Expression of nischarin, an imidazoline 1 receptor candidate protein, in the ventrolateral medulla of newborn rats

The activation of imidazoline 1 (I1) receptors is suggested to stimulate the respiratory drive in newborn rats. Here, we immunohistochemically examined whether nischarin, an I1 receptor candidate protein, is expressed in the ventrolateral medulla, where cardiorespiratory centers are located. Newborn rats (age, 3 to 5 days) were deeply anesthetized with isoflurane; the brainstem was dissected, sectioned sagittally, and labeled with nischarin. Nischarin-associated signals were observed broadly throughout the newborn rat brainstem, including at motor nuclei (motor trigeminal nucleus and facial nucleus), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate nucleus, spinal trigeminal nucleus, and solitary nucleus), and the rostral and caudal ventrolateral medullar regions. In particular, the rostral ventrolateral medulla included a layer of aggregated nischarin expression along the ventral surface, and the layer was in close contact with GFAP-positive processes. In addition, some Phox2b-positive neurons were positive for nischarin in the region. Our results reveal nischarin expression in the newborn rat brainstem and suggest that I1 receptor activation at the level of the ventrolateral medulla contributes to central chemoreception and respiratory control in newborn rats.


Introduction
Imidazoline receptors are divided into three subtypes (I 1 , I 2 , and I 3 ) [1], and the action of I 1 receptors in rostral ventrolateral medulla (RVLM) [2] is considered to exert a central sympathoinhibitory effect and suppress arterial pressure and heart rate [1]. In support of this idea, previous immunohistochemical studies using imidazoline receptor binding protein (IRBP) [3] or nischarin [4] demonstrated the expression of imidazoline receptors in adult rat brainstem. Nischarin is an I 1 receptor candidate protein that was first identified as the mouse homolog of human imidazoline receptor antisera-selected protein (IRAS) [4,5]. IRBP was originally isolated from bovine adrenal chromaffin cell membranes, and IRAS is thought to be either the human homolog of IRBP or a protein closely related to it [5].
In a previous study in newborn rats, we suggested that mean inspiratory flow (V T /T I, where V T is tidal volume and T I is inspiratory time), an index of respiratory drive, is preserved during the administration of dexmedetomidine (an α 2 -adrenoceptor/I 1 receptor agonist [1,6]) through I 1 receptor activation [7]. Dexmedetomidine is used clinically as a sedative and an analgesic and only minimally suppresses respiration in humans [8], including a child with congenital central hypoventilation syndrome [9]. Hence, we hypothesized that I 1 receptor stimulation enhances respiratory drive at the level of the ventrolateral medulla (VLM) in newborn rats at dexmedetomidine administration [7]. However, because I 1 receptor expression has not previously been confirmed in the brainstem of newborn rats [3,10], we here examined immunohistochemically whether I 1 receptor are present in the newborn rat brainstem, including regions related to the respiratory centers of the VLM.

Material and methods
The experimental protocol was reviewed and approved by the Animal Research Committee of the Nippon Dental University, School of Life Dentistry at Tokyo, Japan (17-08; approved in July 2017). The animals were treated in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences [11] and the ARRIVE guideline [12]. All efforts were made to minimize animal pain and distress and the number of animals used.
Pregnant Wistar rats (n = 6) were obtained from CLEA Japan Inc. (Tokyo, Japan) and maintained in the Nippon Dental University's animal center. Newborn rats (age, 3-5 days) ( inhalant liquid, AbbVie GK, Tokyo, Japan); the brainstems were dissected. The tissues were then prepared according to standard techniques and embedded in paraffin. Sagittal sections were cut at a thickness of 5 μm. We applied Nissl staining to every 10th consecutive slide to identify anatomical structures [13].
For immunohistochemical examination, deparaffinated tissue sections were incubated with Deparaffinization/Antigen Retrieval Solution pH6 (catalog no.415281, Nichirei Biosciences, Tokyo, Japan) for 40 min at 95-99 • C for antigen retrieval and washed in PBS. The tissue sections were then incubated at room temperature (RT) in 3% H 2 O 2 to block endogenous peroxidase activity, washed with PBS, and incubated in Blocking One Histo (Nacalai Tesque, Kyoto, Japan) as a blocking treatment.

Results
During this study, we referred to the Purkinje cells in the cerebellar cortex of the cerebellum (anatomical location is shown in the magnified inset in Fig. 1 D) as a positive control to identify nischarin immunoreactivity [16] (Fig. 2 B), applied GFAP as a marker to identify astrocyte [17], and used TH as a marker to identify catecholamine neuron groups [18], including the adrenergic C1 and noradrenergic A1 (or C1/A1 in newborn rat brain [15]) in the VLM (Fig. 1 C, F). The distributions of C1 and A1 neurons nearly match the anatomical locations of the RVLM and the CVLM, respectively [19], or when present as C1/A1 neurons, of the RVLM [20].
We noted extensive nischarin immunoreactivity throughout the newborn rat brainstem (Fig. 1 B, E), including motor nuclei (motor trigeminal and facial nuclei), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate and external cuneate nuclei, solitary nucleus, and spinal trigeminal nucleus), and neurons in the VLM (RVLM and CVLM). In low-power photomicrographs of the VLM, dense immunoreactivity was present (from rostral to caudal) in the most rostral area near the facial nucleus in the RVLM (hereafter, we refer to this region as the rostral RVLM), facial nucleus, inferior olive (dorsal nucleus), and lateral reticular nucleus ( Fig. 1 B, E). In high-power photomicrographs, nischarin immunoreactivity occurred not only in cell bodies but also in axons and dendrites, in the facial nucleus ( Fig. 2  E), inferior olive (dorsal nucleus) (Fig. 2 G), lateral reticular nucleus ( Fig. 2 H), and rostral RVLM (Fig. 1 B, E, and Fig. 3C, D). In contrast, nischarin staining was less prominent and occurred diffusely and    Abbreviation (based on [14,15]): 7N, facial nucleus. sporadically in the neurons comprising the reticular formation in the VLM (Fig. 2 D, F). Furthermore, in the rostral RVLM, the nischarinpositive aggregated neurons seemed to constitute a layer-like structure along the ventral surface (Fig. 3 C, D), where we found abundant GFAPpositive processes (Fig. 3 A, B). Interestingly, the staining pattern of the nischarin-immunoreactive layer at the ventral surface differed from that obtained by staining for TH, where the positive signal was primarily associated with axons ( Fig. 3 E, F). In the rostral RVLM, which corresponds to the anatomical locations of the retrotrapezoid nucleus (RTN) [21] or, when present, the ventral parafacial region [22], we observed Phox2b-positive cells (Fig. 4 B), some of which were also positive for nischarin (Fig. 4 A, B, C).

Discussion
Our results from nischarin immunohistochemistry of the brainstem of newborn rats indicate 1) the possible presence of I 1 receptors in the VLM, and 2) an I 1 -immunoreactive neuronal layer along the ventral surface of the RVLM.
The finding from the present study suggest that I 1 receptors are broadly expressed throughout the newborn rat brainstem. Although our observation was limited to a nonquantitative evaluation of a small area of the lateral medulla, our results were compatible with an immunohistochemical observation of the adult rat brainstem that used IRBP [3] and with the results of a study of adult rat brain, in which imidazoline binding sites occupied 36.5% of the medulla oblongata [23]. In the VLM of newborn rats, various nuclei-namely, the facial nucleus, inferior olive (dorsal nucleus), and the lateral reticular nucleus-showed particularly prominent nischarin immunoreactivity. In rat cell line (PC-12), nischarin is expressed in Map-2-positive neuronal cell bodies and dendrites [16]. In addition, electron microscopy of adult rat brain showed IRBP in nerve termini and astrocytes [24]. Taken together, these previous findings suggest that the nischarin immunoreactivity in the facial nucleus, inferior olive (dorsal nucleus), and lateral reticular nucleus may be composed of axons, dendrites, and astrocytes (Fig. 2 E, G,  H), and this same description can be applied to the layer characterized by I 1 immunoreactivity along the ventral surface of the rostral RVLM ( Fig. 3 C, D). Interestingly, a light microscopic examination of transverse sections of adult rat brain revealed IRBP-immunolabeled processes on the surface of the VRLM, although IRBP expression in the lateral reticular nucleus was low or negligible [3,24].
In the RVLM and CVLM, respiration-related regions coexist with cardiovascular sympathetic and parasympathetic circuitries [19,25]. In particular, the lateral reticular nucleus contains sympathetic neurons [1], and microinjection of clonidine (an α 2 -adrenoceptor/I 1 receptor agonist) into this region decreases blood pressure in adult cats [26]. Hence, the dense nischarin immunoreactivity that we noted in the lateral reticular nucleus of newborn rats (Fig. 1 B, E, Fig. 2 H) is compatible with the earlier observations [1,26]. Whether the areas showing high-density I 1 receptor immunoreactivity change by the species and/or with age, as seen in the catecholamine cell groups in the brainstem [27], is an interesting question.
In the RTN (also known as the parafacial respiratory group/retrotrapezoid nucleus), Phox2b-positive neurons and astrocytes are sensitive to CO 2 and may enhance active expiration and ventilatory responses [17,21,22,25]. Hence, our present results (Fig. 3 A-D and Fig. 4) suggest that I 1 receptors also contribute to the central chemoreception. We observed TH immunoreactivity (Fig. 3 E, F) and a layer of nischarin immunoreactivity (Fig. 3 C, D) along the ventral surface of the rostral RVLM. A previous study in newborn kittens showed TH-positive cell bodies and varicosities near the ventrolateral surface of VLM (longitudinally, approximately from the lateral reticular nucleus to facial nucleus); the authors explained this structure as a tributary of a major longitudinal catecholamine bundle, which courses in a dorsolateral position through the entire brainstem tegmentum [28]. Considering these previous findings in light of our current observations reminds us of the area reticularis superficialis ventrolateralis, which is important for cardiorespiratory control and the dysfunction of which has been implicated in sudden infant death syndrome [29]. Although I 1 agonist has been reported to increase second messengers concentrations in the rat VLM and causes hypotension [4], contribution of I 1 receptors on neurons seems rather complex in the central nervous system [1,5], and in light of our present observations, further investigation regarding the functional role of I 1 receptor in the brainstem is warranted.

Conclusions
Our current results confirm I 1 receptor expression in the VLM, including the rostral RVLM, of newborn rats. Our findings are noteworthy because the RVLM plays crucial roles in central chemoreception and the ventilatory responses.

Author contributions
C.S. and N.S.H. conceived of the present idea. Y.N. carried out the experiment. C.S. wrote the manuscript with input from all authors. T.I. supervised the project. C.S. and R.I. drafted the manuscript, and designed and processed the figures. All authors discussed the results and contributed to the final manuscript.

Funding
Department resources funded the study.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.