Effects And Mechanism Of Orexin-A On The Spontaneous Firing Activity Of Hippocampal CA1 Neurons

Orexins(generally known as hypocretin)including two peptides,orexin-A and orexin-B,are produced in the posterior lateral hypothalamus.There are two types of orexin receptors,orexin-1(OX1)and orexin-2(OX2)receptors,which belong to G-protein-coupled receptors.Orexin-A combines to both OX1 and OX2 receptors with similar affinity,while orexin-B mainly combines with OX2 receptors.Hypothalamic orexinergic neurons extend ascending and descending projections to almost all brain areas.Much evidence has indicated that central orexinergic systems play numerous functions including sleep/wakefulness,energy metabolism,feeding behavior,neuroendocrine and motor control.Hippocampus,as a major component of the limbic system in brain,plays important roles in learning,memory and cognition.According to the cellular morphology and nerve fiber formation of different regions,hippocampus is separated into CA1,CA2,CA3 and CA4 regions.Morphological studies have shown that the hippocampal CA1 regions receive orexinergic innervation originating from the hypothalamus.Both OX1 and OX2 receptors are expressed in the hippocampus.OX1 receptor mRNAs are expressed in the CA1 and CA2 regions,while OX2 receptor mRNAs are most dense in the CA3 regions of the hippocampus.Previous behavioral studies have shown that intracerebroventricular administration of orexin-A modulates learning and memory in rats.Blockade of hippocampal OX1 receptor impairs acquisition and consolidation of spatial memory.However,up to now,little is known about the direct electrophysiological effects of orexin-A on hippocampal CA1 neurons.Aim:To study the effects and mechanism of orexin-A on the spontaneous firing activity of hippocampal CA1 neurons.Methods:Multibarrel single-unit extracellular recordings and immunohistochemistry were employed in the present studies.The male Wistar rats were anaesthetized with urethane and positioned in the stereotaxic frame(Narishige).A craniotomy was performed at the site of 3.0-3.8 mm posterior and 2.0-2.4 mm lateral from the bregma according to the stereotaxic atlas.Three-barrel microelectrodes were pulled by a Stoelting pipette puller with the tip of 3-10?m in diameter and 10-20 M? in resistance.One of the three barrels was filled with 0.5 M sodium acetate with 2%pontamin sky blue dye,using as a recording electrode.The other two were used as drug application barrels.The electrical signals were amplified by a micro-electrode amplifier and the firing rate and coefficient of variation(CV)were analyzed with spike2 software.Paired-samples t-test was used to compare the difference of spontaneous firing before and after drug application.Statistical comparisons between two groups were determined with Mann-Whitney test.Chi-Square test was used to compare the fractions of different firing pattern types between orexin-A responsive and non-responsive neurons.Results:1.In 15 out of the 23 hippocampal CA1 neurons recorded,micro-pressure administration of 0.01mM orexin-A significantly increased the spontaneous firing rate from 2.96±0.85 Hz to 8.45±1.86 Hz(t=4.89;n=15;P<0.001).The average increase was 411.78±125.46%,which was significantly increased(Z=3.87;P<0.001)compared to that of vehicle group(basal:2.71 ±0.86 Hz;normal saline:2.55±0.87 Hz;t=0.56;n=8;P=0.591).Orexin-A did not change the CV significantly(basal:1.23±0.09,orexin-A:1.36±0.11;t=1.20;n=15;P=0.250)in the 15 neurons.2.In 7 out of the 17 hippocampal CA1 neurons recorded,micro-pressure administration of OX1 receptor antagonist SB-334867(0.1 ?M)decreased the spontaneous firing rate from 4.02± 1.08 Hz to 2.11 ±0.58 Hz(t=2.68;n=7;P=0.036),suggesting that endogenous orexinergic systems modulate the firing activity of CA1 neurons through OX1 receptors.However,micro-pressure administration of OX2 receptor antagonist TCS-OX2-29 at 0.1 ?M did not change the basal firing rate significantly(basal:3.11±0.64 Hz;TCS-OX2-29:3.07±0.71 Hz;t=0.11;n=10;P=0.918).3.Furthermore,co-application of SB-334867 completely blocked orexin-A-induced excitation of hippocampal CA1 neurons(basal:3.13±0.90 Hz;SB-334867+orexin-A:3.08±0.79Hz;t=0.09;n=9;P=0.928).4.Pre-application of the PLC pathway inhibitor,U73122,completely blocked orexin-A-induced excitation of CA1 neurons(basal:3.11±0.48 Hz;orexin-A+U73122:3.33±0.66 Hz;t=0.94;n=8;P=0.376),which indicated the possible involvement of PLC pathway in orexin-A-induced increase of firing rate.5.Immunostaining revealed that strongly positive expression of orexin-A was located on both soma and neuropil of the lateral hypothalamus neurons.In hippocampal CA1 regions,orexin-A was expressed on the neuropil.Further double immunostaining showed that OX1 receptors were expressed on both PV negative neurons and PV positive interneurons in hippocampal CA1 regions.Conclusion:Our present in vivo single unit recordings demonstrated that orexin-A increased the spontaneous firing rate of hippocampal CA1 neurons via OX1 receptors.Endogenous orexins are involved in the modulation of spontaneous firing activity of hippocampal CA1 neurons.The PLC pathway may be involved in activation of OX1 receptor-induced excitation of CA1 neurons.The present studies may provide a rational for further investigation of the modulation of orexin on learning and memory as well as its possible involvement in Alzheimer's disease.