Neuronal Connections of a Ventral Brainstem
ELMOND J. JOHNSON
College of Medicine
Several investigations have implicated neurons at
the ventral brainstem in the central chemical regulation of breathing
(1--3). This chemosensitive region lies ventral to the inferior
olives at the level where the hypoglossal rootlets exit the brainstem
and may be reached by fluids superfusing the ventrolateral medullary
surface (VMS) (Fig. 1).
Electrical stimulation of the region of maximal
chemosensitivity (area L) in the cat resulted in a marked increase
in ventilation (2,4). Berndt et al. (5) demonstrated that
superfusion of this area with 2% procaine solution in peripheral
chemoreceptor--denervated animals completely abolished the ventilatory
response to electrical stimulation, causing respiratory arrest.
However, electrical stimulation of the respiratory center in these
animals still elicited an inspiratory response. It appears, therefore,
that these superficial structures might be essential in driving
the respiratory center in anesthetized cats in the absence of peripheral
C-fos and other immediate-early genes, in response to various physiological and pharmacological stimuli, induce concomitant nuclear binding proteins that appear to play a role in signal transduction and to function as intracellular third messengers. Sato et al. (6) described the distribution of the c-fos in cells of the ventral medulla oblongata that were responsive to 15% inspired CO2.
Immunocytochemical evaluation of the tissue revealed
c-fos--positive neurons that were located within 150 ~m
of the VMS in the caudal ventrolateral medulla oblongata (cVLM);
with a majority of cells within the first 50 ~m.
Seven rats weighing 300 to 400 g, anesthetized with
chloralose urethane (200 mg/kg glucochloralose and 400 mg/kg urethane)
were used. The musculi longi capitis and recti capitis ventrales,
as well as the pars basalis os occipitale, the ventral portion of
the atlas, and the processus odontoideus of the epistropheus were
removed. The dura was then opened and reflected laterally to expose
the ventral surface of the medulla oblongata. Rectal temperature
was maintained constant at 370C. The animals were then
placed in the supine position with the head stabilized in a stereotaxic
Following surgical exposure of the ventral brainstem
surface, a solution of 0.02 to 0.05 ML of 4% HRP (Sigma VI) was
injected into the caudal chemosensitive area on the VMS with the
aid of a micromanipulator in a stereotaxic coordinate system. A
1 -ML Hamilton syringe was used. At 48 h postinjection of HRP, the
head of the animal was perfused and 40 ~m tissue sections were processed
for the TMB reaction product according to the protocol of Mesulam
(7). In another series of experiments, wherever anterograde labeling
was noted in the present study, microinjections of HRP were made
to validate the presence of projections to that site, for example
the nucleus ambiguus (NA).
In Fig. 2, the injection site was located in the
cVLM overlying the exit of the hypoglossal (XII) rootlets. Only
those cases where the injection was confined to the VMS were chosen
for this report. A small ipsilateral projection appears to exist
between the NA [or ventral respiratory group (VRG)] and the VMS.
Light deposits of HRP reaction product could also be visualized
in a few small, oval neurons of the ventrolateral nucleus tractus
solitarius (NTS) -- that is, the dorsal respiratory group (DRG),
Ipsilateral to the cVMS injection site, heavy labeling
with HRP reaction product occurred in the nerve XII fibers traversing
the medulla and in the large, multipolar neurons of the hypoglossal
nucleus. This may represent uptake of HRP from the surrounding medium
by nerve XII fibers. Occasional large, multipolar neurons containing
HRP were also noted within the medullary reticular nucleus in the
vicinity of the fibers of XII and in the lateral reticular nucleus
(LRtN) (Fig. 3).
Contralateral to the injection site, HRP reaction product was detected in a few unique areas; the parvocellular region of the lateral reticular nucleus (LRt-PC), the ventrolateral and dorsolateral aspects of the inferior olivary nucleus (10) and along the VMS. Small to medium sized neurons located in the contralateral medial and ventrolateral 10 as well as superficial neurons within the thickened marginal glia (TMG) were variably labeled with HRP. A few large multipolar neurons within the contralateral LRt-PC were heavily retrogradely labeled with HRP and fibers emanating from their somata would be visualized directed toward the injection site (Fig. 4). Mesulam (7) and Kahlia and Wells (8) have demonstrated the usefulness of using TMB as the substrate for HRP histochemistry in studies where the anterograde transport of HRP is desirable. In these studies, a few tortuous fibers and axon terminal fields containing anterogradely labeled HRP could be detected within the NA, LRt-PC, and along the VMS, indicating that reciprocal connections exist between VMS,ambigual, and LRt-PC neurons. Verification studies were subsequently performed by microinjection of HRP into sites demonstrating anterograde labeling. These observations agree with the reports of Norgren (9), who, with the use of tritiated proline or leucine, described projections emanating from the NA that project to the NTS. Indeed, Gallagher and Pert (10) and Walberg (11) have found that extensive reciprocal connections exist between reticular nuclei. Neurovascular elements, variably labeled with HRP, were noted in superficial regions of the medulla. These cells that are in close contact with blood vessel walls are reminiscent of the neurovascular elements described by Scheibel and Scheibel (12) and Scheibel et al. (13).
Regions of respiratory chemosensitivity that respond to cerebrospinal fluid pH changes and inspired carbon dioxide have been described on the VMS. In this investigation, HRP, a neuronal tracer, was utilized to ascertain the possible connections between the VMS and the deeper-lying respiratory structures. In spontaneously breathing, chloralose-urethane anesthetized rats, 0.02 to 0.05 ML of 4% HRP was microinjected into the cVMS via a l-ML Hamilton syringe mounted on a stereotaxic coordinate system. The animals were maintained for a minimum period of 48 h to allow for the axonal transport of HRP. The injection site was located over the hypoglossal rootlets and less than 500 ~m into the caudal VMS. The results reveal that the majority of neuronal connections with the caudal VMS arise (retrogradely) from (1) Ipsilaterally: lateral reticular nucleus, the nucleus ambiguus, and the nucleus tractus solitarius. HRP reaction product was also visualized within the nerve fibers and large multi-polar neurons of the hypoglossal nucleus. (2) Contralaterally: 10 nuclei and the parvocellular region of the LRtN (with a few neurons that have extremely long processes directed towards the midline). Neuronal connections were also identified by anterograde labeling, which indicated the presence of projections arising from the cVMS that may impinge on the respiratory centers. This investigation demonstrates that (a) superficial neuronal elements project to deeper-lying respiratory structures such as the NA and NTS; (b) bilateral connections exist between the LRt-PC nuclei; (c) possible bilateral connections between lateral inferior olivary nuclei and VMS cells, and (d) neurovascular elements may play a role in respiratory control mechanisms within the ventrolateral medulla oblongata. It is therefore possible that the lateral horns of the 10, the parvocellular regions of the LRtN, and superficial neurons within the TMG are involved in higherorder processing within the central respiratory chemoreceptor reflex pathway.
This work was supported by NIH NIGMS Grant No. S 06 GM 08016.
1. Mitchell RA, Loeschcke HH, Massion WH, Severinghaus
JW. Respiratory responses mediated through superficial chemosensitive
areas on the medulla. J Appl Physiol 1963; 18:523-533.