Fabienne Liénard

Olfactory discrimination ability and brain expression of c-fos, Gir and Glut1 mRNA are altered in n-3 fatty acid-depleted rats.

The long-chain polyunsaturated n-3 fatty acids (n-3 PUFA), particularly docosahexaenoic acid (DHA), are abundantly present in the central nervous system and play an important role in cognitive functions such as learning and memory. We, therefore, investigated the effects of n-3 PUFA-depletion in rats (F2 generation) on the learning of an olfactory discrimination task, progressively acquired within a four-arm maze, and on the mRNA expression of some candidate genes, i.e., c-fos, Gir and glucose transporter (Glut1), which could reflect the level of cerebral activity. We observed that DHA contents were dramatically decreased in the olfactory bulb, the piriform cortex and the neocortex of n-3-depleted rats. Furthermore, the n-3 deficiency resulted in a mild olfactory learning impairment as these rats required more days to master the olfactory task compared to control rats. Real-time RT-PCR experiments revealed that the training induced the expression of c-fos mRNA in all the three regions of the brain whereas Gir and Glut1 mRNA were induced only in olfactory bulb and neocortex. However, such an increase was less marked in the n-3-deficient rats. Taken together, these results allow us to assume that the behavioural impairment in n-3-deficient rats is linked to the depletion of n-3 fatty acids in brain regions processing olfactory cues. Data are discussed in view of the possible role of some of these genes in learning-induced neuronal olfactory plasticity.

Learning-stage dependent Fos expression in the rat brain during acquisition of an olfactory discrimination task.

By using Fos immunocytochemistry, we investigated the activation in olfactory-related areas at three stages (the first and fourth days of conditioning and complete acquisition) of an olfactory discrimination learning task. The trained rats (T) had to associate one odour of a pair with water-reward within a four-arm maze whereas pseudo-trained (P) rats were only submitted to the olfactory cues without any reinforcement. In the piriform cortex, both T and P rats exhibited a higher immunoreactivity on the first day, which seemed to indicate a novelty-related Fos expression in this area, but whatever the learning-stage, no significant difference in Fos expression between T and P rats was observed. In hippocampus, Fos expression was significantly different between T and P rats in CA1 and CA3 on the first and fourth days respectively. Thus we showed a differential activation of CA1 and CA3 subfields which might support a possible functional heterogeneity. In the orbitofrontal cortex, Fos immunoreactivity was significantly higher in T rats compared to P rats when mastery of the discrimination task was complete. In contrast, no learning-related Fos expression was found in infralimbic and prelimbic cortices. The present data suggest an early implication of the hippocampal formation and a later involvement of neocortical areas throughout different stages of a progressively acquired olfactory learning task.

Cue valence representation studied by Fos immunocytochemistry after acquisition of a discrimination learning task

The piriform cortex (PCx) and related structures such as hippocampus and frontal cortex could play an important role in olfactory memory. We investigated their involvement in learning the biological value of an odor cue, i.e. predicting reward or non-reward in a two-odor discrimination task. Rats were sacrificed after stimulation by either rewarded or non-rewarded odor and Fos immunocytochemistry was performed. The different experimental groups of rats did not show strongly differentiated Fos expression pattern in either the PCx or the hippocampus. A few differences were noted in frontal areas. In the ventro-lateral orbital cortex, rats, ramdomly rewarded during the conditionning had a higher Fos level in comparison with other groups. In infralimbic cortex, rats, which learned the reward value of the olfactory cue and were water-reinforced the day of sacrifice, showed a higher Fos expression. Data are discussed in view of the olfactory learning paradigm and of the accuracy of the control groups used in the present experimental design. The behavioural conditions leading to Fos expression are further discussed since Fos is a marker of learning-induced plasticity as well as a general activity marker which can be activated by a wide range of stimuli not directly linked to memory.

Angiotensin II receptor subtype antagonists can both stimulate and inhibit salt appetite in rats

In urethane-anaesthetised male Wistar rats iontophoretic application of the angiotensin II (Ang II) type 1 (AT-1) receptor specific nonpeptide antagonist losartan in the septo-preoptic continuum can produce neuronal excitation of short- and long-term duration which has been interpreted as removal of tonic Ang II-induced inhibition. Mineralocorticoid pretreatment, which increases neuronal sensitivity to Ang II to enhance salt appetite, also removes this losartan-induced long-term excitation. These results suggested steroid modulation of the AT-1 receptor and a complex involvement of Ang II in salt appetite. To investigate the role of the inhibitory action of central Ang II on salt appetite, we gave intracerebroventicular injections of a single, low dose (10 ng) of losartan in normal euhydrated rats and this produced, paradoxically, a progressive increase in salt intake (1.6 +/- 0.3 ml/day to 3.5 +/- 0.9 ml/day, n = 15, P < 0.05). Treatment of these salt enhanced rats with DOCA (0.5 mg/day, s.c., for 3 days) further increased the salt appetite, but then a second i.c.v. injection of the same dose of losartan significantly inhibited the enhanced salt appetite (4.7 +/- 0.7 to 1.3 +/- 0.4, n = 9, P < 0.05). These results provide evidence for a complex action of Ang II on the At-1 receptor mediating the generation of salt appetite that appears to involve either at least two functional subtypes of this AT-1 receptor, as already suggested by previous electrophysiological experiments, or one AT-1 receptor with several activation states.

Transient Receptor Potential Canonical 3 (TRPC3) Channels Are Required for Hypothalamic Glucose Detection and Energy Homeostasis

The mediobasal hypothalamus (MBH) contains neurons capable of directly detecting metabolic signals such as glucose to control energy homeostasis. Among them, glucose-excited (GE) neurons increase their electrical activity when glucose rises. In view of previous work, we hypothesized that transient receptor potential canonical type 3 (TRPC3) channels are involved in hypothalamic glucose detection and the control of energy homeostasis. To investigate the role of TRPC3, we used constitutive and conditional TRPC3-deficient mouse models. Hypothalamic glucose detection was studied in vivo by measuring food intake and insulin secretion in response to increased brain glucose level. The role of TRPC3 in GE neuron response to glucose was studied by using in vitro calcium imaging on freshly dissociated MBH neurons. We found that whole-body and MBH TRPC3-deficient mice have increased body weight and food intake. The anorectic effect of intracerebroventricular glucose and the insulin secretory response to intracarotid glucose injection are blunted in TRPC3-deficient mice. TRPC3 loss of function or pharmacological inhibition blunts calcium responses to glucose in MBH neurons in vitro. Together, the results demonstrate that TRPC3 channels are required for the response to glucose of MBH GE neurons and the central effect of glucose on insulin secretion and food intake.

Role of the basolateral amygdala in retrieval of conditioned flavors in the awake rat.

Learned association between odor, taste and further post-ingestive consequence is known as flavor nutrient conditioned preference. Amygdala is supposed to be one of the areas involved in these associations. In the present study, one flavor was associated with a 16% glucose (CS(+)) whereas another flavor was paired with less reinforcing 4% glucose (CS(-)). We showed that CS(+) presentation after conditioning increased Fos expression in the basolateral nucleus of amygdala (BLA). Furthermore, we performed electrophysiological recordings in the BLA in free moving rats. After preference acquisition, rats were exposed to either the CS(+) or the CS(-). The proportion of neurons showing a decreased activity during the CS(-) presentation was significantly higher in conditioned rats compared to controls. Among this neuronal population recorded in conditioned rats, we noticed a significant proportion of neurons that also showed a decreased activity during the CS(+) presentation. Our data indicate an involvement of BLA during retrieval of learned flavors. It also suggests that both flavors might have acquired a biological value through conditioning.

Water versus salty taste and Iontophoretic ANGII responses of septopreoptic neurons in dehydrated and euhydrated awake rats.

Little is known of the influence of gustatory, particularly salt, input on neurons of the forebrain and if the same neurons are sensitive to hydromineral balance humoral stimuli. In awake, nonpremedicated rats we recorded the activity of spontaneously active neurons in the preoptic/anterior hypothalamic area of dehydrated and euhydrated rats while allowing them to ingest water or a hypertonic salt solution (1.6% NaCl) administered to the tongue. The hormones angiotensin and aldosterone, both implicated in hydromineral balance, were applied by iontophoresis to the same neurons. In the dehydrated rats, 27% (15/55) of the spontaneously active neurons responded to a liquid (either water or the NaCl) applied to the tongue; in the euhydrated rats 23% (18/78) responded to the same stimuli. In the dehydrated rats, however, 33% (5/15) of the responding neurons were inhibited when the NaCl solution was applied to the tongue compared with only 5% (1/18) in the euhydrated rats. Iontophoretic application of angiotensin increased the spontaneous activity in 21% of those neurons tested that responded to taste. These results suggest that the state of hydration of an animal is able to change the neuronal response to substances applied to the tongue. Furthermore, it appears that these gustatory-sensitive neurons may also be related to hydromineral balance regulation since they are able to respond to angiotensin.

Animal Models and Methods to Study the Relationships Between Brain and Tissues in Metabolic Regulation

Abstract The constant increase in the number of obese and diabetic patients, which has become a concern of public health, is the consequence of dysregulations in energy homeostasis. Communications between the brain and peripheral tissues play a critical role in this regulation. Studying the brain-periphery axis has become a critical field of research. This chapter lists a panel of concepts, approaches, tools and techniques scientists possess to study the brain-periphery axis in the regulation of energy homeostasis. We focused on techniques used in vivo to stimulate the brain such as the stereotaxy, electrical stimulation, vascular surgery and optogenetic. We described tools and approaches used to study in vivo and in vitro response of neural cells to metabolic stimuli such as electrophysiology, cellular imaging, microdialysis and c-fos mapping. Finally, approaches used to study peripheral behavioral and metabolic responses such as food intake and body weight monitoring and glucose clamps are presented.