Shigenori Kawahara

Classical eyeblink conditioning in glutamate receptor subunit δ2 mutant mice is impaired in the delay paradigm but not in the trace paradigm

In mice lacking glutamate receptor subunit δ2 (GluRδ2–/– mice), cerebellar long‐term depression (LTD) at the parallel fibre–Purkinje cell synapses is disrupted. Unlike the cerebellar LTD‐deficient mice previously used for eyeblink conditioning, however, the abnormalities of the GluRδ2–/– mice are restricted to the cerebellar cortex. In delay eyeblink conditionings (interstimulus interval of 252 and 852 ms), in which the conditioned stimulus (CS) overlaps temporally with a coterminating unconditioned stimulus (US), GluRδ2–/– mice are severely impaired in learning, strongly supporting the hypothesis that cerebellar cortical LTD is essential for delay conditioning. In the trace paradigm, in which a stimulus‐free trace interval of 500 ms intervened between the CS and US, GluRδ2–/– mice learned as successfully as wild‐type mice, indicating that cerebellar LTD is not necessary for trace conditioning. Thus, the present study has revealed a cerebellar LTD‐independent learning in eyeblink conditioning.

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.

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.

Impairment of eyeblink conditioning in GluRδ2-mutant mice depends on the temporal overlap between conditioned and unconditioned stimuli

Mice lacking the glutamate receptor subunit δ2 (GluRδ2) are deficient in cerebellar long‐term depression (LTD) at the parallel fibre–Purkinje cell synapses. We conducted delay and trace eyeblink conditioning with these mice, using various temporal intervals between the conditioned stimulus (CS) and unconditioned stimulus (US). During trace conditioning in which a stimulus‐free trace interval (TI) of 250, 100 or 50 ms intervened between the 352‐ms tone CS and 100‐ms US, GluRδ2‐mutant mice learned as successfully as wild‐type mice. Even in the paradigm with TI = 0 ms, in which the end of CS and onset of US are simultaneous, there was no difference between the GluRδ2‐mutant and wild‐type mice in their acquisition of a conditioned response. However, in the delay paradigm in which the 452‐ms CS overlapped temporally with the coterminating 100‐ms US, GluRδ2‐mutant mice exhibited severe learning impairment. The present study together with our previous work [Kishimoto, Y., Kawahara, S., Suzuki, M., Mori, H., Mishina, M. & Kirino, Y. (2001) Eur. J. Neurosci.,13, 1249–1254], indicates that cerebellar LTD‐independent learning is possible in paradigms without temporal overlap between the CS and US. On the other hand, GluRδ2 and cerebellar LTD are essential for learning when there is CS–US temporal overlap, suggesting that the cerebellar neural substrates underlying eyeblink conditioning may change, depending on the temporal overlap of the CS and US.

mGluR1 in cerebellar Purkinje cells is required for normal association of temporally contiguous stimuli in classical conditioning.

In metabotropic glutamate receptor‐subtype 1 (mGluR1)‐null (mGluR1–/–) mice, cerebellar long‐term depression (LTD) and several forms of memory are impaired. However, because mGluR1 is expressed in various brain regions in wild‐type mice, it has been difficult to identify which type of memory depends on mGluR1 expressed in a given brain region. Furthermore, severe ataxia in mGluR1–/– mice complicated interpretation of the data from non‐cerebellum‐dependent tasks. We have generated mGluR1‐rescue mice, which express mGluR1 only in Purkinje cells (PCs) of their cerebellum, by introducing the mGluR1α transgene into mGluR1–/– mice under the control of a PC‐specific promoter. The mGluR1‐rescue mouse has normal LTD and displays no apparent ataxia. Therefore, this mouse is the first animal model in which effects of mGluR1 deficiency outside PCs can be studied without cerebellar dysfunction. We used three eyeblink conditioning paradigms with different temporal specificities between conditioned stimulus (CS) and unconditioned stimulus (US). Delay conditioning, in which CS and US coterminate, was impaired in mGluR1–/– mice but normal in mGluR1‐rescue mice. However, both strains of mice displayed severe impairment in trace conditionings, in which a stimulus‐free interval of 250 or 500 ms intervened between CS and US. We also examined social transmission of food‐preference and novel‐object‐recognition memory tests. In these tasks, mGluR1‐rescue mice showed normal short‐term but impaired long‐term memory. We conclude that mGluR1 in PCs is indispensable for normal learning of association of temporally contiguous stimuli in associative conditioning. In contrast, mGluR1 in other cell types is required for associating discontiguous stimuli and long‐term memory formation in nonspatial hippocampus‐dependent learning.

Differential Regulation of Synaptic Plasticity and Cerebellar Motor Learning by the C-Terminal PDZ-Binding Motif of GluRδ2

The δ2 glutamate receptor (GluRδ2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluRδ2−/− 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 GluRδ2−/− Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluRδ2−/− Purkinje cells by the virus-mediated expression of the wild-type GluRδ2 transgene (Tgwt) but not by that of mutant GluRδ2 lacking the C-terminal seven residues to which several PDZ proteins bind (TgΔCT7). These results indicated that the C terminus of GluRδ2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluRδ2−/− 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 GluRδ2 in vivo, we generated transgenic mice that expressed TgΔCT7 on a GluRδ2−/− background. Interestingly, although TgΔCT7 restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluRδ2−/− Purkinje cells. Furthermore, although the gross motor discoordination of GluRδ2−/− 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 GluRδ2, whereas other functions may be differentially regulated by other regions of GluRδ2.

Long‐trace interval eyeblink conditioning is impaired in mutant mice lacking the NMDA receptor subunit ε1

To elucidate the role of the N‐methyl‐d‐aspartate (NMDA) ‐type glutamate receptor subunit ε1 (GluRε1) in classical eyeblink conditioning, delay and trace eyeblink conditioning were investigated in GluRε1‐null mutant mice. In delay conditioning and short‐trace interval conditioning with a trace interval of 250 ms, GluRε1 mutant mice attained a normal level of the conditioned response (CR), although acquisition was a little slower than in wild‐type mice. In contrast, GluRε1 mutant mice exhibited severe impairment of the attained level of the CR and disturbed temporal pattern of CR expression in trace conditioning with a longer trace interval of 500 ms. These findings indicate that GluRε1 is essential for long‐trace interval eyeblink conditioning. The impairments of the associative learning with a long temporal separation between the conditioned and unconditioned stimuli observed in the GluRε1 mutant mice could be attributed to an impairment of hippocampal long‐term potentiation in this line of mutant mice.

Impaired delay but normal trace eyeblink conditioning in PLCbeta4 mutant mice.

To elucidate the functional role of phospholipase Cβ4 (PLCβ4), which is highly expressed in the Purkinje cells of the rostral cerebellum, cerebellar long-term depression (LTD) and delay and trace eyeblink conditioning were investigated in PLCβ4-deficient mice. Rostral cerebellar LTD and delay eyeblink conditioning were severely impaired, whereas trace eyeblink conditioning was not. These results indicate that PLCβ4 is essential for LTD in the rostral cerebellum and delay conditioning, but not trace conditioning. Rostral cerebellar LTD may be required as a neural substrate for delay conditioning, but is not required for trace conditioning.

Effects of the noncompetitive NMDA receptor antagonist MK-801 on classical eyeblink conditioning in mice.

N-methyl-D-aspartate (NMDA) receptors are involved in synaptic plasticity and play a critical role in learning and memory. We investigated the effects of the noncompetitive NMDA receptor antagonist (+)MK-801 on classical eyeblink conditioning of mice, using various interstimulus intervals between the conditioned stimulus (CS) and unconditioned stimulus (US). A tone was used for the CS and a periorbital shock was used for the US. In the delay paradigm, in which the US coterminated with the CS or started immediately after CS offset, the effect of (+)MK-801 (0.1mg/kg, i.p.) was a slight impairment in the acquisition of the conditioned response (CR). During subsequent CS-alone trials, the responses of (+)MK-801-injected mice were extinguished as easily as those of saline-injected mice. In the trace paradigm, (+)MK-801 impaired acquisition of the CR with a trace interval of 250 ms more than it did with a trace interval of 100 ms, and more than in the delay paradigm. (+)MK-801 injected after acquisition of 250-ms trace conditioning did not impair expression or extinction of the CR. These results suggest that NMDA receptors are involved in acquisition of the CR during longer trace interval conditioning more than during shorter trace interval conditioning or delay conditioning, and that their contribution to extinction is much smaller than their contribution to acquisition in mouse eyeblink conditioning.

Glutamate induces Cl− and K+ currents in the olfactory interneurons of a terrestrial slug

Abstract Glutamate-induced responses in the procerebral neurons of the terrestrial slug Limax marginatus were examined using the nystatin-perforated patch recording technique applied in the voltage-clamp mode and local application of drugs. The procerebrum contains two types of interneurons with different spontaneous activities, bursting and nonbursting neurons. In the bursting neurons, a puff of glutamate evoked a rapidly desensitizing current followed by a smaller sustained current. The reversal potential of the early component showed that the current was mediated by Cl− ions, while the late component was presumed to be mediated by K+ ions. In the nonbursting neurons, glutamate evoked a sustained current with a strong outward rectification, and the current was mediated by K+ ions. Ibotenate selectively evoked the rapidly desensitizing response in the bursting neurons, whereas quisqualate evoked a non-desensitizing K+ current both in the bursting and nonbursting neurons. The glutamate-induced K+ current had similar characteristics with the spontaneous synaptic activities in the procerebrum neurons, suggesting the possibility that glutamate receptors are involved in the spontaneous oscillatory activity.