The Excitatory Effect Of Orexins On Medial Prefrontal Cortex Neurons In Mice

The micromolecular neuropeptides orexin A and B, also known as hypocretin-1 and -2, are mainly produced by the neurons within the lateral hypothalamic area (LHA). The effects of orexins are mediated by two types of G-protein-coupled receptors, which are called OX1R and OX2R. Although orexin neurons are localized in a subregion of hypothalamus, their projections and orexin receptors are widely distributed in the brain. Accordingly, the peptides are implicated in the regulation of many physiological functions. Much evidence has shown that orexins play an important part in promotion of wakefulness. Additionally, their role in regulation of higher nervous activity has been revealed by recent studies. The prelimbic (PL) area, a part of medial prefrontal cortex (mPFC) in rodent, is critical for higher nervous activity such as cognition and attention, and it is the final target of multiple subcortical arousal systems. It has been found that the mPFC receives the orexinergic projections from LHA and expresses the orexin receptors, whereas only little has been known about the detailed pattern of the distribution of OX1R and OX2R within this region.In the previous work, we have shown that orexin A excites the acutely dissociated pyramidal neurons of the frontal cortical area 1 and 2 (Fr1 and Fr2), via activation of the PKC-PLC signaling cascades and inhibition of K+ currents, providing evidence of the direct excitatory effect of orexins on the PFC. In primary somatosensory cortex, primary motor cortex, primary visual cortex and cingulate cortex, the effect of orexins are found to be highly restricted to sublayer VIb neurons. However, it remained unknown whether the selectivity of the orexins action to sublayer VIb is also present in PL area.In the present study, we firstly observed the distribution of OX1R and OX2R in mice PFC by means of immunohistochemistry. Subsequently, using whole-cell patch-clamp recordings technique, we examined the effect of orexins on neurons in different layers of the PL area. The neurons recorded were visualized by infrared video-microscopy, and most of them were labeled with biocytin to further identify their location. In addition, it was also investigated if OX1R and OX2R were involved in the action of orexins.1. Distribution of OX1R and OX2R in the PFCImmunohistochemistry study of orexin receptors revealed that OX1R and OX2R distributed in a similar pattern in PFC. Most orexin receptors immunoreactive (ORs-IR) cells resembled pyramid neurons, and both somas and processes were stained. In lateral PFC and Fr2 area, immunolabeling was particularly concentrated in layers V and VI, while in anterior cingulate (AC) area and PL area, all of the layers were immunopositive, with layers II/III and V being more strongly labeled than layers I and VI. In contrast, in infralimbic area, another subregion of mPFC, ORs-IR cells were sparsely distributed.2. The excitatory effect of orexin A on PL neurons of layers I～VICurrent-clamp recordings showed that the neurons responsive to orexin A were diffuse throughout layers I～VI, and the higher responsivity were found in layers II/III and V, which were 35.09% (20/57) and 37.93% (22/58) respectively. Bath application of orexin A resulted in sustained depolarization and an increase in action potential frequency of these responsive cells. The excitatory effect was dose-dependent between 100~800 nM. Furthermore, in the presence of TTX, which are known to block synaptic transmission, the depolarizing effect of the peptide was still observed. Altogether, these findings indicate a postsynaptic action of orexin A on PL neurons of all layers.3. The effect of SB-334867 on the orexin A-induced excitationWhen the OX1R antagonist SB-334867 was included in the ACSF, the effect of orexin A was totally blocked on 30.77% neurons (4/13) we tested. On the other 9 cells, the mean depolarization (6.41±0.94 mV) induced by orexin A was notably decreased in compare with that in standard ACSF (10.69±1.53 mV,n=9,p﹤0.01). The inhibitory effect of SB-334867 on orexin A-caused excitation illustrates that the action of orexin A on PL neurons are mediated by OX1R. Additionally, the remaining part of excitation in the presence of SB-334867 suggests OX2R may be also involved in the excitatory effect of the peptide.4. Comparison of the effect of orexin A and B on PL neuronsThere were no statistically differences between the action of orexin A and B when they were applied at 100 nM (orexin A: 4.88±0.20 mV, orexin B: 5.18±0.14 mV, n=4, p﹥0.05 )or 400 nM (orexin A: 7.63±0.66 mV, orexin B: 7.21±0.67 mV, n=11, p﹥0.05). However, at the dose of 800 nM, orexin A-induced depolarization (10.07±0.68 mV) was significantly higher than that induced by orexin B (8.76±0.75 mV, n=17, p﹤0.05). These data shows that the excitatory effect of orexin A is stronger than that of orexin B at a high dose, but similar to that of orexin B at relatively lower doses. Considering the fact that OX1R has a significantly higher affinity for orexin A, whereas OX2R shows a similar affinity for orexin A and B, the results imply both OX1R and OX2R participate in orexins excitation. Moreover, by comparing orexin B-induced depolarization at different doses, we find orexin B also excites PL neurons in a dose-dependent manner.In conclusion, these observations provide morphological and electrophysiological evidence that the direct excitatory effect of orexins is widely spread in PL area of mice. To be specific, in AC area and PL area, ORs-IR cells are diffuse throughout layers I～VI, with stronger immunosignals in layers II/III and V. In addition, by activation of OX1R and OX2R, orexin A and B directly excite PL neurons of layers I～VI, and among these layers, II/III and V show the higher responsivity to the peptides.