SIPA1L1/SPAR1 Interacts with the Neurabin Family of Proteins and is Involved in GPCR Signaling.

Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1; also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy (SRM) in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of G-protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome (FXS).SIGNIFICANCE STATEMENT Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1) is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified G-protein-coupled receptor (GPCR)-signaling regulators. Moreover, SIPA1L1 knock-out (KO) mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.

Perineuronal Nets in the Deep Cerebellar Nuclei Regulate GABAergic Transmission and Delay Eyeblink Conditioning.

Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum.SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions.

Dual involvement of G-substrate in motor learning revealed by gene deletion.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate-deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10-15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

Blockade of the M1 muscarinic acetylcholine receptors impairs eyeblink serial feature-positive discrimination learning in mice.

The serial feature-positive discrimination task requires the subjects to respond differentially to the identical stimulus depending on the temporal context given by a preceding cue stimulus. In the present study, we examined the involvement of the M1 muscarinic acetylcholine receptors using a selective M1 antagonist VU0255035 in the serial feature-positive discrimination task of eyeblink conditioning in mice. In this task, mice received a 2-s light stimulus as the conditional cue 5 or 6 s before the presentation of a 350-ms tone conditioned stimulus (CS) paired with a 100-ms peri-orbital electrical shock (cued trials), while they did not receive the cue before the presentation of the CS alone (non-cued trials). Each day mice randomly received 30 cued and 30 non-cued trials. We found that VU0255035 impaired acquisition of the conditional discrimination as well as the overall acquisition of the conditioned response (CR) and diminished the difference in onset latency of the CR between the cued and non-cued trials. VU0255035 administration to the control mice after sufficient learning did not impair the pre-acquired conditional discrimination or the CR expression itself. These effects of VU0255035 were almost similar to those with the scopolamine in our previous study, suggesting that among the several types of muscarinic acetylcholine receptors, the M1 receptors may play an important role in the acquisition of the conditional discrimination memory but not in mediating the discrimination itself after the memory had formed in the eyeblink serial feature-positive discrimination learning.

Differential regulation of synaptic plasticity and cerebellar motor learning by the C-terminal PDZ-binding motif of GluRdelta2.

The delta2 glutamate receptor (GluRdelta2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluRdelta2-/- mice display ataxia and impaired motor learning. In addition, long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is abrogated, and synapse formation with PFs and climbing fibers (CFs) is severely disturbed in GluRdelta2-/- Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluRdelta2-/- Purkinje cells by the virus-mediated expression of the wild-type GluRdelta2 transgene (Tg(wt)) but not by that of mutant GluRdelta2 lacking the C-terminal seven residues to which several PDZ proteins bind (Tg(DeltaCT7)). These results indicated that the C terminus of GluRdelta2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluRdelta2-/- cerebellum, especially morphological abnormalities at PF and CF synapses, could not be rescued by virus-mediated transient expression. Thus, whether these phenotypes are mediated by the same signaling pathway remains unclear. To address these issues and to further delineate the function of GluRdelta2 in vivo, we generated transgenic mice that expressed Tg(DeltaCT7) on a GluRdelta2-/- background. Interestingly, although Tg(DeltaCT7) restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluRdelta2-/- Purkinje cells. Furthermore, although the gross motor discoordination of GluRdelta2-/- mice was restored, the cerebellar motor learning underlying delayed eyeblink conditioning remained impaired. These results indicate that LTD induction and motor learning are regulated by signaling via the C-terminal end of GluRdelta2, whereas other functions may be differentially regulated by other regions of GluRdelta2.

Orexins increase mRNA expressions of neurotrophin-3 in rat primary cortical neuron cultures.

Orexins and melanin-concentrating hormone (MCH) as orexigenic neuropeptides are present in the lateral hypothalamus, and their receptors are distributed in the cerebral cortex and hippocampus. In the present study, the regulatory effects of orexin-A, orexin-B and MCH on neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) expressions were examined in primary cortical neuron cultures using quantitative real-time PCR. Both orexin-A and orexin-B on 6-day exposure significantly increased the NT-3 mRNA at concentrations of 0.01, 0.1 and 1microM. Orexin-A and B at 1microM led to an increase of twofold or more over the control. However, no such NT-s mRNA increase occurred with exposure to MCH at the same concentrations as orexins. The mRNA expression of BDNF was significantly increased only by orexin-B at 1microM. These findings suggest that orexins, but not MCH, may be an inducer of NT-3 in the cerebral cortex.

Orexin decreases mRNA expressions of NMDA and AMPA receptor subunits in rat primary neuron cultures.

Orexin is one of the orexigenic neuropeptides in the hypothalamus. Orexin neurons in the lateral hypothalamus (LH) project into the cerebral cortex and hippocampus in which the receptors are distributed in high concentrations. Therefore, to elucidate the actions of orexin in the cerebral cortex, we examined its effects on the mRNA expressions of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A, NR2B) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits (GluR1, GluR2) following 6-day application of orexin-A or orexin-B to rat primary cortical neuron cultures. The mRNAs of NR1 and NR2A subunits were significantly decreased by orexin-A and orexin-B at concentrations over 0.1 microM and 0.01 microM, respectively. The mRNA expression of NR2B subunit was also significantly decreased by orexin-A and orexin-B only at the concentration of 1 microM. Moreover, orexin-A and orexin-B at concentrations over 0.01 microM significantly decreased the mRNA expressions of AMPA receptor subunits, GluR1 and GluR2. The present study demonstrated that orexins significantly suppressed RNA expressions of NMDA and AMPA receptor subunits in cortical neuron cultures, suggesting that orexin may regulate the higher functions of the cerebral cortex as well as be involved in energy regulation in the hypothalamus.

Change in hippocampal theta oscillation associated with multiple lever presses in a bimanual two-lever choice task for robot control in rats.

Hippocampal theta oscillations have been implicated in working memory and attentional process, which might be useful for the brain-machine interface (BMI). To further elucidate the properties of the hippocampal theta oscillations that can be used in BMI, we investigated hippocampal theta oscillations during a two-lever choice task. During the task body-restrained rats were trained with a food reward to move an e-puck robot towards them by pressing the correct lever, ipsilateral to the robot several times, using the ipsilateral forelimb. The robot carried food and moved along a semicircle track set in front of the rat. We demonstrated that the power of hippocampal theta oscillations gradually increased during a 6-s preparatory period before the start of multiple lever pressing, irrespective of whether the correct lever choice or forelimb side were used. In addition, there was a significant difference in the theta power after the first choice, between correct and incorrect trials. During the correct trials the theta power was highest during the first lever-releasing period, whereas in the incorrect trials it occurred during the second correct lever-pressing period. We also analyzed the hippocampal theta oscillations at the termination of multiple lever pressing during the correct trials. Irrespective of whether the correct forelimb side was used, the power of hippocampal theta oscillations gradually decreased with the termination of multiple lever pressing. The frequency of theta oscillation also demonstrated an increase and decrease, before and after multiple lever pressing, respectively. There was a transient increase in frequency after the first lever press during the incorrect trials, while no such increase was observed during the correct trials. These results suggested that hippocampal theta oscillations reflect some aspects of preparatory and cognitive neural activities during the robot controlling task, which could be used for BMI.

Systems consolidation requires postlearning activation of NMDA receptors in the medial prefrontal cortex in trace eyeblink conditioning.

The importance of the hippocampus in declarative memory is limited to recently acquired memory, and remotely acquired memory is believed to be stored somewhere in the neocortex. However, it remains unknown how the memory network is reorganized from a hippocampus-dependent form into a neocortex-dependent one. We reported previously that the medial prefrontal cortex (mPFC) is important for this neocortex-dependent remote memory in rat trace eyeblink conditioning. Here, we investigate the involvement of NMDA receptors in the mPFC in this reorganization and determine the time window of their contribution using chronic infusion of an antagonist into the mPFC, specifically during the postlearning consolidation period. The rats with blockade of the mPFC NMDA receptors during the first 1 or 2 weeks after learning showed a marked impairment in memory retention measured 6 weeks after learning, but relearned normally with subsequent conditioning. In contrast, the same treatment had no effect if it was performed during the third to fourth weeks or during the first day just after learning. The specificity of NMDA receptor blockade was confirmed by the reduced long-term potentiation in the hippocampal-prefrontal pathway in these rats. These results suggest that successful establishment of remotely acquired memory requires activation of NMDA receptors in the mPFC during at least the initial week of the postlearning period. Such NMDA receptor-dependent processes may mediate the maturation of neocortical networks that underlies permanent memory storage and serve as a way to reorganize memory circuitry to the neocortex-dependent form.

Impaired motor functions in mice lacking the RNA-binding protein Hzf.

Local protein synthesis in dendrites plays an important role in some aspects of neuronal development and synaptic plasticity. Neuronal RNA-binding proteins regulate the transport and/or translation of the localized mRNAs. Previously, we reported that hematopoietic zinc finger (Hzf) is one of the neuronal RNA-binding proteins that regulate these processes. The Hzf protein is highly expressed in neuronal cells including hippocampal pyramidal neurons and cerebellar Purkinje cells, and plays essential roles in the dendritic mRNA localization and translation. In the present study we demonstrated that mice lacking Hzf (Hzf(-/-) mice) exhibited severe impairments of motor coordination and cerebellum-dependent motor learning. These findings raise the possibility that the post-transcriptional regulation by Hzf may contribute to some aspects of synaptic plasticity and motor learning in the cerebellum.

Purkinje cell activity during classical eyeblink conditioning in decerebrate guinea pigs.

Purkinje cells are the sole output from the cerebellar cortex and play a critical role during classical eyeblink conditioning. The present study revealed for the first time a learning-related change in individual Purkinje cell activity during successive eyeblink conditioning in decerebrate guinea pigs which permitted continuous single unit recording from the simplex lobe of the cerebellar cortex. The pair-conditioned group received paired presentation of the conditioned stimulus (CS) and unconditioned stimulus (US) until the frequency of the conditioned response (CR) exceeded 80%. The control group received a comparable number of the CS and US in a pseudorandom fashion. Responses of Purkinje cells to the CS were classified into four types: excitatory, inhibitory, a combination of the two, or no response. Approximately half of the recorded cells from both groups changed their response type at various conditioning stages. The firing frequency of a Purkinje cell to the CS showed a tendency to decrease in the pair-conditioned group, while it had a tendency to increase in the pseudoconditioned group. This learning-related difference in change of response type was attributable to a difference in the change between the no response and the inhibitory response types. Correlation analysis of the temporal pattern between the neural activity and the CR revealed that most of the cells that developed an inhibitory response by paired conditioning acquired the CR-related temporal pattern. These results suggest that the learning-related Purkinje cells gain an inhibitory response with a temporal pattern correlated with the CR topography.

Role of Muscarinic Acetylcholine Receptors in Serial Feature-Positive Discrimination Task during Eyeblink Conditioning in Mice.

We investigated the role of muscarinic acetylcholine receptors (mAChRs) in eyeblink serial feature-positive discrimination learning in mice using the mAChR antagonist. A 2-s light cue was delivered 5 or 6 s before the presentation of a 350-ms tone paired with a 100-ms periorbital electrical shock (cued trial) but not before the tone-alone presentation (non-cued trial). Mice received 30 cued and 30 non-cued trials each day in a random order. We found that saline-injected control mice were successfully discriminating between cued and non-cued trials within a few days of conditioning. The mice responded more frequently to the tone in cued trials than in non-cued trials. Analysis of conditioned response (CR) dynamics revealed that the CR onset latency was shorter in cued trials than in non-cued trials, despite the CR peak amplitude not differing significantly between the two conditions. In contrast, scopolamine-injected mice developed an equal number of CRs with similar temporal patterns irrespective of the presence of the cue during the 7 days of conditioning, indicating in a failure to acquire conditional discrimination. In addition, the scopolamine administration to the control mice after they had successfully acquired discrimination did not impair the conditional discrimination and expression of pre-acquired CR. These results suggest that mAChRs may play a pivotal role in memory formation in the conditional brain state associated with the feature cue; however they are unlikely to be involved in the development of discrimination after conditional memory had formed in the serial feature-positive discrimination task during eyeblink conditioning.

Time-dependent reorganization of the brain components underlying memory retention in trace eyeblink conditioning.

Many studies have confirmed the time-limited involvement of the hippocampus in mnemonic processes and suggested that there is reorganization of the responsible brain circuitry during memory consolidation. To clarify such reorganization, we chose trace classical eyeblink conditioning, in which hippocampal ablation produces temporally graded retrograde amnesia. Here, we extended the temporal characterization of retrograde amnesia to other regions that are involved in acquisition during this task: the medial prefrontal cortex (mPFC) and the cerebellum. At a various time interval after establishing the trace conditioned response (CR), rats received an aspiration of one of the three regions. After recovery, the animals were tested for their CR retention. When ablated 1 d after the learning, both the hippocampal lesion and the cerebellar lesion group of rats exhibited a severe impairment in retention of the CR, whereas the mPFC lesion group showed only a slight decline. With an increase in interval between the lesion and the learning, the effect of the hippocampal lesion diminished and that of the mPFC lesion increased. When ablated 4 weeks after the learning, the hippocampal lesion group exhibited as robust CRs as its corresponding control group. In contrast, the mPFC lesion and the cerebellar lesion groups failed to retain the CRs. These results indicate that the hippocampus and the cerebellum, but only marginally the mPFC, constitute a brain circuitry that mediates recently acquired memory. As time elapses, the circuitry is reorganized to use mainly the mPFC and the cerebellum, but not the hippocampus, for remotely acquired memory.

The hippocampus plays an important role in eyeblink conditioning with a short trace interval in glutamate receptor subunit delta 2 mutant mice.

Mutant mice lacking the glutamate receptor subunit delta2 exhibit changes in the structure and function of the cerebellar cortex. The most prominent functional feature is a deficiency in the long-term depression (LTD) at parallel fiber-Purkinje cell synapses. These mutant mice exhibit severe impairment during delay eyeblink conditioning but learn normally during trace eyeblink conditioning without the cerebellar LTD, even with a 0 trace interval. We investigated the hippocampal contribution to this cerebellar LTD-independent "0 trace interval" learning. The mutant mice whose dorsal hippocampi were aspirated exhibited severe impairment in learning, whereas those that received post-training hippocampal lesions retained the memory. The wild-type mice showed no impairment in either case. These results suggest that the hippocampal component of the eyeblink conditioning task becomes dominant when cerebellar LTD is impaired.

Characterization of hippocampal theta rhythm in wild-type mice and glutamate receptor subunit delta2 mutant mice during eyeblink conditioning with a short trace interval.

We have shown that glutamate receptor subunit delta2 (GluRdelta2) null mutant mice, which have serious morphological and functional deficiencies in the cerebellar cortex, are severely impaired in delay eyeblink conditioning but not in trace eyeblink conditioning, even with a 0-trace interval. Such 0-trace conditioning does not depend critically on the hippocampus in wild-type mice, but it does in GluRdelta2 mutant mice. Here we examined the hippocampal electroencephalogram (EEG) during 0-trace conditioning in GluRdelta2 mutant and wild-type mice. During the apparatus habituation sessions, the total hippocampal theta activity (4-12 Hz) of GluRdelta2 mutant mice was less than that of wild-type mice. Activity in the higher frequency band (8-12 Hz, type 1) in GluRdelta2 mutant mice was significantly less than it was in wild-type mice, but activity in the lower frequency band (4-8 Hz, type 2) was not. As learning proceeded during the acquisition sessions, the total theta activity decreased in many of the wild-type mice, while this phenomenon was less prominent in GluRdelta2 mutant mice. Further analysis showed that the type 1 activity in wild-type mice increased in the early sessions and then decreased, while that in GluRdelta2 mutant mice did not change. Type 2 activity tended to decrease in both types of mice as the conditioning proceeded. These results indicate that the distribution of hippocampal EEG frequency and its properties during conditioning are different between wild-type and GluRdelta2 mutant mice, suggesting that the cerebellar cortical dysfunction may cause an alteration in the electrophysiological characteristics of the hippocampus.

Role of hippocampal NMDA receptors in trace eyeblink conditioning.

We examined the effects of acute injections of competitive N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV) into the dorsal hippocampus on contextual fear conditioning and classical eyeblink conditioning in C57BL/6 mice. When injected 10 to 40 min before training, APV severely impaired contextual fear conditioning. Thus, APV injection under these conditions was sufficient to suppress hippocampal NMDA receptors. To investigate the role of hippocampal NMDA receptors on eyeblink conditioning, we carried out daily training of mice during 10-40 min after injection of APV. In the delay eyeblink conditioning, in which the unconditioned stimulus (US) is delayed and terminates simultaneously with the conditioned stimulus (CS), APV-injected mice acquired the conditioned responses (CRs) as well as artificial cerebrospinal fluid (aCSF)-injected control mice did. However, in the trace eyeblink conditioning, in which the CS and US were separated by a stimulus-free trace interval of 500 ms, APV-injected mice showed severe impairment in acquisition of the CR. There was no significant difference in pseudo-conditioning between APV- and aCSF-injected mice. These results provide evidence that the NMDA receptor in the dorsal hippocampus is critically involved in acquisition of the CR in long trace eyeblink conditioning.

Scopolamine impairs eyeblink conditioning in cerebellar LTD-deficient mice.

We investigated the effect of the muscarinic acetylcholine receptor (mAChR) antagonist scopolamine on eyeblink conditioning in glutamate receptor subunit 62 null mice, which have severe impairments in cerebellar long-term depression (LTD). Mice were injected intraperitoneally with scopolamine (0.5 mg/kg) or saline, and conditioned using a delay paradigm with tone and periorbital shock but with no overlap between them. The saline-injected mutant mice learned this paradigm normally, as predicted from our previous study. When scopolamine was injected, learning was impaired more severely in the mutant mice than in the wild type mice. Basic sensory and motor performances were not affected. These results suggest that eyeblink conditioning in cerebellar LTD deficient mice depends largely on neural functions susceptible to blocking of mAChRs.

Purkinje cell activity during learning a new timing in classical eyeblink conditioning.

During classical eyeblink conditioning, animals acquire adaptive timing of the conditioned response (CR) to the interstimulus interval (ISI) between the conditioned stimulus (CS) and the unconditioned stimulus (US). To investigate this coding of the timing by the cerebellum, we analyzed Purkinje cell activities during acquisition of new timing after we shifted the ISI. Decerebrate guinea pigs were conditioned to an asymptotic level of learning using a delay paradigm with a 250-ms ISI. A 350-ms tone and a 100-ms electrical shock were used as the CS and US, respectively. As reported previously in other species, Purkinje cells in the simplex lobe exhibited three types of responses to the CS: excitatory, inhibitory, or a combination of the two. After we increased the ISI to 400 ms, the frequency of the CR stayed at an asymptotic level, but the latency of the CR peak became gradually longer. Two types of cells were observed, based on changes in the nature of their response to the CS; one changed its type of response in parallel with learning the new timing, while the other did not. There was no correlation between the type of response before and after we changed the ISI. In some cells, the peak latency of activities became longer or shorter, while the type of response did not change. These results suggest that some Purkinje cells code the timing of the CR, but do not play a consistent role in shaping the CR over a range of ISIs.

Time-limited role of the hippocampus in the memory for trace eyeblink conditioning in mice.

We examined the role of the hippocampus in memory retention after trace eyeblink conditioning in mice. After establishing the conditioned response (CR) in the trace paradigm, mice received a bilateral aspiration of the dorsal hippocampus and its overlying neocortex on the next day (1-day group) or after 4 weeks (4-week group). Control mice received a neocortical aspiration on the same schedule as the hippocampal-lesion group. After 2 weeks of recovery, these groups received additional conditioning for 3 days. Frequency of the CR of the 1-day group was as low as spontaneous values on the first day in the post-lesion session and never reached pre-surgical level during the post-lesion sessions, while that of the control group did reach pre-surgical level during the post-lesion sessions although there was a transient decline just after lesion. In contrast to the 1-day group, the 4-week-hippocampal lesion group retained the CR and showed a further increase, without significant difference from the control group. The temporal pattern of the CR also was unchanged by the hippocampal lesion 4 weeks after learning. These results suggest a time-limited role for the hippocampus in memory retention after trace conditioning in mice: the CR acquired recently requires an intact hippocampus for its retention, but the CR acquired remotely does not. This is similar to the result reported in rabbits. Therefore, the mechanism and time course of memory consolidation after trace eyeblink conditioning may be similar in mice and rabbits.