Imaging olfactory network activity in a semi-intact nose-brain preparation: On the origin of spontaneous theta oscillations in the olfactory bulb

以θ节律（4-8赫兹）的神经元活动振荡发生在许多脊椎动物的神经元群中，最著名的是在与海马区相连的回路中。在嗅觉系统中，已知θ波振荡使老鼠和人类的嗅觉编码正常。它们的起源与呼吸活动有关，呼吸活动主要通过机械敏感、嗅觉和三叉神经通路影响鼻子的感觉输入。我们在半完整的鼻脑制备中观察到嗅球中与呼吸无关的θ振荡，表明这种振荡可以自发发生，没有任何感觉或离心输入。我们的主要工作假设是，呼吸和球活动的呼吸模式利用嗅球网络的内在共振；在鼻脑准备的球中观察到的自发θ振荡是这种共振的相关，并且可以被呼吸样感觉输入所引导。为了研究这一想法，我们计划将鼻脑准备与我们在双光子成像技术方面的专业知识结合起来，在单个神经元和集合水平上探索网络功能，从而揭示自发θ波的起源及其与完整网络中感觉输入的相互作用。自发θ波振荡起源的一个强有力的候选机制是小球旁细胞类型的内在起搏，即外部簇状细胞（ETC），因为已知ETCs在θ波频率范围内产生自发的活动爆发。因此，该项目的第一步是基于已有的共振扫描双光子显微镜系统，在鼻脑制备中充分建立肾小球层内的Ca2+群成像。由于ETC靠近球表面，并且它们的爆发已知会产生大量的Ca2+内流，因此ETC体细胞信号是Ca2+成像的绝佳目标。接下来，我们将研究与局部场电位记录并行的ETCs的自发活动，并通过药理学手段测试它们对θ节律的贡献。最后，我们将探讨嗅觉刺激、呼吸样气流和ETC活动之间的耦合，以及主要二尖瓣细胞和簇状细胞的平行活动。这种方法将使我们能够研究鼻-脑制备过程中活动的生理模式，从而研究体外系统中相关的网络特性。综上所述，我们的目的是阐明嗅球内在θ节律的机制，以及它如何与主要神经元的感觉输入和嗅球输出相互作用。我们的研究也可能有助于理解大脑其他区域的θ波编码。

Oscillations of neuronal activity at the theta rhythm (4-8 Hz) occur in many vertebrate neuronal ensembles, most famously within circuits linked to hippocampal areas. In the olfactory system, theta oscillations are known to enable proper olfactory coding in rats and humans. Their origin has been tied to respiratory activity which can effect sensory input through the nose mainly by mechanosensitive, olfactory and trigeminal pathways. Our observation of respiration-independent theta oscillations in the olfactory bulb in an decerebrated semi-intact nose-brain preparation indicates that such oscillations can occur spontaneously, without any sensory or centrifugal inputs. Our main working hypothesis is that breathing and therewith respiratory patterning of bulbar activity tap an intrinsic resonance of the olfactory bulb network; the spontaneous theta oscillations observed in the bulb of the nose-brain preparation are a correlate of this resonance and can be entrained by respiratory-like sensory input. To investigate this idea, we plan to combine the nose-brain preparation with our expertise in two-photon imaging techniques to explore network function at the single neuron and ensemble level and thereby unravel the origin of spontaneous theta and its interaction with sensory input in an intact network. A strong candidate mechanism for the origin of spontaneous theta oscillations is intrinsic pacemaking in a juxtaglomerular cell type, the external tufted cell (ETC), since ETCs are known to produce spontaneous bursts of activity within the theta frequency range. Thus the first step in this project is to fully establish Ca2+ population imaging within the glomerular layer in the nose-brain preparation, based on a pre-existing resonant scanning two-photon microscope system. Because the ETCs are located close to the bulbar surface and their bursts are known to yield substantial Ca2+ influx, ETC somatic signals are an excellent target for Ca2+ imaging. Next we will investigate the spontaneous activity of ETCs in parallel with local field potential recordings and test their contribution to the theta rhythm by pharmacological means. Finally we will explore the coupling between sensory stimulation with odorants, respiratory-like airflow and ETC activity, as well as the parallel activity of the principal mitral and tufted cells. Such an approach will allow us to investigate physiological patterns of activity in the nose-brain preparation and thus study releavant network properties in an in vitro system.In summary, we aim to elucidate the mechanism of the intrinsic olfactory bulb theta rhythm and how it interacts with both sensory input and olfactory bulb output by the principal neurons. Our investigations might also contribute to the understanding of theta coding in other brain areas.