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. 2008 Apr 20;507(6):1990-2003.
doi: 10.1002/cne.21665.

Genetically expressed transneuronal tracer reveals direct and indirect serotonergic descending control circuits

João Manuel Braz  1 , Allan I Basbaum
Affiliations

Genetically expressed transneuronal tracer reveals direct and indirect serotonergic descending control circuits

João Manuel Braz et al. J Comp Neurol. .

Abstract

Despite the evidence for a significant contribution of brainstem serotonergic (5HT) systems to the control of spinal cord "pain" transmission neurons, attention has turned recently to the influence of nonserotonergic neurons, including the facilitatory and inhibitory controls that originate from so-called "on" and "off" cells of the rostroventral medulla (RVM). Unclear, however, is the extent to which these latter circuits interact with or are influenced by the serotonergic cell groups. To address this question we selectively targeted expression of a transneuronal tracer, wheat germ agglutinin (WGA), in the 5HT neurons so as to study the interplay between the 5HT and non-5HT systems. In addition to confirming the direct medullary 5HT projection to the spinal cord we also observed large numbers of non-5HT neurons, in the medullary nucleus reticularis gigantocellularis and magnocellularis, that were WGA-immunoreactive, i.e., were transneuronally labeled from 5HT neurons. FluoroGold injections into the spinal cord established that these reticular neurons are not only postsynaptic to the 5HT neurons of the medulla, but that most are also at the origin of descending, bulbospinal pathways. By contrast, we found no evidence that neurons of the midbrain periaqueductal gray that project to the RVM are postsynaptic to midbrain or medullary 5HT neurons. Finally, we found very few examples of WGA-immunoreactive noradrenergic neurons, which suggests that there is considerable independence of the monoaminergic bulbospinal pathways. Our results indicate that 5HT neurons influence "pain" processing at the spinal cord level both directly and indirectly via feedforward connections with multiple non-5HT descending control pathways.

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Figures

Fig. 1
Fig. 1
WGA expression in 5HT-immunoreactive raphe neurons in ePet-ZW mice. Cre-mediated excision of the floxed lacZ cDNA initiates WGA expression in 5HT-expressing neurons of the brainstem and midbrain. All raphe nuclei contain the WGA tracer (black): (A) raphe obscurus (Rob), raphe pallidus (RPa), and nucleus reticularis paragigantocellularis (PGi); (B) Raphe magnus (NRM); (C) Midbrain dorsal (DR) and median raphe (MnR). There is dense accumulation of transported WGA in presumptive terminals (arrows) in the lateral (Pbl) and medial (Pbm) parabrachial nuclei (D,E). E: Higher magnification of the boxed area in D. Arrowhead in D points to a transneuronally labeled neuron in the PB. Scale bars = 100 μm in A,B,D,E; 200 μm in C.
Fig. 2
Fig. 2
Intermingled populations of 5HT and non-5HT raphe neurons contain the WGA tracer in ePet-ZW mice. Double labeling for serotonin (5HT, green, column 1) and WGA (red, column 2) illustrates the transneuronal transfer of WGA from 5HT to non-5HT neurons in the dorsal (DR), and median raphe (MnR) and in the nucleus raphe magnus (NRM). Inset: high magnification of the boxed area showing single-labeled neurons that correspond to neurons that are postsynaptic to 5HT neurons. A magenta-green version of this figure is available as a supplementary figure online. Scale bar = 100 μm.
Fig. 3
Fig. 3
Transneuronal transfer of WGA to medullary reticular neurons in ePet-ZW mice. Outside the boundaries of the midline raphe nuclei many non-5HT neurons contain the WGA tracer (black). These were located dorsal and lateral to the raphe magnus, in nucleus reticularis magnocellularis (A) and in the nucleus reticularis gigantocellularis (B). Scale bar 100 μm.
Fig. 4
Fig. 4
Distribution of postsynaptic, WGA-labeled neurons in nucleus reticularis magnocellularis and nucleus reticularis gigantocellularis. Single WGA-labeled neurons (black asterisks) were detected throughout the brainstem, in a region, ≈ 1,400 μm in length, extending from the caudal raphe magnus (1) to the caudal pole of the trigeminal motor nucleus (9). The neurons were located lateral and dorsal to the 5HT/WGA double-labeled neurons (squares) of the raphe magnus. VII: seventh nucleus; 7n: seventh nerve.
Fig. 5
Fig. 5
Cre recombinase expression pattern in ePet-Cre mice. To determine whether or not the Cre recombinase is expressed exclusively in 5HT neurons we crossed the ePet-Cre mice with the ROSA26 Cre reporter mice, in which expression of β-galactosidase (β-gal) is induced in Cre-expressing neurons. Double labeling for β-gal (red in A) and 5HT (green in B) illustrates that all β-gal-positive neurons are 5HT-immunoreactive (yellow in C). Inset: high magnification of the boxed area showing double-labeled neurons. Note that not all 5HT neurons express β-gal, presumably because there is some mosaicism in the ROSA26 Cre reporter mouse. A magenta-green version of this figure is available as a supplementary figure online. Scale bar = 100 μm.
Fig. 6
Fig. 6
Transneuronal transport of WGA in the spinal cord of ePet-ZW mice. Tyramide signal amplification of immunoreactive WGA labeling reveals the transport of the WGA tracer to terminals in superficial laminae of the dorsal horn of the spinal cord. There is no labeling in control experiments, where the primary antibody was omitted (column 2). Scale bars = 500 μm in A,C; 150 μm in B,D.
Fig. 7
Fig. 7
Non-5HT neurons that project to the spinal cord are postsynaptic to medullary 5HT neurons. FluoroGold injection in the spinal cord of ePet-ZW mice (A) retrogradely labels a heterogeneous population of 5HT (green) and non-5HT/WGA (red) neurons throughout the brainstem (FG-positive neurons are white). 5HT neurons in the midbrain dorsal raphe (B,C) and median raphe (D–F) do not project to the spinal cord. In contrast, the medullary raphe nuclei (notably the raphe magnus; G–I) contain large numbers of 5HT neurons that project to the spinal cord (G–I). Arrows point to single-labeled WGA neurons that project to the spinal cord, but are not 5HT. These lie downstream of the “primary” 5HT neurons. A magentagreen version of this figure is available as a supplementary figure online. Scale bars = 600 μm in A; 100 μm in B–I.
Fig. 8
Fig. 8
Serotonin inputs to neurons of the nucleus reticularis gigantocellularis that project to the spinal cord. FluoroGold injections in the spinal cord of ePet-ZW mice retrogradely label large numbers of neurons (white in A) in the nucleus reticularis gigantocellularis (RGc). Double labeling for WGA (red in B) shows that most RGc neurons (80%) that receive 5HT inputs project to the spinal cord. A magenta-green version of this figure is available as a supplementary figure online. Scale bar = 100 μm.
Fig. 9
Fig. 9
The PAG-RVM pathway does not include 5HT neurons. FluoroGold injections in the RVM retrogradely labeled large numbers of neurons (white in A) in the ventrolateral periaqueductal gray (vlPAG). However, double-labeling for WGA (green in B) shows that none of these receive direct or indirect inputs from 5HT (i.e., primary) neurons. Scale bar = 100 μm.
Fig. 10
Fig. 10
Brainstem noradrenergic neurons do not receive 5HT inputs in ePet-ZW mice. Double labeling for WGA (red) and tyrosine hydroxylase (green) illustrate that noradrenaline- and serotonin-containing neurons constitute distinct populations. Moreover, the lack of WGA labeling in TH-positive neurons indicates that NA-immunoreactive neurons are not postsynaptic to 5HT (primary) neurons. A: rostral PAG; A10. B: A5 cell group. C: A1 cell group. D: locus coeruleus (A6) and subcoeruleus (A7). 7n, seventh nerve; PGi, nucleus reticularis paragigantocellularis; DR, dorsal raphe. A magenta-green version of this figure is available as a supplementary figure online. Scale bar = 100 μm.
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