Elsevier

Brain Research

Volume 1247, 9 January 2009, Pages 79-91
Brain Research

Research Report
Proximal colon distension induces Fos expression in oxytocin-, vasopressin-, CRF- and catecholamines-containing neurons in rat brain

https://doi.org/10.1016/j.brainres.2008.09.094Get rights and content

Abstract

Little is known about the chemical coding of the brain neuronal circuitry activated by nociceptive signals of visceral origin. We characterized brain nuclei activated during isovolumetric phasic distension of the proximal colon (10 ml, 30 s on/off for 10 min) in conscious male rats, using Fos as a marker of neuronal activation and dual immunohistochemistry to visualize co-localization of Fos expression and oxytocin (OT), arginine-vasopressin (AVP), corticotrophin-releasing factor (CRF) or tyrosine hydroxylase (TH). Proximal colon distension, compared with sham distension, induced a robust increase in Fos-like immunoreactive (IR) neurons in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and accessory neurosecretory nuclei of the hypothalamus, nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), and to a lower extent, in the locus coeruleus (LC) and Barrington nucleus. Fos-IR neurons in the PVN after colon distension were identified in 81% of OT-IR, 18% AVP-IR and 16% CRF-IR neurons, while in the SON it represented 36% of OT-IR and 16% AVP-IR. Catecholaminergic cell groups in the pons (LC) and medulla (VLM, NTS) were also activated by proximal colon distension. Of the TH-IR neurons in VLM and NTS, 74% and 42% respectively were double labeled. These results indicate that colon distension stimulates OT-, AVP- and CRF-containing hypothalamic neurons, likely involved in the integration of colonic sensory information to modulate autonomic outflow and pain-related responses. Activation of medullary catecholaminergic centers might reflect the afferent and efferent limbs of the functional responses associated to visceral pain.

Introduction

Enhanced colonic and rectal sensitivity to distension is frequently encountered as a clinical manifestation of irritable bowel syndrome (IBS) (Bouin et al., 2002, Drossman et al., 2002, Verne et al., 2003). In an attempt to develop experimental models that recapture this feature, the distension of the rectum and distal colon (colorectum) in rats has been extensively used to assess visceral pain and the underlying mechanisms of colonic sensitivity (Mayer et al., 2008, Ness and Gebhart, 1990). Processing of signals from visceral organs recruits neurons and transmitters in the brain that are important mechanisms regulating adaptive autonomic, neuroendocrine and pain-related responses. We previously used a model of proximal colon distension in conscious rats to study the neuronal pathways activated during noxious visceral mechanical stimulation (Martinez et al., 1998, Martinez et al., 2006). This model uses a chronically implanted balloon in the proximal half of the colon, thus avoiding acute procedures of balloon insertion under anesthesia that cause interfering effects on neuronal activation (Martinez et al., 2006, Ness and Gebhart, 1988). After phasic distension of the proximal colon, neuronal activation, demonstrated by Fos-like immunoreactivity (IR), was found significantly increased in specific brain areas and spinal cord lumbosacral segments (Martinez et al., 1998, Martinez et al., 2006). Main brain nuclei activated by distension of the proximal colon were located in the hypothalamus [paraventricular nucleus (PVN) and supraoptic nucleus (SON)] and the brainstem [nucleus tractus solitarius (NTS), ventrolateral medulla (VLM) and locus coeruleus (LC)–Barrington nucleus complex] (Martinez et al., 2006). These areas represent, respectively, centers of integration of neuroendocrine responses and the origin of efferent autonomic pathways involved mainly in the control of visceral and cardiovascular functions (Madden and Sved, 2003, Ness et al., 1999, Pan et al., 1999, Sawchenko et al., 1996, Sawchenko and Swanson, 1982, Weston et al., 2003). The PVN contains multiple neurotransmitters and neuronal hormones, including corticotropin-releasing factor (CRF), oxytocin (OT) and arginine-vasopressin (AVP), directly involved in responses to stress and visceral functions (Leng et al., 1999, Sawchenko et al., 1996, Swanson and Sawchenko, 1983). Moreover, the PVN receives prominent inputs from catecholaminergic neurons in the brainstem, such as the VLM and LC, which are involved in sympathetic regulation of cardiovascular and visceral functions (Cunningham and Sawchenko, 1988), and were also activated during colonic distension (Card et al., 2006, Martinez et al., 2006, Pan et al., 1999).

Although colonic distension is a commonly used experimental model to study visceral sensitivity, the related neurotransmitters associated to the brain pathways activated during the process have not been explored. In the present study, we used a dual immunohistochemical method for coexistence of the c-fos gene protein and specific neuropeptides (namely OT, AVP and CRF) or the neurotransmitter-synthesizing enzyme for catecholamines, tyrosine hydroxylase (TH), to localize and characterize the chemical coding of the neurons activated by proximal colon distension in conscious rats.

Section snippets

Results

Animals recovered well from the surgical procedure involved in the positioning the balloon into the proximal colon and resumed normal feeding and defecation behaviors during the 48 h period thereafter. Isovolumetric phasic distension of the proximal colon by inflating the balloon with 10 ml of air for 30 s on/30 s off for 10 min resulted in a mean intra-balloon pressure of 87 ± 4 mmHg (distension pressure oscillated between 78 and 100 mmHg). This mechanical stimulation in awake unrestrained rats

Discussion

Phasic isovolumetric distension of the proximal colon for a 10-min period at pressures within a noxious range (∼ 90 mmHg) in freely moving rats evoked a marked neuronal activation in specific populations of brain neurons as assessed by Fos expression 1 h later. The most intense Fos expression was observed in the NTS, AP, PVN and SON, while a more modest increase, although still clear, was found in the VLM and the LC–Barrington nucleus complex, a pattern consistent with that reported previously

Animals preparation

Male Sprague–Dawley rats (280–350 g; Harlan, San Diego, CA) were maintained under conditions of controlled temperature (20–22 °C), illumination (12:12 h light/dark cycle, starting from 0600 h) and humidity with food (Purina Lab Chow, Purina Mills, Inc., Richmond, IN) and water ad libitum. All experiments were performed under the VA animal component of research protocol number 95-085-10.

The implantation of a flaccid latex balloon (5 × 1 cm) attached to polyethylene tubing (PE-50; ID, 0.58 mm; OD,

Acknowledgments

This work was supported by the National Institute of Arthritis, Metabolism and Digestive Diseases; Center Grant DDK-P30 41301 (VM, Pilot and Feasibility Award; Animal Core), R01 Grant DK-33061 (YT), P50 AR 049550 (YT, LW) and VA Career Scientist Award (YT). We thank Dr Wylie Vale (Salk Institute, La Jolla, CA) for the generous supply of the rat CRF antibody.

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    Present address: Department of Cell Biology, Physiology and Immunology, Unit of Physiology, Veterinary School, Universidad Autonoma de Barcelona, 08193-Bellaterra, Barcelona, Spain.

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