus of the vagus. Bar = 100 km.
VAGAL, INPUT TO PBN PROJECTING
NEURONS
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PAGE
FIG. 2. (A) A light micrograph of a transverse section through the pons showing the extent of the WGA-HRP injection site in DC 396. Abbreviations as in Fig. 1A. Bar= 1 .O mm. (B) An electron micrograph showing HRP retrogradely labeled neurons in the AP resulting from the injection illustrated in A. The labeled neurons are indicated by an asterisk (*), while the crystals characteristic of the TMB reaction for HRP are indicated by arrowheads. The open block arrows indicate the extensive extracellular spaces typical of the AP. G= Golgi apparatus, N=Nissl substance, Nu -Nucleus. Bar= 5.0 km. (C) The area included within the rectangle in B is shown here at a higher magnification. TMB crystals are indicated by block arrows. Bar= 1 .O pm. (D) An axon terminal (AT) containing round, clear and dense-cored (arrowhead) vesicles is shown contacting (arrow) a labeled soma in the AP of DC 396. The TMB reaction product in the soma is indicated by a solid block arrow. Bar=0.5 pm. (E) An axon terminal (AT) containing round, clear vesicles and forming a synapse (arrow) upon a labeled dendrite (Dn) is shown in the AP of DC 396. The TMB reaction product is indicated by a solid block arrow. Bar=OS pm.
FIG. 3. (A) A labeled axon terminal (AT) containing round, clear vesicles and a single dense-cored vesicle (arrowhead) is shown in close apposition to a labeled soma (S) in the AP of DC 400. The TMB reaction product is indicated by solid block arrows. (B) A labeled axon terminal (AT) containing round, clear and dense-cored vesicles (arrowheads) is shown in close apposition to a labeled dendrite (Dn) in the AP of DC 416. Solid block arrows indicate the TMB reaction product. (C) A heavily labeled axon terminal (AT) is seen in apposition to a labeled dendrite (Dn) in the AP of DC 416. The TMB reaction product is indicated by solid block arrows, while the small arrow indicates the region of an obliquely sectioned synaptic density between the labeled preand postsynaptic elements. (D) A labeled axon terminal (AT) is shown synapsing upon a large labeled dendrite (Dn) in the AP of DC 416. Solid block arrows indicate the TMB reaction product in both the terminal and the dendrite. (E) The axon terminal in D is shown here at a higher magnification. The small arrows indicate the location of the postsynaptic densities, while the TMB reaction product is indicated by the solid block arrows. Bars =O.S pm.
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VAGAL INPUT TO PBN PROJECTING NEURONS
vibratome sections (100 to 125 pm) were cut through the AP. These sections were processed using a modification of Mesulam’s (36) TMB technique for revealing HRP activity at the electron microscopic level. The region including the AP and the NTS were cut out of each section. The excised tissue was osmicated, stained with saturated aqueous many1 acetate, and embedded in TAAB resin. Thick (1 .O pm) sections were cut and examined in the light microscope to determine the location of the AP. Using camera lucida drawings of these sections as a guide, blocks were retrimmed to include only the AP and NTS. Thin sections were then cut from the blocks, stained with lead citrate, and examined in a Zeiss EM 10 electron microscope for the presence of labeled axon terminals in contact with retrogradely labeled neurons. As a control between 20-40 p1 of 30% HRP was unilaterally injected into the nodose ganglion in three cats. Tissue from these cases was processed as described above for the doubly injected animals. Sections through the AP were examined in the electron microscope for the presence of any transsynaptic labeling of
RESULTS
In five cases the distribution of neurons in the AP retrogradely labeled from the PBN was studied. The results from one cat, DC 369, will be presented in detail because these are representative of our findings in general. The central zone of the injection extended throughout the length of the PBN and included the medial and lateral parabrachial nuclei (PB, and PB, , respectively) and the Kolliker-Fuse nucleus (KF). The peripheral zone of the injection site extended into the region of the locus ceruleus and the dorsal division of the principal sensory nucleus of the trigeminal nerve, while at rostral levels, a small amount of HRP spread into the region of the dorsal nucleus of the lateral lemniscus. In the AP, retrogradely labeled neurons were found bilaterally, but in far greater numbers ipsilateral to the injection site. Labeled neurons were most abundant in the caudal 2/3’sof the area (Fig. 1B and C), while very few labeled cells were found throughout the rostral % of the AP (Fig. 1D). In addition, labeled neurons were distributed bilaterally, though with an ipsilateral predominance, throughout the length of the NTS (Fig. IB-D). While present in most of the subnuclei of the NTS, HRP-positive cells were most numerous in the subnucleus gelatinosus (SNG) and the medial subnucleus. No evidence of anterograde terminal labeling was found in either the APortheNTS. Four of five animals which received unilateral injections of WGA-HRP into the PBN and which were subsequently processed for electron microscopy demonstrated retrograde labeling in the AP. Representative findings from one of these four cases (DC 396) are illustrated in Fig. 2. The injection site in this animal, as in the other three positive cases included both the PB, and PB, , as well as the KF (Fig. 2A). Some spread of the HRP into the locus ceruleus occurred. In the AP, retrogradely labeled neurons could be identified easily by the presence in the cytoplasm of darkly staining crystalline reaction product (Fig. 2C). The somata of labeled neurons were small (7 to 12 pm in diameter), and were scattered, singly or in groups of two or three, throughout the neuropil (Fig. 2B). The nuclear envelope of labeled neurons was frequently infolded and the cytoplasm contained the usual complement of organelles, including rough endoplasmic reticulum, Golgi apparatus and lysosomes. Labeled somata received synapses (Fig. 21)) typically from axon terminals which contained round, clear vesicles or a combination of round, clear and dense-cored vesicles. Terminals containing pleomorphic vesicles were not observed in these preparations. HRP reaction product was also found in dendrites in the AP (Fig. 2E). Axon terminals forming
synapses with labeled dendrites contained round, clear vesicles or both round, clear and dense-cored vesicles, although terminals containing only round, clear vesicles were more commonly encountered than those with a mixed population of vesicles (Fig. 3E). The diameter of round, clear vesicles ranged from 30 to 50 nm, while dense-cored vesicles were 60 to 100 nm in diameter. No labeled axon terminals were encountered in the AP or NTS in any of the four cases comprising this portion of the study. In all three cases in which WGA-HRP was injected into the PBN and HRP was injected into the nodose ganglion, many labeled terminals were seen in the AP in apposition to labeled profiles, most of which were dendritic in nature. Results from two of these cases (DC 400 and DC 416) are illustrated in Fig. 3. The injection sites in both of these animals were similar to those shown in Figs. 1 and 2. The HRP injections were centered on the PBN, with some spread into surrounding structures such as the dorsal principal sensory and motor nuclei of the trigeminal nerve, and the dorsal nucleus of the lateral lemniscus. Labeled terminals contained round, clear vesicles (Fig. 3D) or a combination of round, clear and dense-cored vesicles (Fig. 3A and B). In Fig. 3A the labeled axon terminal is seen in close apposition to a labeled soma while in Fig. 3B the labeled terminal is apposed to a labeled dendrite. No synaptic densities are associated with the membranes which are included in the plane of section in these examples. In Fig. 3C, however, a heavily labeled axon terminal is apposed to a lightly labeled dendrite. An obliquely sectioned synaptic density can be observed at the margin of the terminal. In Fig. 3D and E, a labeled axon terminal is shown which clearly forms an asymmetrical synaptic contact with a large labeled dendrite. In control animals in which HRP was injected into the nodose ganglion alone, anterograde terminal labeling was seen in the AP and NTS at the EM level, but no labeling was seen in any ~stsynaptic structures, DISCUSSION
The existence of a projection from the AP to the PBN has been reported from several laboratories (34, 45, 49). Due to inconsistent findings it was argued in one study in cat (27) that HRP labeling observed in the AP following injections of the enzyme into the PBN could not be construed to be proof of a connection between the two structures. In contrast to that study, we consistently observed HRP neuronal labeling in the AP at the light microscopic level, using both bright- and darkfield illumination. Our electron microscopic studies confirmed that HRP reaction product was located in neurons and their processes in the AP. Moreover, a number of other studies at the light microscopic level have shown a large number of retrogradely labeled neurons in the AP following injection of HRP into the PBN in both cat (34) and rat (45,49). In fact, in rat it has been suggested (49) that the majority of neurons in the AP project to the PBN. The present study has confiied a major efferent pathway from the AP and the NTS to the PBN in cat. Retrogradeiy labeled neurons were observed ~oughout the rostr~audal extent of the NTS in most subnuclei including the commissural, medial, lateral, intermediate and interstitial nuclei and in the SNG. The largest number of retrogradely labeled neurons were observed in the medial subnucleus and the SNG. In the AP retrogradely labeled neurons were mainly confined to the caudal %‘s of this region while the rostra1 % contained few or no retrogradely labeled neurons. The distinct caudo-rostral ~s~bution within the AP observed in this study has not been reported previously in cat, but has been described in rat (45). Retrogradely labeled cell bodies in the AP which project to the PBN were found to be small in size, ranging from 7-12 pm in
STRAIN, GWYN, RUTHERFORD
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diameter, and occurred singly or in groups in the neuropil. These data correspond to a previous description of normal neurons in the AP of the cat (28). Previous light microscopic studies in monkey (3,19), cat (l&24) and rat (32) have reported a vagal projection to the AP. In cat it has been reported that vagal projections to the AP arise from the trachea, lungs, heart and stomach (25). Using a combined retrograde and anterograde electron microscopic HRP labeling technique, the present study has demonstrated that vagal afferent fibers synapse directly upon neurons in the AP which in turn project to the PBN. Such vagal axon terminals contain round, clear vesicles or mixed populations of clear vesicles and dense-cored vesicles. No pleomorphic vesicle containing terminals were observed in the present study, although the existence of terminals containing pleomorphic vesicles has been reported in the AP of the rat (30). Many labeled vagal terminals were seen apposed to labeled neuronal somata or dendrites. By comparison, far fewer labeled vagal terminals were observed forming distinct synaptic contacts with labeled postsynaptic elements, although such synaptic relationships were seen in each animal examined. Thus, while there is clear evidence of a vagal projection to neurons in the AP which project to the PBN in the cat, the magnitude of the projection is uncertain. The ultrastructure of vagal sensory terminals in the AP is similar to that previously described for vagal terminals in the NTS. Following vagotomy or HRP labeling, vagal axon terminals in the
AND LOSIER
subnucleus gelatinosus of the NTS (32,33), the medial subnucleus of the NTS (20) and the commissural subnucleus of the NTS (10) were found to contain mainly round, clear vesicles and a few dense-cored vesicles. In an autoradiographic study in rat, it was reported that vagal sensory terminals in the caudal NTS contained small round, clear vesicles and large dense-cored vesicles (46). That study further showed that vagal terminals formed predominantly axodendritic synapses, a feature which was also observed in the present study. In the rat, a very small number of retrogradely labeled neurons were found in the PBN following an injection of HRP into the AP [see (45), Fig. 4B]. In a study in the cat in which a silver degeneration technique was employed, a projection from the PBN to the NTS dorsolateral to the tractus solitarius was shown; however, no projection to the AP was described or illustrated (47). In the present study, following injections of WGA-HRP into the PBN in a total of ten cats, no anterograde labeling was seen in the AP at either the light or electron microscopic level. We conclude that at least in the cat the AP is not reciprocally connected to the PBN. ACKNOWLEDGEMENTS
The authors thank Ms. Christine Anjowski for typing the manuscript and Mr. Frank Sasinek for assistance with the photography. This work was supported by a grant from the M.R.C. of Canada.
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