The distribution of oxytocin and the oxytocin receptor in rat brain: relation to regions active in migraine

Oxytocin (OT) is a nine amino acid peptide (nonapeptide) structurally similar to peptide arginine vasopressin, an antidiuretic hormone, which differs from OT in two of the nine amino acid residues. These two hormones exhibit in their structure pronounced evolutionary stability [1]. Numerous studies have been performed on the expression of OT in the brain using e.g. radio-immunoassays, autoradiography, electrophysiology, OT receptor-lacZ reporter mouse, OT binding sites studies and immunohistochemistry. These have shown that OT, together with vasopressin, are synthesized in magnocellular neurosecretory cells in the supraoptic (SO or SON) and paraventricular nuclei (Pa or PVN) in the hypothalamus [2‐8]. In addition, some OT is released from centrally projecting parvocellular neurons of the Pa. These contain however much less peptide levels than do the magnocellular neurons [9]. Groups of oxytocinergic cells outside of SO/Pa are called “accessory neurons”. The magnocellular neurons of SO and Pa project to the posterior pituitary gland where they release OT into the bloodstream thus controlling endocrine events associated with reproduction in both males and females [10]. In addition, OT is released from the cell bodies and dendrites of these neurons [11]. It is still valid that OT neurons are confined to the hypothalamus, even though there seems to be a vast functional diversity. In contrast, the expression of the oxytocin receptor (OTR) is abundant throughout the brain [10], however, the functional role is still unclear.

A steadily increasing number of publications on the molecular, physiological, pharmacological, and behavioral significance of OT, the underlying neuroanatomical and neurochemical basis have been studied to a much lesser extent. This is especially true for OT projections within brain [10].

OT plays a central role in initiating the onset of maternal behavior. In addition to its involvement in uterine contraction, lactation and psychosocial processes, OT has also been implicated in many central processes, including learning and memory, anxiety, addiction, feeding behavior, sexual control and processing of social information [12]. Compared to the vasopressin system, there are fewer sex differences in OT synthesis and expression in the brain and, if any of such, sex differences are specific to brain regions and species [13]. In males, and females, OT neurons are continuously active. In males, OT is involved in reproductive functions such as ejaculation, in cardiovascular homeostasis and other peripheral activities [14]. Significantly, OT is implicated as an endogenous modulator of pain originating from the trigeminal nociceptive system [15].

The ability to artificially synthesize and intranasal administer OT has catalyzed fast-growing line of research on clinical and healthy populations. Acute, single-dose trials are not representative of long-term treatment. Because certain functional impairments have lifelong impacts on quality of life (e.g., schizophrenia, stress disorders), it is improbable that a single administration will constitute an effective treatment plan [16, 17]. Daily OT administration offers greater ecological validity in assessing the neuropeptide’s efficacy as a treatment [16]. In addition, a systematic review of the available literature showed that OT had a reliable effect as defined by increasing “pain” tolerance in 29 of 33 animal studies [18]. The results suggested that OT acts as an analgesic for acute “pain” in animals. Research with humans offers consistent evidence to suggest that OT decreases “pain” sensitivity [18].

Migraine is currently rank sixth in worldwide prevalence, 15% of the population suffers migraine, 3 times more women than men, and is posing a heavy burden on individuals and society [19]. While much remains to be unraveled regarding the neurobiology of migraine, emerging evidence links the hypothalamus with the “migraine generator” in the dorsal rostral pons as well as with the spinal trigeminal nuclei and sensory trigeminovascular system (Fig. 1a), a key pathway in transmission of headache pain [21]. An important focus of migraine research has been identifying possible neuromodulators, primarily neuropeptides, involved in migraine pathophysiology [22].

Circumstantial evidence has pointed us to propose that OT might be involved in migraine, for example the increase of circulating OT during pregnancy correlates with decrease the frequency of headache, and breastfeeding results in higher levels of circulating OT and less postpartum migraine reoccurrence [23]. Clearly, men also suffer from migraine, but so far no differences in OT levels between mothers and fathers have been demonstrated (reviewed [24]).

Therefore, the aim of the present study was to comprehensively map the distribution of OT and OTR in the male rat brain in order to provide an qualitative overview of their localization in fibres and cells to add to the discussion of migraine/headache pathogenesis in general and to compare with their localization with that of calcitonin gene-related peptide (CGRP) and CGRP receptors in the brain [25].

The study followed the guidelines of the European Communities Council (86/609/ECC) and was approved by the Regional Ethical Committee on Animal Research, Malmö/Lund, Sweden (M17–15).

Ten Wistar male rats were euthanized by CO₂ inhalation followed by decapitation. The brains were carefully dissected, cut sagittal in the midline and placed in 4% paraformaldehyde (PF) in phosphate buffer for 4 h, followed by incubation overnight in Sörensen’s phosphate buffer (pH 7.2) containing 10% and 25% sucrose in turn. This method of PF fixation allows for a proper fixation up to 2–3 mm from the surface of the brain and inwards [26]. Since we examine a volume of 1 mm lateral to the midline (+ 0.5 − + 1.5 mm), a proper fixation of the tissue used for the immunohistochemical investigation is achieved. The tissue was embedded in Yazulla embedding medium (30% egg albumin, 3% gelatin) and cryo-sectioned at 12 μm. The sections were stored at − 20 °C until use.

The 6th edition of The Rat Brain in Stereotaxic Coordinates by Paxinos and Watson [20] and HE staining of sagittal sections spanning over 0.5 mm to 1.5 mm lateral to the midline were used to identify the different areas subjected to the detailed study of OT and OTR distribution. The methods used in the present study are the same as published earlier [25]. However, it is not the same brains as the ones published.

The results of our OT/OTR mapping study is summarized in Fig. 1c. We have compared this distribution with brain areas identified in imaging studies of humans experiencing a migraine attack [27] (Fig. 1a). In addition, we have correlated our findings with that from our recent study on CNS localization of CGRP, another neuropeptide associated with migraine pathophysiology [25] (Fig. 1b).

Migraine occurs in 15% of the general population, but its pathophysiology is not well understood. Imaging results have pointed towards the hypothalamus as a tentative initiation region for the migraine attack [27] and the attacks are often linked to symptoms like stress, depression, diurnal rhythms and autonomic dysfunctions, all of which point towards involvement of the hypothalamus. OT is an important hypothalamic neuropeptide/neurohormone with widespread actions in the periphery and CNS. Recent studies indicate that OT may be involved in migraine and that its administration may relieve acute attacks [15, 23, 30, 31]. In the present study, we demonstrate that OT and OTR are present in a number of brain regions associated with migraine, thus strengthening our insight into possible involvement of OT in migraine attacks.

Historically, OT neurons have been divided into two types, magnocellular and parvocellular OT cells, which project to the posterior pituitary [32‐34]. In addition to magno- and parvocellular OT systems, there may be more types of OT neurons which cannot be characterized just by their size [35]. OT is transported to the posterior pituitary gland and secreted into the circulation from the pituitary nerve terminals to regulate numerous peripheral tissues. OT secreted from the pituitary does not enter the brain in significant amounts [36] and a major component of migraine pathophysiology – the trigeminovascular system – is a likely target of circulating OT as it lacks a blood-brain barrier (BBB) [37].

A central role of OT is that both OT and vasopressin are released within the brain by centrally projecting parvocellular neurons (fibers) and also from the soma and dendrites of magnocellular neurons [9, 38, 39]. Synthesis of OT from its precursor “prooxytophysin”, and the secretion of OT are modulated by estrogen, however, the magnocellular OT neurons do not express any of the classical estrogen receptors.

The most fascinating feature of the OT neuron is the long-range axonal projections [40], amply demonstrated in the present work. It has been shown that magnocellular OT neurons project to more than 50 forebrain regions, in addition to their well-known terminations in the posterior pituitary. Parvocellular OT projections give rise to central projections to the SO to influence magnocellular OT activity and simultaneously innervate the dorsal spinal cord to attenuate acute pain perception [41]. In the present study, we demonstrate using immunohistochemistry a delicate network of fibers expressing OT in many areas of the rat brain, however, the positive fibers in the cerebral cortex showed the most remarkably stunning organization, resembling the distribution of CGRP receptors [25].

Whereas neurotransmitters are ‘private’ messages from one neuron to another, neuropeptides, like OT, are messages between populations of cells — messages that can act across substantial distances linking widely dispersed cell groups, but which lack temporal accuracy or much spatial refinement [9]. To produce its actions in the brain, OT must reach and activate its main target, the OTR. This means that it is not the distribution of OT fibers in the brain that determines behavior, but the distribution of OTR [9]. When comparing migraine active regions seen in imaging studies of migraine patients [27] and the localization of OT/OTR, it shows that there is overlap in several parts of the brain.

In a recent review, the literature on the overlap OT/OTR in the rat brain has been summarized using autoradiography, in situ hybridization and immunohistochemistry [40]. The authors concluded that there is a complete overlap between OT fibers and OTR, but a mismatch appears in a few areas in the brain. Mismatch in the present study using immunohistochemistry was primarily confined to the cortex (OT expression) and hippocampus (OTR expression). The reason for this is unclear but, in many parts, the OT fibers distribute to the ependymal cells of the ventricles, suggestive of cerebral spinal fluid secretion, and to intracerebral microvessels, sometimes fibers reaching through the endothelium and hence suggestive of a secretion to the circulation [10].

Recent experiments have demonstrated that OTR are expressed in trigeminal ganglion neurons; some of which co-express CGRP, which is indicative of their presence on primarily nociceptive trigeminal neurons (Warfvinge, unpublished). Furthermore, OTR are up-regulated 1n trigeminal neurons following inflammatory and/or noxious stimulation.

Effects of OT on the trigeminal pathway have recently been elucidated by [42, 43]. Thus, the acute dura mater stimulation elicits neuronal activation in the trigeminocervical complex (TCC) in the brainstem [44]. This response in the TCC is blunted by spinal (local) OT administration [43]. In addition, the application of a OTR blocker reduced this response, indicating involvement of an OTR at the TCC level. Since the TNC/TCC is protected by the BBB it is reasonable that OT should come from a CNS site like the long slender neuronal fibers from the SO/Pa. The neuroanatomy provided presently support the functional data [42, 43]. We have shown that there are OT positive fibers projecting from the hypothalamic neurons to different parts of the brainstem.

The current study examined the CNS distribution of OT and its receptor with a focus on migraine-related regions and areas expressing CGRP/receptors. In many regions, in particular regions harboring the “migraine generator” in the brainstem, we found correlation between the three types of mappings. We propose that a central role for OT will be a key to understanding more about migraine pathophysiology.