Neural pathways mediating control of reproductive behavior in male Japanese quail

Abstract

The sexually dimorphic medial preoptic nucleus (POM) in Japanese quail has for many years been the focus of intensive investigations into its role in reproductive behavior. The present study delineates a sequence of descending pathways that finally reach sacral levels of the spinal cord housing motor neurons innervating cloacal muscles involved in reproductive behavior. We first retrogradely labeled the motor neurons innervating the large cloacal sphincter muscle (mSC) that forms part of the foam gland complex (Seiwert and Adkins-Regan [1998] Brain Behav Evol 52:61-80) and then putative premotor nuclei in the brainstem, one of which was nucleus retroambigualis (RAm) in the caudal medulla. Anterograde tracing from RAm defined a bulbospinal pathway, terminations of which overlapped the distribution of mSC motor neurons and their extensive dorsally directed dendrites. Descending input to RAm arose from an extensive dorsomedial nucleus of the intercollicular complex (DM-ICo), electrical stimulation of which drove vocalizations. POM neurons were retrogradely labeled by injections of tracer into DM-ICo, but POM projections largely surrounded DM, rather than penetrated it. Thus, although a POM projection to ICo was shown, a POM projection to DM must be inferred. Nevertheless, the sequence of projections in the male quail from POM to cloacal motor neurons strongly resembles that in rats, cats, and monkeys for the control of reproductive behavior, as largely defined by Holstege et al. ([1997], Neuroscience 80:587-598).

Figure 1

Photomicrographs of retrogradely labeled motor neurons in the ventral horn (vh) of the sacral spinal cord following injections of CTB 488 (A, longitudinal section) or CTB 555 (B, C, transverse sections) into the ipsilateral foam gland. In A multiple injections were made into the gland, in B only a single injection in the gland was made, while in C as much of the gland as possible on one side was injected. Note in A the medial extension of dendrites at this ventral level of the cord. In contrast, note in B and C the very extensive dorsally directed dendrites that extend into the white matter and towards the medial aspect of the lateral funiculus (lf) and neck of the dorsal horn (dh). The asterisk denotes marginal, not dorsal root ganglion cells. Scale bar = 200 μm.

Figure 2

A–G. A caudorostral series of schematic transverse sections through the brainstem of the quail showing the location of neurons (1 dot = 1 neuron) retrogradely labeled by an injection of CTB into the sacral spinal cord. Those on the right of each section are ipsilateral to the injection. Note in G that the absence of labeled neurons in ICo also pertains to the ipsilateral side. In H are shown photomicrographically some of the labeled neurons in RAm (Scale bar = 100 μm).

Figure 3

A. Nissl counterstained right transverse hemisection through the caudal medulla showing the diagonal distribution of RAm neurons. Scale bar = 200 μm. B. An extracellular multiunit recording of respiratory-related activity in RAm. Although the phase of respiration was not measured, it was clear from visual inspection of the anesthetised bird that the rhythmical bursts of discharges over the 12 seconds corresponded to expiration.

Figure 4

Photomicrographs of anterogradely labeled fibers and putative terminations (red) and retrogradely labeled neurons (green) in the right half of the sacral spinal cord following an injection of CTB 555 into RAm and of CTB 488 into the ipsilateral foam gland in the same animal. A and B are longitudinal sections, C and D are transverse sections. A is turned 90 degrees clockwise and shows only labeled fibers and putative terminations (the foam gland was not injected) throughout the ventral horn adjacent to the lateral funiculus (lf). B shows both fibers and cell bodies with their extensive medially directed dendrites at this ventral level of the cord. In C dorsally directed green dendrites can be seen approximating red axons in the medial part of the lateral funiculus (arrow). vl: ventral funiculus. Green cell bodies are inadvertently oversaturated photographically, but a reduction of their saturation also reduced the visibility of their distal dendrites – see text. Scale bars = 100 μm.

Figure 5

B–E. Schematic representations of the location of ICo and DM cell bodies (1 dot = 1 cell body) retrogradely labeled by an injection of CTB centered on RAm (shown in black in A). Note that the majority of labeled neurons in DM are ipsilateral to the injection, and in B and C are split into a numerous medial group and a small group lateral to MLd. Rostral to MLd (D and E) DM cells form a continuous band that extends as far as the caudal border of the diencephalon. Large dots represent labeled neurons of the mesencephalic nucleus of the trigeminal nerve (Mes V) inadvertently labeled by dorsal spread from the RAm injection (see Wild and Krützfeldt, 2012). F and G are photomicrographs of labeled DM neurons, those in F forming a continuous band rostral to MLd and those more caudally in G showing dorsally directed processes (arrows) into ICo. Scale bars = 100 μm.

Figure 6

Photomicrographs showing, in A, an injection of CTB 555 in ICo medial to MLd; in B, a terminal field (red) in RAm resulting from the injection shown in A and retrogradely labeled neurons (green) resulting from an injection of CTB 484 in the sacral spinal cord in the same case. Note the presence of a few red retrogradely labeled cells within the terminal field, indicating reciprocity of the ICo-RAm projections (see Wild et al., 2009). C: Predominantly ipsilateral terminations in the central gray (GCt) resulting from the injection in A. Scale bars = 200μm.

Figure 7

Photomicrographs of neurons retrogradely labeled by an injection in ICo, such as is shown in figure 6A. Those in A are in POM, those in B are located ventral to POM at about the same rostrocaudal level, those in C are in the lateral hypothalamus bilaterally, those in D are in the more caudal hypothalamus, including VMN, and those in E are in the ventromedial arcopallium. Scale bars = 100μm.

Figure 8

A–F: Schematic hemisections depicting the projections (short fine lines and dots) resulting from a BDA injection in POM, shown as black in A and photomicrographically in figure 9. Arrows in E and F point to fibres and terminations lateral to MLd. The two areas enclosed by dashed lines in C receive little or no labeling. The more dorsal area is DM, the more ventral one receives input from the arcopallium – see figure 9. Scale bars = 1 mm.

Figure 9

A: Photomicrograph of an iontophoretic BDA injection in POM (just ventral to the anterior commissure, CA) that gave rise to the projections depicted in figure 8. B: anterograde and retrograde labeling in ICo following an injection of CTB centered on POM. Note the two relatively unlabeled areas (asterisks), one dorsal to the other. The dorsal one is DM, the ventral one an area receiving projections from the ventromedial arcopallium (see text and figures 8 and 10E). Scale bars = 100 μm.

Figure 10

A–D: Anterograde (red) and retrograde (green) labeling in ICo and DM following an injection of CTB 555 into POM and an injection of CTB 484 into RAm in the same case. Note how in A the retrogradely labeled cells are interspersed among the anterogradely labeled fibers and terminations, whereas in B–D they form the tightly clustered DM nucleus that does not admit the anterogradely labeled fibers, but is surrounded by them. E: DM (red) retrogradely labeled from an injection of CTB 555 in RAm, dorsal to and separate from a terminal field (green) in ICo resulting from an injection of CTB 484 into the medial arcopallium. Scale bar = 100 μm.

Figure 11

Schematic depiction of the projections defined in the present study (solid lines). Note that DM is shown as a specific subnucleus within ICo, although caudally DM neurons are scattered within ICo rather than being tightly clustered. Dashed lines indicate possible projections that require further definition.