Numerous GABAergic afferents to locus ceruleus in the pericerulear dendritic zone: possible interneuronal pool

Abstract

Most nuclei in the CNS are composed of principal neurons that project to other areas and interneurons that serve to integrate information among afferents. The noradrenergic brain nucleus locus ceruleus (LC) has appeared to be an exception to this general rule, because the LC is composed almost entirely of noradrenergic principal neurons. Here, we report that numerous small neurons in the peri-LC region become retrogradely labeled after focal injections of wheat germ agglutinin-apo (inactivated) horseradish peroxidase conjugated to colloidal gold, or pseudorabies virus (PRV), into the nuclear core of the rat LC. A substantial number of these neurons were routinely found within the dendritic field of the LC, in the area surrounding the compact cell-dense region classically defined as LC. Double labeling revealed that a large percentage of these cells stained for GABA. Ultrastructural analyses revealed axodendritic and axosomatic contacts between PRV-labeled afferents and LC neurons labeled with tyrosine hydroxylase immunohistochemistry. In addition, PRV-labeled neurons or axons were immunopositive for GABA in ultrastructural localizations. Analysis of the synaptology of immunopositive profiles demonstrated that these LC afferents in the peri-LC region receive several non-LC synaptic inputs. These results indicate that a population of small GABAergic neurons in the peri-LC dendritic zone may provide interneuronal integration for LC noradrenergic neurons.

Figure 6.

Method used to identify peri-LC region for ultrastructural analyses. Systematic analysis of sections from Vibratome and ultramicrotome allowed unambiguous identification of peri-LC region in EM analyses. A, B, Photomicrographs of a transilluminated quasi-horizontal 40 μm thick Vibratome section through the LC at 35 hr survival after a focal microinjection of PRV in the LC nucleus. This section is doubly stained for PRV (DAB, brown) and TH (silver-intensified gold, black). The trapezoid defined in A illustrates the rostroventral peri-LC region sectioned for subsequent ultrastructural analyses, shown at higher magnification in B. Note the numerous small PRV-labeled neurons among TH dendrites in the peri-LC. Rostroventral is down, medial is left; the fourth ventricle is at the top left. Arrows indicate the same cells in both A and B. C, Toluidine blue-counterstained 0.35 μm semi-thin section from the trapezoid identified in A. Arrows indicate the same cells as in A and B. D, Ultrathin (70 nm) section adjacent to the semi-thin section shown in C. Scale bars: A, 100 μm; B, C, 50 μm; D, 10 μm.

Figure 1.

LC neurons have extensive extranuclear dendrites. Dark-field photomicrographs of frontal sections through the rat LC stained with an antibody against dopamine β hydroxylase (DBH) showing extensive extranuclear NE dendrites of LC neurons, arranged from caudal (A) to rostral (D). These levels match those in Figure 2. Previous ultrastructural analyses of the extranuclear DBH+ processes demonstrated that >90% are dendrites (Shipley et al., 1996). Ventral is down, medial is left for all panels; the fourth ventricle is at the top left. Scale bar, 500 μm.

Figure 2.

Injection of WGA-Au into LC reveals local afferents. Photomicrographs of frontal sections through the LC showing a WGA-Au injection (A, B, black) confined to within the mid-LC and caudal LC nucleus. NE neurons in LC are stained with an antibody against TH (brown). Note the focal WGA-Au injection restricted to the LC nucleus. Note also the retrogradely labeled darkly stained neurons among the LC dendrites, most apparent among the LC extranuclear dendrites in C and D. The distribution of these neurons is primarily coextensive with that of the LC dendrites. Scale bar, 200 μm.

Figure 3.

GABAergic neurons and terminals in peri-LC. A, Fluorescence photomicrograph of a frontal section through the level of the mid-LC showing GAD+ (presumably GABAergic; Alexa Fluor 594; red) and TH+ (Alexa Fluor 488; green) neurons and processes in the peri-LC and LC nuclear core. This section was stained to emphasize the GAD+ staining of somata (no Triton X-100 pretreatment). Note the numerous GAD+ neurons adjacent to the ventromedial NE neurons and the dense GAD+ terminals in both peri-LC and the LC nucleus (more clearly revealed in B). Medial is to the left, dorsal is up. The fourth ventricle is at the top left. B, Dark-field photomicrograph through the same area as in A from a Vibratome section processed to emphasize GAD+ fibers and terminals (0.3% Triton X-100 pretreatment). Note the dense GAD+ innervation in the peri-LC and the LC nuclear core. LC neurons are visible as dark unstained somata in the upper right. The orientation for both panels is identical. Scale bar, 50 μm.

Figure 4.

LC afferents in peri-LC zone stain for GAD. Photomicrographs of frontal sections through the ventral LC and ventromedial peri-LC showing neurons labeled for GAD and the retrograde tracer WGA-Au after injection into the LC nuclear core. Arrows indicate neurons stained for both WGA-Au and GAD. A, B, Bright-field photomicrographs of mid- (A) and rostral (B) peri-LC areas stained for both GAD (brown) and the retrograde tracer WGA-Au (black particulate) in a case with a focal WGA-Au injection in the LC nuclear core. C, D, High-power photos taken from areas in A and B. Note that WGA-Au-labeled LC afferents (filled with black particles) often also stained for GAD (diffuse brown stain). Arrows indicate neurons stained for both WGA-Au and GAD. For all panels, ventral is down, medial is left. Scale bars: (in A) A, B, 250 μm; C, D, 50 μm.

Figure 5.

Peri-LC labeling with PRV. Photomicrographs of frontal sections through the LC at 23 hr (A-D) or 35 hr (A′-D′) after focal microinjections of the trans-synaptic retrograde tracer PRV in the caudal LC nuclear core. A, A′, Injection sites in LC indicated by co-injected CTb. CTb was used for this because PRV is quickly transported from the site of injection, so that the injection site is best delineated with a co-injected agent. O'Donnell et al. (1997) demonstrated that CTb is taken up from a larger area than PRV after simultaneous injection, so the zone of PRV uptake would be smaller than that evident for CTb. Note the dense injection focused in the LC nuclear core in both cases. B-D, B′-D′, Sections through the caudal (B and B′), mid- (C and C′) and rostral (D and D′) LC at 23 or 35 hr of survival (B-D and B′-D′, respectively) after the injections indicated in A and A′. Note the greater number of small, labeled neurons in the peri-LC zone at 35 compared with 23 hr, particularly at rostral levels (D and D′). These findings indicate that there are two paths for labeling in the peri-LC: a nuclear path mediated by projections of peri-LC neurons into the LC nuclear core and a dendritic path mediated by peri-LC neuronal connections onto extranuclear LC dendrites. For all panels, ventral is down, medial is left; the fourth ventricle is at the top left. Scale bar, 200 μm.

Figure 7.

Ultrastructure of GABAergic LC afferents. EM photomicrograph of a neuron in the rostroventral peri-LC immunolabeled for PRV (stained with DAB) and GABA (stained with silver-intensified gold). High-power images in B and C are taken from areas indicated in the low-power image in A. Note the virions in the nucleus (N) at arrows in A and the nuclear inclusions in A and B, indicative of PRV infection (at arrowheads). Note also the cytoplasmic virions (indicated by arrows in B and C), and abundant GABA staining in the cytoplasm. Scale bars: A, 2 μm; B, C, 0.5 μm.

Figure 8.

GABAergic terminals contacting PRV- or TH-labeled dendrites. A, High-power EM photomicrograph of an area in the rostroventral peri-LC immunolabeled for PRV (stained with DAB) and GABA (stained with silver-intensified gold). The dendrite in the center contains PRV virions (arrowheads) and receives a symmetric synapse (long arrow) from a GABA+ terminal-containing pleomorphic vesicles. This is consistent with inhibitory influences of GABA neurons onto LC dendrites in the peri-LC region. B, High-power EM photomicrograph from an area in the rostroventral peri-LC immunolabeled with TH (stained with DAB) and GABA (stained with silver-intensified gold). The TH+ dendrite receives symmetric synaptic contacts (long arrows) from a GABA+ terminal. Note also that both the TH dendrite and the GABA terminal contain viral particles (arrowheads). This indicates PRV transfer from TH dendrites to GABA+ afferent terminals in the peri-LC region. The TH dendrite also receives an asymmetric synaptic input (short arrow) from an unlabeled terminal (ut). C, Similar section as in B, showing multiple synaptic contacts of a GABA terminal with a TH+ dendrite (arrows). Although the immunoperoxidase reaction in the postsynaptic dendrite makes classification of synapse symmetry difficult, it appears that two of the three contacts are symmetrical, whereas the postsynaptic density of the third contact is more prominent. Serial analysis would be required to make a definitive determination of the synapse morphology. Asterisks identify glial processes interposed between TH and GABA dendrites. D, High-power EM photomicrograph from an area in the rostroventral peri-LC immunolabeled with TH (stained with DAB) and GABA (stained with silver-intensified gold). A GABA terminal synapses onto a TH dendrite that contains PRV capsids (arrowheads). DAB reaction product underlies the symmetrical synaptic contact (long arrow). Scale bars, 0.5 μm.

Figure 9.

Peri-LC GABA neurons receive numerous synaptic inputs. High-power EM photomicrographs from the rostroventral peri-LC dendritic zone immunolabeled for GABA (stained with silver-intensified gold). A, A GABAergic dendrite receives synaptic input from GABA and non-GABA terminals. The contact from the GABA terminal is symmetrical in character (long arrow), whereas the unlabeled terminal (ut) forms an asymmetrical contact (short arrow). B, A GABAergic dendrite receives asymmetric synapses (short arrows) from unlabeled non-GABA terminals (ut). A labeled GABA terminal also forms a symmetrical contact with an unlabeled dendrite (ud). Both the GABAergic dendrite and GABAergic terminal contain PRV virions (arrowheads). Scale bars, 0.5 μm.