Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat

Abstract

The tuberomammillary nucleus (TMN) is the major source of histaminergic innervation of the mammalian brain and is thought to play a major role in regulating wake-sleep states. We recently found that sleep-active neurons in the ventrolateral preoptic nucleus (VLPO) provide a major input to the TMN, but the specificity of this projection and the neurotransmitters involved remain unknown. In this study, we examined the relationship of VLPO efferents to the TMN using both retrograde and anterograde tracing, combined with immunocytochemistry. We found that the descending projection from the VLPO selectively targets the cell bodies and proximal dendrites of the histaminergic TMN. In addition, VLPO axons could be traced into the brainstem, where they provided terminals in the the serotoninergic dorsal and median raphe nuclei, and the core of the noradrenergic locus coeruleus. Approximately 80% of the VLPO neurons that were retrogradely labeled by tracer injections including the TMN were immunoreactive either for galanin or for glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA. Virtually all of the galaninergic neurons in the VLPO were also GAD positive. Our results indicate that the VLPO may provide inhibitory GABAergic and galaninergic inputs to the cell bodies and proximal dendrites of the TMN and other components of the ascending monoaminergic arousal system. Because these cell groups are simultaneously inhibited during sleep, the VLPO sleep-active neurons may play a key role in silencing the ascending monoaminergic arousal system during sleep.

Fig. 1.

A series of bright-field photomicrographs illustrating various histological markers for identifying the ventrolateral preoptic nucleus. a shows the appearance of the VLPO in Giemsa-stained sections as a small triangular cluster of neurons along the ventral surface of the brain, with its lateral edge bordering the nucleus of the horizontal limb of the diagonal band.b illustrates the Fos-immunoreactive neuronal nuclei in the VLPO after a 1 hr period spent predominantly asleep. Inc, the VLPO is clearly demarcated as a galanin-immunoreactive cell group in a colchicine-pretreated animal.d shows an injection site of biotinylated dextran (BD) in case VLPO 11; the section is counterstained with Giemsa. The borders of the injection site correspond closely with the location of the cluster of Fos-positive and galanin-positive neurons seen in b and c. Scale bar (shown in b): a, 500 μm; b–d, 300 μm.

Fig. 2.

Summary of findings from a fluorogold injection into the core of the TMNv with no involvement of adjacent structures. a, Fluorescence photomicrograph of a caudal hypothalamic section stained immunocytochemically for adenosine deaminase, as visualized using a rhodamine filter cube. Immunoreactive neurons delineate the TMNv (arrow). b, Fluorescence photomicrograph of the same field, demonstrating Fluorogold fluorescence as seen through a UV filter cube, showing that the center of the injection is limited to and essentially demarcates the TMNv. c, Camera lucida drawing of a caudal preoptic section showing retrogradely labeled cells (eachasterisk represents one cell) produced by the injection in b. In this case, the vast majority of retrogradely labeled neurons were concentrated in the ventral portion of the lateral preoptic area. This pattern of labeling extended roughly 300 μm rostrally and 100 μm caudally from the level drawn. Scale bar (shown in c): a, b, 400 μm; c, 800 μm.

Fig. 3.

Summary of findings from three animals with CTB injections into the posterolateral hypothalamus demonstrating the topographical projections to this area from the preoptic area.a, Injection sites from these cases plotted onto a representative schematic of the TMNv in the caudal hypothalamus.b–d, Bright-field photomicrographs of preoptic sections at the level of the VLPO from these cases demonstrating the resultant pattern of retrograde label. Note that the VLPO contained a cluster of retrogradely labeled neurons after an injection that included the cell-dense core of the TMNv (J-39). However, few retrogradely labeled neurons were seen in the VLPO after injection of retrograde tracer into structures dorsomedially (J-10) or dorsolaterally (J-9) adjacent to the core of the TMNv. Scale bar, 700 μm.

Fig. 4.

A summary diagram illustrating biotinylated dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c, c′,d, and d′ (in light gray).Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).

Fig. 5.

A series of camera lucida drawings illustrating the pattern of axonal labeling in case VLPO 11 (see Fig. 1 for photomicrograph of injection site). Note dense and selective innervation of all parts of the histaminergic tuberomammillary nucleus as well as the selectivity of innervation of the dopaminergic ventral tegmental area, serotoninergic dorsal, median, pontine, and medullary raphe nuclei, cholinergic pedunculopontine and laterodorsal tegmental nuclei, and noradrenergic locus coeruleus.

Fig. 5.

A series of camera lucida drawings illustrating the pattern of axonal labeling in case VLPO 11 (see Fig. 1 for photomicrograph of injection site). Note dense and selective innervation of all parts of the histaminergic tuberomammillary nucleus as well as the selectivity of innervation of the dopaminergic ventral tegmental area, serotoninergic dorsal, median, pontine, and medullary raphe nuclei, cholinergic pedunculopontine and laterodorsal tegmental nuclei, and noradrenergic locus coeruleus.

Fig. 6.

A series of photomicrographs to illustrate the innervation of the TMN histaminergic neurons by the VLPO.a shows a dark-field photomicrograph of anterogradely labeled VLPO axons in the TMN (indicated by arrows). The same area is shown at higher magnification in a bright-field photomicrograph in b, to demonstrate the large numbers of labeled axons and terminals. A single retrogradely labeled neuron (arrow) is seen in the TMN. cdemonstrates the relationship of these axons to TMN cell bodies, in a different experiment in which the TMN neurons were stained immunocytochemically (brown) for adenosine deaminase. Individual black axons and terminals can be seen closely associated with immunoreactive cell bodies and dendrites (Nomarski optics). The neuron indicated by the arrow is illustrated at higher magnification and in a slightly different focal plane in d, receiving multiple appositions from a single axon. e shows a bright-field photomicrograph of a coronal section through the caudal hypothalamus demonstrating the center of an injection of gold-conjugated CTB in case J-78. This section was stained first for gold particles with a silver intensification procedure (black), followed by adenosine deaminase immunocytochemistry (brown) to identify histaminergic neurons. Note that the injection spreads dorsally above but manages to fill the rostral TMNv. This injection continues caudally into the heart of the TMNv. f and g show high-power bright-field Nomarski photomontages of VLPO neurons from case J-78, showing individual, retrogradely labeled neurons (black granular precipitate) that are GAD-immunoreactive (brown; f) or galanin-immunoreactive (brown; g). Photomontage was necessary to combine different focal planes to keep the CTB granules in focus. h–j illustrate the labeling of VLPO neurons with antisera against both galanin (h) and GAD (i). Note that because GAD antiserum also stains many axon terminals in the VLPO, this image is shown at much higher contrast to highlight the GAD-ir cell bodies, which appear as a cluster in the VLPO rather than as discrete cell bodies. Other GAD-positive cell bodies outside the VLPO are indicated by arrowheads. A double exposure inj demonstrates the double-labeled neurons in the VLPO as a bright gold color, whereas single-labeled neurons in the supraoptic nucleus (for galanin, arrow) and in the lateral preoptic area for (GAD, arrowheads) demonstrate that the antisera used do not cross-react. See Table 2. Scale bars:a, 250 μm; b, 50 μm;c, 25 μm; d, 10 μm; (shown ing for e–g): e, 650 μm;f, g, 20 μm; (shown in h forh–j): h–j, 100 μm.

Fig. 7.

Electron micrographs illustrating the relationship of anterogradely labeled terminals from the VLPO with neurons within the TMN core. The anterograde label appears as an electron-dense precipitate filling axons and terminals but outlining mitochondria and vesicles. In a–c, the sections have been stained with a post-embedding immunocytochemical method, using 10 nm colloidal gold-labeled antibodies. Small, dark gold particles may be seen (arrows) over these terminals, which are therefore also GABA-immunoreactive. In a, d, ande, the labeled terminal makes a symmetric synapse with unlabeled large dendrites (arrowheads). Scale bar, 0.2 μm for all panels.