Cerebellar function in consolidation of a motor memory

Several forms of motor learning, including classical conditioning of the eyeblink and nictitating membrane response (NMR), are dependent upon the cerebellum, but it is not known how motor memories are stored within the cerebellar circuitry. Localized infusions of the GABA(A) agonist muscimol were used to target putative consolidation processes by producing reversible inactivations after NMR conditioning sessions. Posttraining inactivations of eyeblink control regions in cerebellar cortical lobule HVI completely prevented conditioning from developing over four sessions. In contrast, similar inactivations of eyeblink control regions in the cerebellar nuclei allowed conditioning to develop normally. These findings provide evidence that there are critical posttraining memory consolidation processes for eyeblink conditioning mediated by the cerebellar cortex.     

小脑皮层在兔瞬膜条件反射过程中的调制作用

以音调结合气流刺激兔角膜的训练建立瞬膜条件反射,在条件反射率刚达90%,连续出现三组的学习初始阶段,电解损毁小脑半球第六小叶皮层使 D-I 核的学习相关性电活动和瞬膜条件反射消除,但不影响“非条件”反射,而在经一周巩固训练的动物,损毁小脑皮层上述区域不发生影响。D-I 核的细胞自发电活动在学习初期和记忆巩固时期也有所不同。在学习后期,D-I 核的细胞自发电活动频率减低,和在学习初期与损毁小脑皮层后的频率变化相似。实验结果表明:在瞬膜条件反射过程中,以小脑皮层为主导,对瞬膜条件反射的产生和D-I 核的学习相关性电活动具有调制作用。随着记忆巩固过程,D-I 核脱离皮层的控制而发展成为这一学习模式的记忆痕迹基础部位。     

A New Rapid Protocol for Eyeblink Conditioning to Assess Cerebellar Motor Learning

Mice with spontaneous and induced mutations causing cerebellar phenotypes have provided key insights into how motor-related memories are stored in cerebellar circuits. Delayed eyeblink conditioning is a form of associative motor learning that depends on the cerebellum. However, neurochemical investigation of the underlying mechanisms has been hampered by the long training period (usually several days) required to establish conditioning. Here, we report a new rapid-training protocol that reliably induced delayed eyeblink conditioning within a single day. The associative memory formation depended on the expression of the δ2 glutamate receptor (GluD2) in cerebellar Purkinje cells. It lasted for several weeks, but could be erased by extinction sessions in a single day. In addition, using the rapid protocol, we found that eyeblink conditioning could be induced in juvenile mice at postnatal day 21, and that the Sindbis-virus-mediated expression of GluD2 could rescue the impaired eyeblink conditioning in GluD2-null mice in vivo.     

Double dissociation of amygdala and hippocampal contributions to trace and delay fear conditioning

A key finding in studies of the neurobiology of learning memory is that the amygdala is critically involved in Pavlovian fear conditioning. This is well established in delay-cued and contextual fear conditioning; however, surprisingly little is known of the role of the amygdala in trace conditioning. Trace fear conditioning, in which the CS and US are separated in time by a trace interval, requires the hippocampus and prefrontal cortex. It is possible that recruitment of cortical structures by trace conditioning alters the role of the amygdala compared to delay fear conditioning, where the CS and US overlap. To investigate this, we inactivated the amygdala of male C57BL/6 mice with GABA (A) agonist muscimol prior to 2-pairing trace or delay fear conditioning. Amygdala inactivation produced deficits in contextual and delay conditioning, but had no effect on trace conditioning. As controls, we demonstrate that dorsal hippocampal inactivation produced deficits in trace and contextual, but not delay fear conditioning. Further, pre- and post-training amygdala inactivation disrupted the contextual but the not cued component of trace conditioning, as did muscimol infusion prior to 1- or 4-pairing trace conditioning. These findings demonstrate that insertion of a temporal gap between the CS and US can generate amygdala-independent fear conditioning. We discuss the implications of this surprising finding for current models of the neural circuitry involved in fear conditioning.     

Neonatal maternal separation alters adult eyeblink conditioning and glucocorticoid receptor expression in the interpositus nucleus of the cerebellum

Neonatal maternal separation alters learning and memory. Glucocorticoids also modulate adult learning and memory, and neonatal maternal separation alters forebrain glucocorticoid receptor (GR) concentrations. We used eyeblink classical conditioning to assess the effect of neonatal maternal separation on associative learning. We assessed delay eyeblink conditioning, GR expression, and total neuron number in the interpositus nucleus, a critical site of plasticity in eyeblink conditioning, in adult rats that had undergone either standard animal facilities rearing, handling for 15 min, or maternal separation for either 15 or 60 min per day on postnatal days 2-14. At 2-3 months of age, delay eyeblink classical conditioning was assessed. Brains were processed for GR immunohistochemistry, and GR expression in the interpositus nucleus was assessed using a computer-based densitometry system. Neuron counts and nuclear volumes were obtained from an alternate series of thionin-stained sections. Maternal separation significantly impaired eyeblink conditioning in male but not female rats. Handling and maternal separation did not significantly affect interpositus neuron number and volume. However, prolonged maternal separation significantly increased GR expression in the posterior interpositus in males, and increases were correlated with eyeblink conditioning. In female rats, maternal separation and handling did not significantly alter interpositus neuron number, volume, or GR protein expression, and GR expression did not correlate with eyeblink conditioning. Thus, neonatal maternal separation produces adult deficits in eyeblink conditioning and alterations in GR expression in its neural substrate in a sex-dependent manner.     

Sleep modulates the neural substrates of both spatial and contextual memory consolidation

It is known that sleep reshapes the neural representations that subtend the memories acquired while navigating in a virtual environment. However, navigation is not process-pure, as manifold learning components contribute to performance, notably the spatial and contextual memory constituents. In this context, it remains unclear whether post-training sleep globally promotes consolidation of all of the memory components embedded in virtual navigation, or rather favors the development of specific representations. Here, we investigated the effect of post-training sleep on the neural substrates of the consolidation of spatial and contextual memories acquired while navigating in a complex 3D, naturalistic virtual town. Using fMRI, we mapped regional cerebral activity during various tasks designed to tap either the spatial or the contextual memory component, or both, 72 h after encoding with or without sleep deprivation during the first post-training night. Behavioral performance was not dependent upon post-training sleep deprivation, neither in a natural setting that engages both spatial and contextual memory processes nor when looking more specifically at each of these memory representations. At the neuronal level however, analyses that focused on contextual memory revealed distinct correlations between performance and neuronal activity in frontal areas associated with recollection processes after post-training sleep, and in the parahippocampal gyrus associated with familiarity processes in sleep-deprived participants. Likewise, efficient spatial memory was associated with posterior cortical activity after sleep whereas it correlated with parahippocampal/medial temporal activity after sleep deprivation. Finally, variations in place-finding efficiency in a natural setting encompassing spatial and contextual elements were associated with caudate activity after post-training sleep, suggesting the automation of navigation. These data indicate that post-training sleep modulates the neural substrates of the consolidation of both the spatial and contextual memories acquired during virtual navigation.     

Reevaluating the Role of the Hippocampus in Delay Eyeblink Conditioning

The role of the hippocampus in delay eyeblink conditioning (DEC) remains controversial. Here, we investigated the involvement of the hippocampus in DEC with a soft tone as the conditioned stimulus (CS) by using electrolytic lesions or muscimol inactivation of guinea pig dorsal hippocampus. Interestingly, when a soft tone was used as a CS, electrolytic lesions of the hippocampus significantly retarded acquisition of the conditioned response (CR), and muscimol infusions into hippocampus distinctly inhibited the acquisition and expression of CR, but had no significant effect on consolidation of well-learned CR. In contrast, both electrolytic lesions and muscimol inactivation of hippocampus produced no significant deficits in the CR when a loud tone was used as the CS. These results demonstrate that the hippocampus is essential for the DEC when the delay task was rendered more difficult.     

Maturation of membrane properties of neurons in the rat deep cerebellar nuclei

Patch clamp recordings of neurons in the adult rat deep cerebellar nuclei have been limited by the availability of viable brain slices. Using a new slicing technique, this study was designed to explore the maturation of membrane properties of neurons in the deep cerebellar nuclei (DCN)—an area involved in rat eyeblink conditioning. Compared to whole‐cell current–clamp recordings in DCN in rat pups at postnatal day 16 (P16) to P21, recordings from weanling rats at P22–P40 revealed a number of significant changes including an increase in the amplitude of the afterhyperpolarization (AHP)—an index of membrane excitability which has been shown to be important for eyeblink conditioning—a prolonged interval between the first and second evoked action potential, and an increase in AHP amplitude for hyperpolarization‐induced rebound spikes. This is the first report of developmental changes in membrane properties of DCN which may contribute to the ontogeny of eyeblink conditioning in the rat. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1268–1276, 2014     

A computational study of synaptic mechanisms of partial memory transfer in cerebellar vestibulo-ocular-reflex learning

There is a debate regarding whether motor memory is stored in the cerebellar cortex, or the cerebellar nuclei, or both. Memory may be acquired in the cortex and then be transferred to the cerebellar nuclei. Based on a dynamical system modeling with a minimal set of variables, we theoretically investigated possible mechanisms of memory transfer and consolidation in the context of vestibulo-ocular reflex learning. We tested different plasticity rules for synapses in the cerebellar nuclei and took robustness of behavior against parameter variation as the criterion of plausibility of a model variant. In the most plausible scenarios, mossy-fiber nucleus-neuron synapses or Purkinje-cell nucleus-neuron synapses are plastic on a slow time scale and store permanent memory, whose content is passed from the cerebellar cortex storing transient memory. In these scenarios, synaptic strengths are potentiated when the mossy-fiber afferents to the nuclei are active during a pause in Purkinje-cell activities. Furthermore, assuming that mossy fibers create a limited variety of signals compared to parallel fibers, our model shows partial memory transfer from the cortex to the nuclei.     

豚鼠齿状回颗粒细胞在追踪性眨眼条件反应巩固过程中的活动

海马在追踪性眨眼条件反应的巩固过程中发挥重要作用,但解剖学上与其紧密联系的齿状回在此过程中的作用尚不清楚. 实验拟观察齿状回颗粒细胞在追踪性眨眼条件反应巩固过程中的放电活动,阐明齿状回在此海马依赖任务中所发挥的作用. 条件反射组动物 (n=8) 首先接受 200 ms 声音条件刺激,间隔 600 ms 后,再被给予 200 ms 吹气非条件刺激,多次重复配对,建立追踪性眨眼条件反应. 对照组动物 (n=8) 接受非配对出现的上述两种刺激. 采用在体单细胞外记录技术,研究习得条件反应豚鼠的齿状回颗粒细胞在条件反应巩固过程中的放电活动. 结果显示:a. 通过 14 天的训练,条件反射组动物均建立了追踪性眨眼条件反应,而非配对组动物则没有建立该条件反应;b. 齿状回颗粒细胞在追踪性眨眼条件反应的巩固过程中表现出不同的活动模式,如在声音条件刺激、间隔期或吹气非条件刺激出现后活动的增强. 这些结果提示:齿状回可能参与巩固追踪性眨眼条件反应所需的神经环路,其颗粒细胞在追踪性眨眼条件反应巩固过程中可能编码不同的信息.     

Roles of diverse glutamate receptors in brain functions elucidated by subunit-specific and region-specific gene targeting

Glutamate receptor (GluR) channels play a major role in fast excitatory synaptic transmission in vertebrate central nervous system. We revealed the molecular diversity of the GluR channel by molecular cloning and investigated their physiological roles by subunit-specific gene targeting. NMDA receptor GluRepsilon1 KO mice showed increase in thresholds for hippocampal long-term potentiation and hippocampus-dependent contextual learning. The mutant mice performed delay eyeblink conditioning, but failed to learn trace eyeblink conditioning. GluRepsilon1 mutant suffered less brain injury after focal cerebral ischemia. NMDA receptor GluRepsilon2 KO mice showed impairment of the whisker-related neural pattern formation and suckling response, and died shortly after birth. Heterozygous (+/-) GluRepsilon2 mutant mice were viable and showed enhanced startle response to acoustic stimuli. GluRdelta2, a member of novel GluR channel subfamily we found by molecular cloning, is selectively expressed in the Purkinje cells of the cerebellum. GluRdelta2 KO mice showed impairments of cerebellar synaptic plasticity and synapse stability. GluRdelta2 KO mice exhibited impairment in delay eyeblink conditioning, but learned normally trace eyeblink conditioning. The phenotypes of NMDA receptor subunits and GluRdelta2 mutant mice suggest that diverse GluR subunits play differential roles in the brain functions.     

Vasopressin analog delays extinction of classically conditioned bradycardia

Albino rabbits were subjected to Pavlovian (classical) conditioning and extinction of concomitant heart rate and eyeblink responses. Sixty minutes before each of three extinction sessions animals were treated with 5 or 20 μg/kg of deamino-dicarba-arginine-8-vasopressin or saline. Vasopressin treatment delayed extinction of bradycardiac conditioned responses but did not affect concomitant eyeblink conditioned responses. It was concluded that classically conditioned autonomic responses may be useful tools for studying the effects of peptides on learning.     

Cholinergic facilitation of trace eyeblink conditioning in aging rabbits

The hippocampus is importantly involved in learning and memory, and is severely impacted by aging. In in vitro hippocampal slices, both the post-burst afterhyperpolarization (AHP) and spike-frequency accommodation are reduced in hippocampal pyramidal neurons after hippocampally-dependent trace eyeblink conditioning, indications of increased cellular excitability. The AHP results from the activation of outward potassium currents, including sI(AHP) and muscarine-sensitive I(M). The AHP is significantly increased in aging hippocampal neurons, potentially contributing to age-associated learning deficits. Compounds which reduce the AHP and spike-frequency accommodation could facilitate learning in normal aging or in age-associated dementias such as Alzheimer's disease. The cholinesterase inhibitor metrifonate enhances trace eyeblink conditioning by aging rabbits and reduces the AHP and accommodation in hippocampal CA1 neurons in a dose-dependent manner. These reductions are mediated by muscarinic cholinergic transmission as they are blocked by atropine. Hippocampal neurons from metrifonate treated but behaviorally naive rabbits were more excitable and not desensitized to the effects of metrifonate since the AHP and accommodation were further reduced when metrifonate was bath applied to the neurons. These observations suggest that the facilitating effect of chronic metrifonate on acquisition of hippocampally dependent tasks is mediated at least partially by increasing the baseline excitability of CA1 pyramidal neurons. The issue of whether learning can be facilitated with muscarinic cholinergic agonists, in addition to cholinesterase inhibitors, was addressed by training aging rabbits during intravenous treatment with the M1 agonist CI1017. A dose-dependent enhancement of acquisition was observed, with rabbits receiving 1.0 or 5.0 mg/ml CI1017 showing comparably improved learning rates as those receiving 0.5 mg/ml or vehicle. Sympathetic side effects, mainly excess salivation, were seen with the 5.0 mg/ml dose. Post-training evaluations suggested that the effective doses of CI1017 were enhancing responsivity to the tone conditioned stimulus. These studies suggest that muscarinic cholinergic neurotransmission is importantly involved in associative learning; that learning in aging animals may be facilitated by enhancing cholinergic transmission; and that the facilitation may be mediated through actions on hippocampal neurons.     

Interaction between Purkinje cells and inhibitory interneurons may create adjustable output waveforms to generate timed cerebellar output

We develop a new model that explains how the cerebellum may generate the timing in classical delay eyeblink conditioning. Recent studies show that both Purkinje cells (PCs) and inhibitory interneurons (INs) have parallel signal processing streams with two time scales: an AMPA receptor-mediated fast process and a metabotropic glutamate receptor (mGluR)-mediated slow process. Moreover, one consistent finding is an increased excitability of PC dendrites (in Larsell's lobule HVI) in animals when they acquire the classical delay eyeblink conditioning naturally, in contrast to in vitro studies, where learning involves long-term depression (LTD). Our model proposes that the delayed response comes from the slow dynamics of mGluR-mediated IP3 activation, and the ensuing calcium concentration change, and not from LTP/LTD. The conditioned stimulus (tone), arriving on the parallel fibers, triggers this slow activation in INs and PC spines. These excitatory (from PC spines) and inhibitory (from INs) signals then interact at the PC dendrites to generate variable waveforms of PC activation. When the unconditioned stimulus (puff), arriving on the climbing fibers, is coupled frequently with this slow activation the waveform is amplified (due to an increased excitability) and leads to a timed pause in the PC population. The disinhibition of deep cerebellar nuclei by this timed pause causes the delayed conditioned response. This suggested PC-IN interaction emphasizes a richer role of the INs in learning and also conforms to the recent evidence that mGluR in the cerebellar cortex may participate in slow motor execution. We show that the suggested mechanism can endow the cerebellar cortex with the versatility to learn almost any temporal pattern, in addition to those that arise in classical conditioning.     

Neurochemical mechanisms of food aversion conditioning consolidation in snail helix lucorum

Effects of cycloheximide, protein synthesis inhibitor as well as serotonin receptor antagonist and NMDA receptor antagonist, on food aversion conditioning consolidation were studied in snail Helix lucorum. Food aversion conditioning was absent in snails after application of cycloheximide. Repeated training produced no food aversion conditioning for the same type of food in these snails without cycloheximide application. Food aversion conditioning was absent in snails after metiotepin, nonselective serotonin receptors antagonist, or after MK-801, NMDA glutamate receptors antagonist, applications. At the same time, repeated training produced facilitated food aversion conditioning for the same type of food in these snails. Our experiments were the first which showed that effect on different molecular mechanisms evoked reversible or irreversible disruption of long-term memory consolidation during the same learning. It was suggested that suppression of retrieval produced reversible effect whereas disruption of memory storage initiated irreversible effect on long-term memory consolidation.