Friday, December 09, 2011

Orexin controversy

In the past few days, two very interesting works exploring the role of orexin/hypocretin (ORX) in driving the autonomic nervous system came out.
Both paper explore the effects that ORX evokes when injected in the Rostral Ventromedial Medulla, an area of the brainstem containing the Raphe Pallidus (RPa) and the Raphe Magnus (RMg).
The first one, leading author Domenico Tupone, from Shaun Morrison's lab, shows very convincingly that ORX is indeed capable of activating the brown adipose tissue sympathetic nerve activity (BAT SNA), but that such activation is dependent on the presence of a small degree of spontaneous activity of the BAT SNA itself. In other words, ORX potentiates an already existing sympathetic drive to the BAT, but seems insufficient to trigger a response independently. Interestingly, little is the effect observed on other autonomic variable such as arterial pressure and heart rate, both showing a modest increase in response to the ORX injection in the Rpa.
The second one, from Leanne Luong and Pascal Carrive, shows no clear effect on the BAT resulting from ORX injection, but a powerful cardiovascular response, both in terms of arterial pressure, heart rate.

Since the two findings seems contradictory, it can be useful to focus on the experimental model used by the two groups:
Tupone's paper described the results of experiments conducted on anaesthetized rats (CD Sprague-Dawley) while Luong's paper make use of free behaving rats (Wistar-Kyoto). The difference in the experimental model account also for the difference in the volume used in the microinjection procedure. Tupone uses 60nl versus the 400 nl used in Luong's paper. Anyone that has conducted experiment in these two different model, knows that the use of anaesthetized model allow the researcher to pinpoint a specific area with an higher degree of precision that what is obtainable in a free-behaving rat. Said so, 400 nl seems a little bit on the too much side, maybe allowing the injected ORX to diffuse to proximal areas. Moreover, the injections conducted in free- behaving animals required to manipulate the animals, inducing a stress response that needs coiuld contaminate the pureness of the response. Another important difference in the techniques used by the two groups is the tool used to measure BAT activation. Tupone's paper shows direct recording of sympathetic nerves innervating the brown adipose tissue pad, while Luong'paper used an infrared thermocamera to infer the BAT temperature. More in details, the difference from the (shaved) trunk and the (shaved) interscapular area is taken as the net results of a (possible) BAT activation.

In the discussion section , Luong and Carrive suggest a possible explanation for the difference findings: the authors suggest that, since Tupone's work shows that ORX acts by potentiating preexisting activity of the BAT (in the paper induced by cold stimulation of the skin), such activity may not be present in free behaving animals at thermoneutrality. The explanation sound reasonable, but keep an interesting questions open:

In Luong's paper, the manipulation of the animal for the microinjection procedure should still activate BAT thermogenesis because of the stress response (saline-injected animals still show an increase in interscapular temperature). So, the BAT could have had some degree of activation when ORX was injected and therefore, according to Tupone's data, it should still have shown an increase in thermogenesis. Tupone's paper only deal with cold-evoked activation of the BAT, so it can also be possible that skin thermal afferences are critical in determining if ORX increases BAT activity, while other kind of centrally-mediated increase in BAT thermogenesis (like prostaglandin E2, or CRF) are not.

Still in Luong's paper, the powerful increase in Tail temperature that is observed from 30 min to 90 minutes after the injection would deserve more attention. It could be due to drug diffusion to other more distal areas, but in my opinion still looks like an ORX induced effects. The authors suggest that may be caused by contemporary thermogenesis in other district, but how? Since the differential does not drop, thermogenesis must for sure be active somewhere, but the traditional signal that is supposed to mediate tail vasodilatation (the increase in core temperature) is, in my opinion, not observed here. So what causes such vasodilatation?

With all these interesting questions open, I am tempted to order some ORX and have a look by myself at all these interesting effects. Maybe in the new year.

Here references and abstracts:

J Neurosci. 2011 Nov 2;31(44):15944-55
Authors: Tupone D, Madden CJ, Cano G, Morrison SF

Orexin (hypocretin) neurons, located exclusively in the PeF-LH, which includes the perifornical area (PeF), the lateral hypothalamus (LH), and lateral portions of the medial hypothalamus, have widespread projections and influence many physiological functions, including the autonomic regulation of body temperature and energy metabolism. Narcolepsy is characterized by the loss of orexin neurons and by disrupted sleep, but also by dysregulation of body temperature and by a strong tendency for obesity. Heat production (thermogenesis) in brown adipose tissue (BAT) contributes to the maintenance of body temperature and, through energy consumption, to body weight regulation. We identified a neural substrate for the influence of orexin neurons on BAT thermogenesis in rat. Nanoinjection of orexin-A (12 pmol) into the rostral raphe pallidus (rRPa), the site of BAT sympathetic premotor neurons, produced large, sustained increases in BAT sympathetic outflow and in BAT thermogenesis. Activation of neurons in the PeF-LH also enhanced BAT thermogenesis over a long time course. Combining viral retrograde tracing from BAT, or cholera toxin subunit b tracing from rRPa, with orexin immunohistochemistry revealed synaptic connections to BAT from orexin neurons in PeF-LH and from rRPa neurons with closely apposed, varicose orexin fibers, as well as a direct, orexinergic projection from PeF-LH to rRPa. These results indicate a potent modulation of BAT thermogenesis by orexin released from the terminals of orexin neurons in PeF-LH directly into the rRPa and provide a potential mechanism contributing to the disrupted regulation of body temperature and energy metabolism in the absence of orexin.

PMID: 22049437 [PubMed - in process]

Source: Neuroscience, Available online 7 December 2011
Leanne N.L. Luong, Pascal Carrive
The rostral medullary raphe region is an important target of hypothalamic orexin neurons, however little is known of the effect of orexin in this key autonomic and somatic premotor region. Here we tested the effect of orexin-A (3 and 30 pmol) microinjected in the medullary raphe, on heart rate, mean arterial pressure, tail skin blood flow, body temperature, and behaviour in freely moving, awake rats. Heart rate, mean arterial pressure and body activity were recorded by radio-telemetry. Changes in tail skin blood flow and body temperature, as well as potential interscapular brown adipose tissue thermogenesis were recorded indirectly by infrared thermography of the skin of the tail, lumbosacral back and interscapular back areas, respectively. Compared to saline, orexin-A (30 pmol) evoked significant and long lasting increases in heart rate (+99 bpm), mean arterial pressure (+11 mmHg) and body activity (grooming, not locomotor activity). However, it did not reduce tail skin blood flow more than saline and there was no significant increase in body temperature. A small, though significant, thermogenic effect was observed in the interscapular region, but this effect is more likely to have originated from activity in neck and shoulder muscles than brown adipose tissue. Thus orexin projections to the rostral medullary raphe can mediate significant cardiovascular changes, but does not seem to affect tail skin vasomotor tone or brown adipose tissue in the awake rat. This important brainstem relay may contribute to the cardiovascular changes associated with arousal and various forms of stress that are associated with activation of orexin neurons.


Microinjections of orexin (3 and 30 pmol) were made in the medullary raphe of awake rats. ▶The effects were recorded with radiotelemetry and infrared thermography. ▶Orexin 30 pmol increased heart rate and mean arterial pressure and evoked grooming but not running. ▶Orexin 30 pmol did not cause tail skin vasoconstriction nor brown adipose tissue activation.