Thursday, December 22, 2011

A new neural correlate of consciousness?

Over at Neuroskeptic an interesting post on this paper. The overall idea of using pavlovian conditioning to test the presence of consciousness is very appealing, even if it is shown in the same paper that the Aplysia would be conscious too (and that seems unlikely, but maybe it is just a cultural bias). 
To take this approach one step forward, wouldn't be interesting to explore which widespread, conscious-related neuronal group (orexin, locus coeruleus, TMN and so on) is necessary for the trace conditioning to happen? 

Front Psychol. 2011;2:337. Epub 2011 Dec 6.

Sea slugs, subliminal pictures, and vegetative state patients: boundaries of consciousness in classical conditioning.


Cognition and Brain Sciences Unit, Medical Research Council Cambridge, UK.


Classical (trace) conditioning is a specific variant of associative learning in which a neutral stimulus leads to the subsequent prediction of an emotionally charged or noxious stimulus after a temporal gap. When conditioning is concurrent with a distraction task, only participants who can report the relationship (the contingency) between stimuli explicitly show associative learning. This suggests that consciousness is a prerequisite for trace conditioning. We review and question three main controversies concerning this view. Firstly, virtually all animals, even invertebrate sea slugs, show this type of learning; secondly, unconsciously perceived stimuli may elicit trace conditioning; and thirdly, some vegetative state patients show trace learning. We discuss and analyze these seemingly contradictory arguments to find the theoretical boundaries of consciousness in classical conditioning. We conclude that trace conditioning remains one of the best measures to test conscious processing in the absence of explicit reports.

Tuesday, December 20, 2011

How many serotoninergic neurons out there?

Published a few days ago, today is futured in the EJN blog.
 A very useful article, that probably opens lots of new question.

Projections and interconnections of genetically defined serotonin neurons in mice:

Brain serotonin neurons are heterogeneous and can be distinguished by several anatomical and physiological characteristics. Toward resolving this heterogeneity into classes of functional relevance, subtypes of mature serotonin neurons were previously identified based on gene expression differences initiated during development in different rhombomeric (r) segments of the hindbrain. This redefinition of mature serotonin neuron subtypes based on the criteria of genetic lineage, along with the enabling genetic fate mapping tools, now allows various functional properties, such as axonal projections, to be allocated onto these identified subtypes. Furthermore, our approach uniquely enables interconnections between the different serotonin neuron subtypes to be determined; this is especially relevant because serotonin neuron activity is regulated by several feedback mechanisms. We used intersectional and subtractive genetic fate mapping tools to generate three independent lines of mice in which serotonin neurons arising in different rhombomeric segments, either r1, r2 or both r3 and r5, were uniquely distinguished from all other serotonin neurons by their expression of enhanced green fluorescent protein. Each of these subgroups of serotonergic neurons had a unique combination of forebrain projection targets. Typically more than one subgroup innervated an individual target area. Unique patterns of interconnections between the different groups of serotonin neurons were also observed and these pathways could subserve feedback regulatory circuits. Overall, the current findings suggest that activation of subsets of serotonin neurons could result in topographic serotonin release in the forebrain coupled with feedback inhibition of serotonin neurons with alternative projection targets.
Thumbnail image of graphical abstract
Brain serotonin neurons are heterogeneous and can be distinguished by several anatomical and physiological characteristics. Toward resolving this heterogeneity into classes of functional relevance, subtypes of mature serotonin neurons were previously identified based on gene expression differences initiated during development in different rhombomeric (r) segments of the hindbrain.

Dreams and the Locus Coeruleus

Is the reduced noradrenergic tone that causes dreams to be bizarre? The monoaminergic theory of sleep has always been around, but it does not mean is wrong.
What I can say is that, whatever the reason for sleep is, I like to see it localized in the hypothalamus - brainstem axis, more than in the Hippocampus.

The involvement of noradrenaline in rapid eye movement sleep mentation.:

Front Neurol. 2011;2:81
Authors: Gottesmann C

Noradrenaline, one of the main brain monoamines, has powerful central influences on forebrain neurobiological processes which support the mental activities occurring during the sleep-waking cycle. Noradrenergic neurons are activated during waking, decrease their firing rate during slow wave sleep, and become silent during rapid eye movement (REM) sleep. Although a low level of noradrenaline is still maintained during REM sleep because of diffuse extrasynaptic release without rapid withdrawal, the decrease observed during REM sleep contributes to the mentation disturbances that occur during dreaming, which principally resemble symptoms of schizophrenia but seemingly also of attention deficit hyperactivity disorder.

PMID: 22180750 [PubMed - in process]

New Orexin Antagonist

If you work on Orexin, you'll understand why it would be so great to have available some new small molecules that work as antagonist.
Here is the last one. Hoping it will be available for lab use soon.

Discovery process and pharmacological characterization of a novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders.:

Bioorg Med Chem Lett. 2011 Sep 15;21(18):5562-7

Authors: Di Fabio R, Pellacani A, Faedo S, Roth A, Piccoli L, Gerrard P, Porter RA, Johnson CN, Thewlis K, Donati D, Stasi L, Spada S, Stemp G, Nash D, Branch C, Kindon L, Massagrande M, Poffe A, Braggio S, Chiarparin E, Marchioro C, Ratti E, Corsi M

The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled receptors, namely the OX(1) and the OX(2) receptor. These receptors are widely distributed, though differentially, in the rat brain. In particular, the OX(1) receptor is highly expressed throughout the hypothalamus, whilst the OX(2) receptor is mainly located in the ventral posterior nucleus. A large body of compelling evidence, both pre-clinical and clinical, suggests that the orexin system is profoundly implicated in sleep disorders. In particular, modulation of the orexin receptors activation by appropriate antagonists was proven to be an efficacious strategy for the treatment of insomnia in man. A novel, drug-like bis-amido piperidine derivative was identified as potent dual OX(1) and OX(2) receptor antagonists, highly effective in a pre-clinical model of sleep.

PMID: 21831639 [PubMed - indexed for MEDLINE]

Stress and glutamate

An interesting review on the interaction between stress, glucocorticoids and glutamate just came out on Nature Review of Neuroscience.
The review focuses on the changes that stress induces in high order area of the CNS, but I can't help asking myself: what are the effects on the autonomic nervous system? Are similar in what is here described for the Hippocampus? In other words, does stress affect long term maintenance of "homeostasis"? I am sure that interesting results would be uncovered by such line of research.

The stressed synapse: the impact of stress and glucocorticoids on glutamate transmission:

Nature Reviews Neuroscience 13, 22 (2012).

Authors: Maurizio Popoli, Zhen Yan, Bruce S. McEwen & Gerard Sanacora

Mounting evidence suggests that acute and chronic stress, especially the stress-induced release of glucocorticoids, induces changes in glutamate neurotransmission in the prefrontal cortex and the hippocampus, thereby influencing some aspects of cognitive processing. In addition, dysfunction of glutamatergic neurotransmission is increasingly considered to be a

Friday, December 16, 2011

Cold Heart

Hypothermia and cardiac function are strongly connected, so much that the conservation of a functional cardiac physiology is critical in any attempt to artificially induce deep hypothermia. Hoping this paper can give us more tools to preserve cardiac function in such condition.

Cold-impaired cardiac performance in rats is only partially overcome by cold acclimation.:

J Exp Biol. 2011 Sep 15;214(Pt 18):3021-31
Authors: Hauton D, May S, Sabharwal R, Deveci D, Egginton S

The consequences of acute hypothermia include impaired cardiovascular performance, ultimately leading to circulatory collapse. We examined the extent to which this results from intrinsic limitations to cardiac performance or physiological dysregulation/autonomic imbalance, and whether chronic cold exposure could ameliorate the impaired function. Wistar rats were held at a 12 h:12 h light:dark (L:D) photoperiod and room temperature (21°C; euthermic controls), or exposed to a simulated onset of winter in an environmental chamber by progressive acclimation to 1 h:23 h L:D and 4°C over 4 weeks. In vivo, acute cold exposure (core temperature, T(b)=25°C) resulted in hypotension (approximately -20%) due to low cardiac output (approximately -30%) accompanying a bradycardia (approximately -50%). Cold acclimation (CA) induced only partial compensation for this challenge, including increased coronary flow at T(b)=37°C (but not at T(b)=25°C), maintenance of ventricular capillarity and altered sympathovagal balance (increased low:high frequency in power spectral analysis, PSA), suggesting physiological responses alone were insufficient to maintain cardiovascular performance. However, PSA showed maintenance of cardiorespiratory coupling on acute cold exposure in both groups. Ex vivo cardiac performance revealed no change in intrinsic heart rate, but a mechanical impairment of cardiac function at low temperatures following CA. While CA involved an increased capacity for β-oxidation, there was a paradoxical reduction in developed pressure as a result of adrenergic down-regulation. These data suggest that integrated plasticity is the key to cardiovascular accommodation of chronic exposure to a cold environment, but with the potential for improvement by intervention, for example with agents such as non-catecholamine inotropes.

PMID: 21865514 [PubMed - indexed for MEDLINE]

Thursday, December 15, 2011

Neurosteroidogenesis Is Required for the Physiological Response to Stress: Role of Neurosteroid-Sensitive GABAA Receptors

This paper brings the concept of long feedback to a new level

Neurosteroidogenesis Is Required for the Physiological Response to Stress: Role of Neurosteroid-Sensitive GABAA Receptors:

The hypothalamic-pituitary-adrenal (HPA) axis, which mediates the body's response to stress, is largely under GABAergic control. Here we demonstrate that corticotropin-releasing hormone (CRH) neurons are modulated by the stress-derived neurosteroid, tetrahydrodeoxycorticosterone (THDOC), acting on subunit-containing GABAA receptors (GABAARs). Under normal conditions, THDOC potentiates the inhibitory effects of GABA on CRH neurons, decreasing the activity of the HPA axis. Counterintuitively, following stress, THDOC activates the HPA axis due to dephosphorylation of KCC2 residue Ser940, resulting in a collapse of the chloride gradient and excitatory GABAergic transmission. The effects of THDOC on CRH neurons are mediated by actions on GABAAR subunit-containing receptors since these effects are abolished in Gabrd–/– mice under both control and stress conditions. Interestingly, blocking neurosteroidogenesis with finasteride is sufficient to block the stress-induced elevations in corticosterone and prevent stress-induced anxiety-like behaviors in mice. These data demonstrate that positive feedback of neurosteroids onto CRH neurons is required to mount the physiological response to stress. Further, GABAAR subunit-containing receptors and phosphorylation of KCC2 residue Ser940 may be novel targets for control of the stress response, which has therapeutic potential for numerous disorders associated with hyperexcitability of the HPA axis, including Cushing's syndrome, epilepsy, and major depression.

Wednesday, December 14, 2011

Of rats and men

I have always considered rats so much more developed socially than mice.  And I dream of an experimental setup that would permit to evaluate the rats social response within the pack.

Animal behaviour: Rats rescue others in distress

Basic Rest Activity Cycle on the ramp

This year, an old concept introduced by Nathaniel Kleitman (see Kleitman N., Basic Rest-activity cycle - 22 years later. Sleep 5(4), 311-317 for the most recent review) was brought back to life by two very interesting paper by Bill Blessing. The original Kleitman's idea has been revisited and incorporated into a behavioral theoretical frame in which the thermogenic activity of the brown adipose tissue acts as a key determinant in the setting the level of activity of the animal. The final concept that William Blessing is try to endorse is that the appearance of long period of active behavior are conditioned by the correct functioning of the thermogenetic autonomic system. In other words, if the brain does not get warm up, it will not function well enough to consent a long period of activity.
I can't help but being supportive of research lines like this, that are leaded by a coherent integrative view of the organism functions, therefore inviting everyone to read the two discussed papers.

Physiol Behav. 2011 Nov 15;
Authors: Blessing W, Mohammed M, Ootsuka Y

Laboratory rats, throughout the 24hour day, alternate between behaviorally active and non active episodes that Kleitman called the basic rest-activity cycle (BRAC). We previously demonstrated that brown adipose tissue (BAT), body and brain temperatures and arterial pressure and heart rate increase in an integrated manner during behaviorally active phases. Studies show that eating is preceded by increases in body and brain temperature, but whether eating is integrated into the BRAC has not been investigated. In the present study of chronically instrumented, unrestrained Sprague-Dawley rats, peaks in BAT temperature occurred every 96±7 and 162±16min (mean±SE, n=14 rats) in dark and light periods respectively, with no apparent underlying regularity. With food available ad libitum, eating was integrated into the BRAC in a temporally precise manner. Eating occurred only after an increase in BAT temperature, commencing 15±1min (mean±SE) after the onset of an increase, with no difference between dark and light phases. There was no preprandial or postprandial relation between intermeal interval and amount eaten during a given meal. Remarkably, with no food available the rat still disturbed the empty food container 16±1min (p>0.05 versus ad libitum food) after the onset of increases in BAT temperature, and not at other times. Rather than being triggered by changes in levels of body fuels or other meal-associated factors, in sedentary laboratory rats with ad libitum access to food eating commences as part of the ultradian BRAC, a manifestation of intrinsic brain activity.
PMID: 22115948 [PubMed - as supplied by publisher]
Authors: Ootsuka Y, Kulasekara K, de Menezes RC, Blessing WW

Brown adipose tissue (BAT) thermogenesis occurs episodically in an ultradian manner approximately every 80-100 min during the waking phase of the circadian cycle, together with highly correlated increases in brain and body temperatures, suggesting that BAT thermogenesis contributes to brain and body temperature increases. We investigated this in conscious Sprague-Dawley rats by determining whether inhibition of BAT thermogenesis via blockade of beta-3 adrenoceptors with SR59230A interrupts ultradian episodic increases in brain and body temperatures and whether SR59230A acts on BAT itself or via sympathetic neural control of BAT. Interscapular BAT (iBAT), brain, and body temperatures, tail artery blood flow, and heart rate were measured in unrestrained rats. SR59230A (1, 5, or 10 mg/kg ip), but not vehicle, decreased iBAT, body, and brain temperatures in a dose-dependent fashion (log-linear regression P < 0.01, R(2) = 0.3, 0.4, and 0.4, respectively, n = 10). Ultradian increases in BAT, brain, and body temperature were interrupted by administration of SR59230A (10 mg/kg ip) compared with vehicle, resuming after 162 ± 24 min (means ± SE, n = 10). SR59230A (10 mg/kg ip) caused a transient bradycardia without any increase in tail artery blood flow. In anesthetized rats, SR59230A reduced cooling-induced increases in iBAT temperature without affecting cooling-induced increases in iBAT sympathetic nerve discharge. Inhibition of BAT thermogenesis by SR59230A, thus, reflects direct blockade of beta-3 adrenoceptors in BAT. Interruption of episodic ultradian increases in body and brain temperature by SR59230A suggests that BAT thermogenesis makes a substantial contribution to these increases.

PMID: 21813867 [PubMed - indexed for MEDLINE]

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.

Thursday, December 08, 2011

Neo-Darwinism, the Modern Synthesis and selfish genes: are they of use in physiology?

I like to open this blog with this deep and interesting reading from Denis Noble, the president of the IUPS, recently published on the Journal of Physiology.
Here is the abstract:

Neo-Darwinism, the Modern Synthesis and selfish genes: are they of use in physiology?:
This article argues that the gene-centric interpretations of evolution, and more particularly the selfish gene expression of those interpretations, form barriers to the integration of physiological science with evolutionary theory. A gene-centred approach analyses the relationships between genotypes and phenotypes in terms of differences (change the genotype and observe changes in phenotype). We now know that, most frequently, this does not correctly reveal the relationships because of extensive buffering by robust networks of interactions. By contrast, understanding biological function through physiological analysis requires an integrative approach in which the activity of the proteins and RNAs formed from each DNA template is analysed in networks of interactions. These networks also include components that are not specified by nuclear DNA. Inheritance is not through DNA sequences alone. The selfish gene idea is not useful in the physiological sciences, since selfishness cannot be defined as an intrinsic property of nucleotide sequences independently of gene frequency, i.e. the ‘success' in the gene pool that is supposed to be attributable to the ‘selfish' property. It is not a physiologically testable hypothesis.