The Djungarian or Siberian dwarf hamster, Phodopus sungorus (hereinafter referred to as Phodopus), is a widely used model organism for studying seasonal dependency of functional body parameters such as reproduction ingestive behavior and obesity as well as aggression [1, 2, 3, 4, 5, 6, 7, 8]. All these involve the components of the autonomic system, i.e., parasympathetic and intramural neurons and, in particular, the sympathetic system.

Phodopus features robust changes of neural, physiological and behavioral parameters in response to photoperiodic variations. The animals are long-day breeders, exhibit gonadal regression and body weight and fur color changes when exposed to short-day conditions [1]. The species is special since animals lose weight in winter by decreasing body mass on a metabolic basis [9].

There is consensus that the crucial seasonal cue is day-length translated into a hormonal signal, i.e., melatonin secretion from the pineal gland into the blood and cerebrospinal fluid. A clear relation was found between day-length and, among other parameters, gonadal function as a response to the melatonin signal’s daily duration. In animals with short gestation periods such as Phodopus, melatonin acts anti-gonadotropic [2, 10].

The pineal gland is a member of the circadian timing system that consists of several brain structures. The endogenous clock is located in the suprachiasmatic nuclei and exerts its influence on pineal function by multisynaptic pathways [11, 12]. The functionally most important route includes sympathetic regulation of pineal melatonin synthesis via noradrenaline released from the superior cervical ganglia (SCG) neurons, usually denominated as postganglionic neurons. Neurons of the SCG are innervated by fibers originating from perikarya located in the lateral horn of lower cervical and upper thoracic spinal cord segments, i.e., the sympathetic neurons of the intermediolateral nucleus usually denominated as sympathetic preganglionic neurons (SPN). This has been shown previously in several species by many studies using retrograde tracing techniques [13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26], without claiming completeness. See also the reviews [27, 28] and the discussion below for more details.

The SPN provide the connection between the central and the autonomic nervous system. They are the last central component, are under the descending and local influence of the CNS, and innervate postganglionic neurons. Among various other functions, the sympathetic trunk couples the pineal organ to the hypothalamus and is thus a pivotal part of the system that generates and maintains pineal-dependent circadian rhythms in mammals.

As part of a long-standing program, we studied structures and pathways mediating photoperiodic control of body functions in Phodopus, particularly the hypothalamus, retinohypothalamic tract, suprachiasmatic nucleus, superior cervical ganglia and pineal gland [29, 30, 31, 32, 33, 34, 35, 36, 37]. Since Reuss [36] focused on the distribution of calcitonin gene-related peptides in the spinal cord referred to SPN in Phodopus without detailed quantification, the present research was conducted to provide a detailed analysis location and distribution of SPN projecting to the SCG. We also sought to find the possible presence of selected neuroactive substances in or neighboring identified SPN. We, therefore, labeled SPN by retrograde neuronal tracing upon injection of the neuronal tracer, Fluoro-Gold (FG) [38], into the SCG of anesthetized animals and analyzed these neurons in sections from the perfusion-fixed spinal cord. We then used immunofluorescent staining of selected spinal sections containing SPN by antibodies directed to either arginine-vasopressin, neuronal nitric oxide-synthase, neuropeptide Y, neurotensin, oxytocin or substance P.

Fourteen (adult male) Djungarian hamsters (Phodopus sungorus) with body weights of 40–55 g were used. The animals stem from a breeding colony descending basically from hamsters provided by Dr. Klaus Hoffmann (Institute of Reproductive Medicine, University of Münster, Germany). They were reared and held under long-day conditions (light:dark 16:8) with food and water ad libitum in the Department of Anatomy and Cell Biology animal facility. The procedures concerning animals complied with German and European laws to protect animals and were approved by the county-government office (Bezirksregierung Rheinhessen-Pfalz). All efforts were made to minimize animal suffering.

For the tracer application, hamsters were anesthetized with tribromoethanol (0.3 g/kg b.wt., intraperitoneal, Merck, Darmstadt, Germany). All surgical procedures were performed aseptically. Following exposure of the right SCG under a surgical microscope, 150 nL of a 5% Fluoro-Gold (FG, see Table 1, Ref. [16, 31, 37, 38, 39, 40, 41]) solution dissolved in distilled water were slowly pressure-injected unilaterally into the SCG using a glass micropipette (tip diameter 1 $\mu$m). The injection methods were optimized in our laboratory and used in several anterograde and retrograde tracing studies. This included backfilling of the glass capillary, the careful cleaning of the tip, and pulling back a small amount of fluid before insertion.

Following a seven days survival, animals were anesthetized and perfused transcardially with room-temperature phosphate-buffered 0.9% saline (PBS) followed by ice-cold periodate-lysine-paraformaldehyde solution [42]. The right atrium was cut to enable venous outflow. Spinal cords were exposed by laminectomy and cut into segments, using the midpoint between the entry of two adjacent ventral roots as rostral and caudal limits. Segments were removed, marked on one side, postfixed for one hour and stored overnight at 4 ${}^{\circ }$C in phosphate-buffered 20% sucrose. Tissue was sectioned serially at 30 $\mu$m on a freezing microtome in either frontal or horizontal plane and collected in PBS into two or three parallel sets of sections. From each of eight animals, one cross-sectioned set was directly mounted in order of appearance on gelatinized glass slides, dried, cleared in xylene, coverslipped with Merckoglas (Merck, Darmstadt, Germany), and analyzed using a Leitz Orthoplan microscope with a Ploemopak epifluorescence unit (filter set D). FG-labeled cell bodies were quantified about their location. Counts were corrected according to Abercrombie [43]. Values are presented as means $\pm$ SD.

For immunohistochemistry, sections containing retrogradely labeled cells were incubated free-floating in antisera directed against either arginine-vasopressin (AVP), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), neurotensin (NT), oxytocin (OT), or substance P (SP) in phosphate-buffered saline to which 0.1% Triton-X and 1% normal swine serum (PBSST) were added. The binding was visualized using Cy3 or Cy2 coupled to IgG-F(ab)${}_2$-fragments directed against the host of the respective primary antiserum.

For each antibody, a total of at least thirty sections per segment stemming from at least six animals were incubated. Further sections were used for antigen distribution and antibody specificity tests. In some cases, FG-fluorescent labeling appeared weaker following free-floating immunohistochemical incubation. Details on antibodies and tracing materials are given in Table 1.

To control for unspecific tracing due to the possible spread of the tracer, three additional animals received FG-application to the respective region without injection into the SCG. In these cases, spinal neurons were not labeled.

Control incubations were carried out by (1) using antibodies preabsorbed with 10–50 $\mu$M concentrations of the respective antigen (Bachem, Bubendorf, Switzerland), (2) using rabbit preimmune-serum instead of the antibodies, and (3) omitting secondary antibodies for control for autofluorescence. The results of controls all demonstrated the specificity of the antibodies.

Following injection of FG into the SCG and retrograde axonal transport, labeled neurons were found ipsilateral at the lateral edge of the spinal gray matter, Rexed-lamina VII, also known as Substantia intermedia lateralis or Zona intermedia. Cell counting in cross-sections showed that individual preganglionic neurons were located in segments C8 to Th5. The number of labeled cells per animal amounted to 486 $\pm$ 99 (n = 8). They were located predominantly in the intermediolateral nucleus pars principalis (IMLp; 326 $\pm$ 66; 67% of total SPN/animal) and its extension into the white matter, i.e., pars funicularis (IMLf; 152 $\pm$ 34; 31%). About 1% of the cells were found in the central autonomic area (CA; Rexed-lamina X; 5 $\pm$ 1; Fig. 1A), a part of the gray matter surrounding the central canal (marked by asterisks in Fig. 2D,E). A few cells (4 $\pm$ 1; less than 1%) were seen in the intercalated region (IC), the gray matter intermediate zone connecting IML and CA. The sampling regions are depicted in a spinal cross-section counterstained for substance P that also demonstrates the typical appearance of the gray and white matters of the spinal cord (Fig. 1A). Soma sizes ranged 15–20 $\mu$m in diameter in the fixed tissue, which is known to shrink by about 30 percent. Arborization was often aligned in a mediolateral direction. There were no quantitative differences when survival times of 4–14 days were tested in preceding experiments. However, dendritic arborization was better impregnated by FG with longer survival times. Typical examples of labeled sympathetic preganglionic neurons in cross or horizontal sections are shown in Fig. 3.

Fig. 1.

Regional and segmental distribution of sympathetic preganglionic neurons (SPN) projecting to the superior cervical ganglion (SCG). (A) Cross-section taken from spinal segment Th2 counterstained for substance P-immunofluorescence demonstrates the location of the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf), the central autonomic area (CA) surrounding the central canal (*) and the intercalated region (IC). Note that white matter appears dark in present fluorescence imaging. (B) Segmental distribution of identified sympathetic preganglionic neurons (SPN) observed upon Fluoro-Gold injection into the superior cervical ganglion and retrograde neuronal transport. The total numbers of SPN in each the IMLp (left) and the IMLf (right), respectively, were taken as one-hundred percent (mean (n = 8) $\pm$ SD).

Fig. 2.

Neuronal nitric oxide synthase (nNOS) in the spinal cord autonomic regions concerning retrogradely labeled neurons. (A) FG-containing sympathetic preganglionic neurons in the Th1 segment and, in the same cross-section, nNOS-immunoreactivity (B). (C) nNOS in the IML (cross-section from Th2). (D,E) In a section taken from Th3, the central autonomic area (CA) exhibited numerous nNOS-perikarya (E), which FG did not label. (D) demonstrates a retrogradely labeled neuron close to the central canal (*), which does not contain nNOS, while other cell bodies shown in the same section in (E) exhibit nNOS but not (F,G). Another example taken from the intercalated region (IC) shows partial overlapping of FG-tracing (F) and nNOS-immunoreactivity (G) in the same section.

Fig. 3.

Sympathetic preganglionic neurons projecting to the superior cervical ganglion, as identified by retrograde neuronal tracing with Fluoro-Gold (FG), in sections of the spinal cord. (A) Ethidium-bromide fluorescent staining of a cross-section showing the lateral horn of Th1. (B) FG-labeled cells in the same section. Arrows depict the most laterally localized SPN to ease orientation. (C) Labeled cells in both parts of the IML in a cross-section of the C8 segment. (D) Labeled cells in a horizontal section of upper thoracic segments. (E) FG-labeled cells in IMLp in a Th1 horizontal section demonstrating the location of labeled neurons in clusters. (F) FG-labeled cells in IMLp in a Th1 cross-section, and rich arginine-vasopressin (AVP)-immunoreactivity in the same section. (G) AVP is often found in the vicinity of FG-neurons. Examples are marked “1” and “2”. Note that following free-floating immunohistochemical incubation, FG-labeling may appear weaker. Abbr.: EBr, ethidium bromide; GM, gray matter; WM, white matter; IMLp, intermediolateral nucleus pars principalis; IMLf, intermediolateral nucleus pars funicularis.

The segmental distribution of labeled cells in both parts of the IML is given in Fig. 1B, where the average total numbers of SPN in each the IMLp (326) and the IMLf (152), respectively, were taken as one-hundred percent. In both regions, the segments Th1 to Th3 contained about 95% of the labeled cells. In horizontal 30-$\mu$m-thick slices, the IML often appeared as a segmental cell column with clusters of up to ten neurons alternating with less dense cellular patches (Fig. 3D,E). Other cell somata of the gray matter, smaller in diameter, probably represent interneurons.

Neuronal NOS was the only substance tested that showed clear-cut immunolabeling of identified SPN. The incubation of spinal sections in respective antibodies demonstrated nNOS perikarya of the IML regions (Fig. 2A–C). It was found that 319 $\pm$ 69, corresponding to 65% of the retrogradely labeled neurons per animal, exhibited nNOS (Fig. 2A,B). In the IMLp, numbers were 166 $\pm$ 30 (51% of SPN in this region) and IMLf 112 $\pm$ 26 (74% of respective SPN). Cell bodies containing FG but no detectable nNOS (Fig. 2D,E), as well as perikarya exhibiting nNOS (Fig. 2E) that were not traced retrogradely were also observed, in particular in the CA (Fig. 2D,E; asterisks mark central canal) where nNOS-neurons outnumbered SPN. Colocalization of FG and nNOS was in the central autonomic region found to a lesser extent (Fig. 2F,G) although nNOS cell bodies were regularly observed.

A few identified SPN exhibited very weak neuropeptide Y-immunofluorescence (Fig. 4A–D; thick arrow in D) just above the background but different from appearance in sections stained with pre-absorbed antibodies. More intensely stained fibers were seen in SPN regions (Fig. 4B,D). These fibers opposed identified SPN in some cases, but their origin was not readily apparent under the microscope.

Fig. 4.

Sympathetic preganglionic neurons and neuropeptide-immunoreactivity in a horizontal section of Th1. Beaded neuropeptide Y (NPY)-fibers (B,D) in the vicinity of retrogradely labeled sympathetic preganglionic neurons in the IML (A,C), which only in some cases exhibited scarce NPY-immunoreactivity (thick arrow in D). Scattered neurotensin (NT)-fibers were seen in the gray matter (F) in a horizontal section, lacking distinct association with FG-labeled SPN (E). Asterisks in E, F mark the location of two FG-neurons in both images taken from the same section. In contrast, SPN in the IML were covered with dense substance P (SP)-immunoreactive structures (G,H) in a Th1 cross-section.

Other neuropeptides tested presently were found only in fibers in the spinal segments with a close spatial relationship to identified SPN. Arginine-vasopressin was present in scattered fine fibers throughout the gray matter with an apparent accumulation at SPN (Fig. 3F,G; “1” and “2” indicated lightly labeled SPN). A very similar distribution was found for oxytocin-fibers in the lateral horn. In contrast, in the dorsal horn and intermediate zone, gray matter slightly more OT was observed than AVP. Thin varicose neurotensin-fibers were present throughout the gray matter, appeared to be concentrated in the lateral horn but did not show appositions to SPN (marked by asterisks in Fig. 4E,F).

The densest fiber staining by far was seen for substance P. The respective immunolabeling was strong in laminae I/II of the dorsal horn. The lateral horn covered the whole region where identified SPN were located (Figs. 1A,4G,H).

The fluorescent appearance of SPN was often weakened following free-floating incubation for immunohistochemistry (see Figs. 3E,4A,G).

The present research sought to characterize the main site of neuroendocrine-autonomic integration in the photoperiodic rodent, Phodopus. It describes the location of spinal SPN innervating the superior cervical ganglion and demonstrates the expression pattern of six selected neuroactive substances in the respective spinal region.

Phodopus is a seasonal breeder. Reproduction and other body parameters are regulated by photoperiod and controlled by mechanisms involving several brain sites, including the hypothalamus and pineal gland [1]. The main relay downstream to the hypothalamus is spinal SPN, where intrinsic, supraspinal and indirect peripheral (sensory) inputs converge and influence the distinct output signals that regulate sympathetic postganglionic neurons. In the context considered here, the rostralmost sympathetic ganglion, the superior cervical ganglion, is of particular interest since its neurons provide sympathetic innervation of heart and head structures such as eye, ear, choroid plexus, carotid body, salivary glands and blood vessels.

Postganglionic neurons also synapse at pinealocytes to regulate melatonin synthesis. This pathway is the route translating day-length into a melatonin secretion signal [44]. The SCG also conveys the autonomic stress response and contributes to related cardiovascular diseases [45]. It is also part of the neuroimmune connection providing the sympathetic innervation of, e.g., nasal-associated lymphoid tissue (NALT) [46]. Notably, there is also evidence for reducing SPN as part of autonomic dysfunction in Parkinson´s and Lewy-body diseases [47, 48].

The exact location of sympathetic preganglionic neurons innervating the superior cervical ganglion is thus eligible for further investigations utilizing experimental manipulations such as a specific lesion, electric stimulation or iontophoretic application of neuroactive substances in this rodent model organism.

In conclusion, the data presented here reveal that sympathetic preganglionic neurons innervating the superior cervical ganglion are located in ipsilateral clusters predominantly in the intermediolateral nucleus and the lateral funiculus of segments Th1 to Th3 in the Phodopus spinal cord, in general accordance to findings in other mammalian species. Many of these neurons produce nitric oxide as an intrinsic neuroactive gaseous substance. Some contain low amounts of neuropeptide Y. Immunohistochemical tests further revealed that other substances such as arginine-vasopressin and oxytocin are present in scarce fibers, mainly in close vicinity to identified SPN, neurotensin in fibers of passage. In contrast, substance P was observed in high density in the area of autonomic neurons. Fiber systems are most probably of supraspinal origin and relay and integrate information from hypothalamic and other sites in SPN en route to sympathetic ganglia.

AVP, arginine-vasopressin; C, cervical segment; CA, central autonomic area; DH, dorsal horn; FG, Fluoro-Gold; IC, intercalated region; IMLf, intermediolateral nucleus pars funicularis; IMLp, intermediolateral nucleus pars principalis; IR, immunoreactivity; nNOS, neuronal nitric oxide synthase; NPY, neuropeptide Y; NT, neurotensin; OT, oxytocin; SCG, superior cervical ganglion; SP, substance P; SPN, sympathetic preganglionic neurons; Th, thoracic segment; VH, ventral horn.

SR designed and performed the experiments, analyzed the data, and wrote the paper.

Animals were reared and held in the animal facility of the Department of Anatomy and Cell Biology, University of Mainz, Germany. All animal experiments were carried out in accordance with the guidelines of the European Community Council Directives 86/609/EEC and approved by the county-government office (Bezirksregierung Rheinhessen-Pfalz; 177-07/961-30).

The author thanks Ursula Disque-Kaiser and Angelika Thomas-Semm for technical assistance. This study was conducted while the author was Head of the Functional Neuroanatomy Group at the Department of Anatomy and Cell Biology, University Medical Center, Mainz. Thanks also to the anonymous reviewers for excellent and helpful criticism of the article.

This study was supported by grants from Deutsche Forschungsgemeinschaft (Re 644) and Naturwissenschaftlich-Medizinisches Forschungszentrum, Mainz.

The author declares no conflict of interest.