Kölliker–Fuse neurons send collateral projections to multiple hypoxia-activated and nonactivated structures in rat brainstem and spinal cord

Abstract

The Kölliker–Fuse nucleus (KFN) in dorsolateral pons has been implicated in many physiological functions via its extensive efferent connections. Here, we combine iontophoretic anterograde tracing with posthypoxia c-Fos immunohistology to map KFN axonal terminations among hypoxia-activated/nonactivated brainstem and spinal structures in rats. Using a set of stringent inclusion/exclusion criteria to align visualized axons across multiple coronal brain sections, we were able to unequivocally trace axonal trajectories over a long rostrocaudal distance perpendicular to the coronal plane. Structures that were both richly innervated by KFN axonal projections and immunopositive to c-Fos included KFN (contralateral side), ventrolateral pontine area, areas ventral to rostral compact/subcompact ambiguus nucleus, caudal (lateral) ambiguus nucleus, nucleus retroambiguus, and commissural–medial subdivisions of solitary tract nucleus. The intertrigeminal nucleus, facial and hypoglossal nuclei, retrotrapezoid nucleus, parafacial region and spinal cord segment 5 were also richly innervated by KFN axonal projections but were only weakly (or not) immunopositive to c-Fos. The most striking finding was that some descending axons from KFN sent out branches to innervate multiple (up to seven) pontomedullary target structures including facial nucleus, trigeminal sensory nucleus, and various parts of ambiguus nucleus and its surrounding areas. The extensive axonal fan-out from single KFN neurons to multiple brainstem and spinal cord structures (“one-to-many relationship”) provides anatomical evidence that KFN may coordinate diverse physiological functions including hypoxic and hypercapnic respiratory responses, respiratory pattern generation and motor output, diving reflex, modulation of upper airways patency, coughing and vomiting abdominal expiratory reflex, as well as cardiovascular regulation and cardiorespiratory coupling.

Appendix: Critique of methodologies

Microinjection

BDA is a proven sensitive and reliable anterograde tracer. With sufficient postinjection survival time (1–2 weeks), even very fine axonal terminals can be sufficiently filled for microscopic observation and long distance tracing. Retrograde labeling has also been reported but only after large injections. In the present study, labeled neurons were observed in areas surrounding the injection site (Fig. 1a) but rarely in other regions. However, BDA is not functionally specific and the KFN is functionally heterogeneous, i.e., besides the well-known pneumotaxic function, KFN is also known to participate in cardiovascular control, anti-nociception, and control of feeding and drinking. To increase the likelihood of labeling neurons that were respiratory-related, we first mapped the KFN with electrical microstimulation for loci that produced respiratory inhibition with the lowest stimulation intensity. Although such electrical microstimulation could activate neurons far from the tip of the stimulating electrode as a result of direct axonal activation (Histed et al. 2009), previous studies have shown that electrical or chemical microstimulation (with glutamate) of neurons in this structure cause similar profound changes in respiratory patterns (Chamberlin and Saper 1992; Cohen 1971; Dutschmann and Herbert 2006; Haji et al. 1998). Thus we believe that the electrical microstimulation-evoked respiratory inhibition in the present study was due to activation of KFN neurons in the vicinity of the electrode tip. Hence, BDA was injected only into such low-threshold regions. With this functionally guided method, we found that the centers of injections in all experimental animals were within the boundaries of KFN and the diffusion of the injectant into neighboring areas was quite limited in most cases. In contrast, failure to identify such low-threshold regions resulted in misplaced injections in three animals. Data from misplaced injections were not included in this report.

In light of the above precautions, we believe that the labeled axons were predominantly from respiratory-related KFN neurons because: (1) the KFN contains the highest density of respiratory-related neurons that send axons to ventrolateral pons and medulla (Ezure and Tanaka 2006; Song et al. 2006); (2) ascending projections from medullary respiratory-related structures predominantly terminate at this structure (Gaytan et al. 1997; Kalia 1977); (3) the highly selective termination of labeled terminals in well-established brainstem respiratory-related structures with c-Fos positive response to hypoxia as demonstrated in this study; (4) BDA injections at control sites neighboring the KFN resulted in very different projection/innervation patterns of the labeled axons. Although nonspecific labeling of axons of other functional modalities (e.g., cardiovascular) from KFN and a minority of axons from neighboring medial and lateral parabrachial nuclei or A7 region (especially for the A7 region) cannot be ruled out especially with pressure injection, useful information about the multi-functional role of the KFN can still be derived from this study.

Axonal tracing

Traditionally, retrograde tracing with a combination of fluorescent tracers of two or three different colors is commonly used to reveal the collateral innervations of a single neuron or axon. However, current fluorescent imaging techniques cannot differentiate the labeling of one neuron by more than three fluorescent tracers. As a result, no more than three innervations can be revealed at a time. In addition, this technique requires 2–3 microinjections at different brain structures, which is rather traumatic to the animal. Because of potential spread of the injectants into neighboring areas, distinct innervations of adjacent target structures cannot be reliably resolved by this method. When more than three structures or when two or more neighboring structures are innervated by branches from such an axon, single-axon anterograde tracing is the only currently available method to reveal these multiple innervations.

On the other hand, long distance anterograde tracing of individual axons over multiple brain sections is generally a challenging task because each section may contain many labeled axons that are difficult to align across consecutive sections. This difficulty has bedeviled early studies using pressure injection, each of which could potentially label hundreds of descending fibers even for a structure with sparse neuronal density such as KFN. To circumvent this difficult, such tracing was performed only in animals with iontophoretic injection in the present study. The number of descending axons labeled after iontophoretic injection varied greatly from five to over one hundred per animal depending on the duration and current intensity of the iontophoresis and the tip diameter of the micropipette. To avoid any ambiguity in the tracing, stem axons that were selected for long distance tracing and reported herein were all relatively large (1.0–2.3 μm diameter) and isolated, i.e., without any neighboring axons of comparable size in each and every section. Such large stem axons typically stood out readily among much smaller axonal branches and were readily identified in coronal sections since they traveled rostrocaudally throughout much of their trajectories from ventrolateral pons to caudal medulla and spinal cord. If the identified axon shifted significantly from its preceding coordinates on the coronal plane or if other axons of comparable size emerged in any section, the tracing was abandoned. These stringent inclusion/exclusion criteria ensured the unequivocal matching of the same stem axon over consecutive brain sections.

In contrast, thinner branches or collaterals arising from such stem axons in any section were typically traced only within the same section to avoid ambiguity; reconstruction of those branches from two or more consecutive sections was performed only when no neighboring fibers with similar diameter and direction of projection were observed in each section. For this reason, some branches could not be traced to their ultimate targets. Fortuitously, unlike the stem axons that projected lengthwise rostrocaudally, such branches or collaterals tended to be much shorter and confined to the coronal planes, with the extensions in sagittal planes rarely spanning beyond three consecutive sections.

With the above precautions, only 1–2 axons of relatively large diameters met our stringent inclusion/exclusion criteria and were traced for various distances in each animal (totally 10 axons in 7 animals). Many of the large-diameter axons could not be traced for a long distance because they followed a more zigzag path that was not always perpendicular to the coronal plane, or simply because there were multiple large-diameter axons in their proximity that confounded the tracing. On the other hand, none of the medium-sized axons was successfully traced for sufficient distance. Nevertheless, branches and collaterals were still observed arising from such axons. In light of these observations, we believe the collateral innervations of multiple target structures might be common (if not universal) among descending axons of both medium and large diameters although many of them might not reach the caudal medulla.