Putting mice into hibernation causes a major loss of synapses
Putting mice into hibernation causes a major loss of synapses
让小鼠进入冬眠会导致突触大量丢失
Our leading hypothesis for how our memories are stored is that when you learn something, the connections among neurons involved get stronger and physically larger, and that constitutes the memory. The trouble is that these connections significantly change over time—they’re plastic. “If you compare the arrangement of these connections on day one with the same on day four or five, it’s very, very different,” says Kazumasa Tanaka, a neuroscientist at the Okinawa Institute of Science and Technology Graduate University in Japan. 关于记忆如何存储，目前主流的假说是：当你学习某样东西时，相关神经元之间的连接会变得更强、物理体积更大，从而构成了记忆。问题在于，这些连接会随着时间推移发生显著变化——它们具有可塑性。日本冲绳科学技术大学院大学的神经科学家田中一正（Kazumasa Tanaka）表示：“如果你比较第一天和第四或第五天这些连接的排列方式，会发现它们非常、非常不同。”
To learn how a memory that can last for years can sit on hardware that shifts every few days, Tanaka’s team made the shift a bit more dramatic. In a recent Science study, they induced a hibernation-like state in mice, which basically erased the state of more than half of their synapses. And yet the mice apparently have kept their memories. 为了探究记忆为何能持续数年，却存储在每隔几天就会发生变化的“硬件”上，田中团队将这种变化变得更加剧烈。在最近发表于《科学》杂志的一项研究中，他们诱导小鼠进入了一种类似冬眠的状态，这基本上抹去了小鼠超过一半的突触状态。然而，小鼠似乎依然保留了它们的记忆。
Hibernation on demand
按需冬眠
Hibernation is a specialty of squirrels, hamsters, and bears, but the neural circuit that triggers it is conserved across mammals, and is present in species that never hibernate in the wild—like mice. In June 2020, a team of researchers led by Takeshi Sakurai, a neuroscientist at the University of Tsukuba and a collaborator on Tanaka’s study, developed a technique to artificially activate this hibernation circuit. This can be done by activating a population called Q neurons in a region of the hypothalamus. 冬眠是松鼠、仓鼠和熊的特长，但触发冬眠的神经回路在哺乳动物中是保守的，即使是在野外从不冬眠的物种（如小鼠）体内也存在。2020年6月，由筑波大学神经科学家、田中研究的合作者樱井武（Takeshi Sakurai）领导的研究团队开发出一种人工激活该冬眠回路的技术。通过激活下丘脑区域中被称为“Q神经元”的群体，即可实现这一过程。
The result is a state dubbed QIH, for Q-neuron-induced hypothermia and hypometabolism. “With our protocol, we can bring mice’s body temperature down to somewhere around 20° Celsius, and their heart rate and breathing rate decrease significantly as well,” Tanaka explains. 这种状态被称为QIH，即“Q神经元诱导的体温过低和代谢减低”。田中解释说：“通过我们的方案，我们可以将小鼠的体温降至20摄氏度左右，它们的心率和呼吸频率也会显著下降。”
Whether that counts as real hibernation depends on which hibernator serves as a reference. Bears in a hibernation state reduce their metabolic demand but don’t change their body temperature, which remains around 36° or 37° Celsius. On the other extreme, some species of squirrels enter super deep hibernation where their body temperature can go very close to freezing. “Artificial hibernation sits somewhere in the middle of that spectrum,” Tanaka says. 这是否算作真正的冬眠，取决于以哪种冬眠动物作为参考。熊在冬眠状态下会降低代谢需求，但体温不会改变，仍保持在36或37摄氏度左右。而在另一个极端，某些种类的松鼠会进入极深度的冬眠，体温甚至接近冰点。田中说：“人工冬眠处于这个光谱的中间位置。”
The most important thing about QIH, though, is that it can be switched on and off at will. In Tanaka’s experiments the mice spent 48 hours in the hibernation-like state and then woke up. For their synapses, these 48 hours worked like Thanos’ snap. 不过，QIH最重要的一点是它可以随意开启和关闭。在田中的实验中，小鼠在类似冬眠的状态下度过了48小时，然后被唤醒。对于它们的突触而言，这48小时就像灭霸打了一个响指。
Synaptic purge
突触清洗
To find out how many synapses are lost during QIH, Tanaka’s team implanted tetrodes, bundles of fine electrodes, into the hippocampus of freely moving mice. These let them record individual neurons firing. They found that activity dropped by about 70 percent once hibernation set in. The brain tissues of some animals were imaged with a technique called serial block-face scanning electron microscopy before hibernation, during it, and days after returning to consciousness. It turned out the hibernation eradicated more than half of the synapses—in principle, this should erase most of the memories. 为了弄清楚在QIH期间丢失了多少突触，田中团队在自由活动的小鼠海马体中植入了四极管（一种细电极束）。这使他们能够记录单个神经元的放电情况。他们发现，一旦进入冬眠，神经活动就会下降约70%。研究人员利用一种称为“连续切片扫描电子显微镜”的技术，对部分动物在冬眠前、冬眠中以及苏醒数天后的脑组织进行了成像。结果显示，冬眠消除了超过一半的突触——按理说，这应该会抹去大部分记忆。
“If you accept that memory traces reside in the efficacy of individual synapses, if you lose more than half of the synaptic connections, of course what you’d expect is impairment of the memory afterwards,” Tanaka says. But the team found no such impairment. 田中说：“如果你认为记忆痕迹存在于单个突触的效能中，那么当你失去超过一半的突触连接时，你当然会预期记忆会受到损害。”但研究团队并没有发现这种损害。
Before hibernation, the mice had been trained on two standard memory tasks. One was contextual fear conditioning, in which an animal learns to associate a particular box with a mild electric shock. The second was a plus-maze task in which the mouse learns to navigate its way to a reward. Performance in both tasks depends on memories stored in the hippocampus, which the team confirmed by creating a lesion in the region after training, which caused the memories to disappear. 在冬眠前，小鼠接受了两种标准记忆任务的训练。一种是情境恐惧条件反射，动物学习将特定的盒子与轻微电击联系起来。第二种是十字迷宫任务，小鼠学习导航以获得奖励。这两项任务的表现都依赖于海马体中存储的记忆，研究团队通过在训练后对该区域造成损伤导致记忆消失，证实了这一点。
When other mice, ones that were put into QIH, were aroused, though, they performed on these tasks just as well as the mice that did not hibernate. “What we found in these two different behavioral paradigms is the memory was completely intact,” Tanaka claims. The survival of these memories through the purge of the synapses was also confirmed by brain activity recordings. Place cells, hippocampal neurons that fire when an animal occupies a particular spot, still fired in the same locations after arousal. And a decoder that read out the population activity could reconstruct where the mouse was just as accurately as before. 然而，当其他进入QIH的小鼠被唤醒时，它们在这些任务上的表现与未冬眠的小鼠一样好。田中声称：“我们在两种不同的行为范式中发现，记忆是完全完整的。”这些记忆在突触清洗中幸存下来，也得到了脑活动记录的证实。位置细胞（当动物处于特定位置时会放电的海马神经元）在苏醒后仍在相同位置放电。而读取群体活动的解码器，也能像以前一样准确地重建小鼠的位置。
Returning synapses
突触回归
Watching the same dendrites over eight days revealed that the synapses that vanished during hibernation reappeared after arousal and 82 percent of them came back at the same spot on the same dendrite they had occupied before, far above what chance would produce. The synapses that vanished weren’t a random sample either. 在八天内观察同一树突发现，冬眠期间消失的突触在苏醒后重新出现，其中82%回到了它们之前占据的同一树突的同一位置，这远高于随机概率。消失的突触也不是随机样本。
Scientists have developed a technique called eGRASP, which makes a connection glow green only where a neuron tagged during learning is connected to another neuron tagged during the same learning event. The team used this to look specifically at engram synapses, specialized synaptic connections between memory-storing neurons. It turned out that engram synapses sitting alone on a dendrite were eliminated by hibernation; engram synapses arranged in tight spatial clusters were preserved. 科学家开发了一种名为eGRASP的技术，它能使连接处发出绿光，前提是学习过程中被标记的神经元与同一学习事件中被标记的另一个神经元相连。研究团队利用该技术专门观察了“记忆印迹突触”（engram synapses），即存储记忆的神经元之间专门的突触连接。结果发现，孤立在树突上的记忆印迹突触被冬眠消除了；而排列在紧密空间簇中的记忆印迹突触则被保留了下来。
Why clustering protects them is not yet understood, though. “Mechanism-wise, we don’t know,” Tanaka says. “That is one of the ongoing projects in the laboratory.” What the team could do is look at the architecture of these clusters. Multisynaptic boutons Tracing the surviving clusters, Tanaka’s team found that a third of the clustered engram synapses were attached to something called a multisynaptic bouton. “Usually a single presynaptic terminal makes a synaptic connection with a single postsynaptic spine—a one-to-one relationship,” Tanaka explains. “In multisynaptic boutons, one presynaptic terminal makes synaptic connections with multiple postsynaptic spines.” 然而，为什么聚类能保护它们尚不清楚。田中说：“从机制上讲，我们还不知道。这是实验室正在进行的项目之一。”研究团队目前能做的是观察这些簇的结构。多突触扣结（Multisynaptic boutons）：在追踪幸存的簇时，田中团队发现三分之一的簇状记忆印迹突触附着在一种称为“多突触扣结”的结构上。田中解释说：“通常，单个突触前末梢与单个突触后棘形成突触连接——这是一种一对一的关系。而在多突触扣结中，一个突触前末梢与多个突触后棘形成突触连接。”