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

Morphological characterization of the bursting and nonbursting neurones in the olfactory centre of the terrestrial slug limax marginatus

We investigated the morphological characteristics of physiologically characterized neurones in the procerebrum (PC) of the terrestrial slug Limax marginatus. The electrophysiological characteristics of the neurones were determined using the perforated patch recording technique. By comparing the shapes and sizes of somata, we found that the somata of bursting neurones were larger and had more oblong shapes than those of nonbursting neurones, indicating that the bursting and nonbursting neurones in the PC could be discriminated on the basis of the sizes and shapes of their soma. Injection of fluorescent dyes into the bursting and nonbursting neurones revealed morphological differences in the neurites. The bursting neurones had extensive projections within the cell body layer, and the major neurites of the bursting neurones were oriented along the dorsal-ventral axis. In contrast, the nonbursting neurones had neurites extending into the neuropile layers, where terminals of both sensory and presumed output neurones exist

Image analysis of olfactory responses in the procerebrum of the terrestrial slug Limax marginatus.

Neural oscillations have been found to occur in the olfactory centers of some vertebrates and invertebrates, including the procerebrum of the terrestrial slug Limax marginatus. Using optical recording with the potential-sensitive dye di-4-ANEPPS, we analyzed the spatiotemporal pattern of procerebral neural activities in response to odorants applied to an in vitro brain-superior tentacle preparation. The odor of rat chow, on which the slugs were normally fed, increased the frequency of the oscillation. Garlic odor, which induced aversive behavior in the slug, caused a transient increase in oscillation frequency during stimulation, followed by a second increase in oscillation frequency when the stimulus was terminated. Wave propagation from the distal to the proximal region of the procerebrum was accelerated in parallel with modulation of the frequency. The cycle-by-cycle average of the optical signals showed that a large area of the cerebral ganglia, including the procerebrum, was depolarized during the initial increase in frequency. During the second increase, however, the net depolarization was most prominent in the terminal mass of the procerebrum. These results suggest that the level of depolarization generated by interactions among the neurites projecting to the terminal mass, such as the neurites of the nonbursting neurons, may control neural oscillations in the procerebrum

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

NMDA receptor-dependent processes in the medial prefrontal cortex are important for acquisition and the early stage of consolidation during trace, but not delay eyeblink conditioning.

Permanent lesions in the medial prefrontal cortex (mPFC) affect acquisition of conditioned responses (CRs) during trace eyeblink conditioning and retention of remotely acquired CRs. To clarify further roles of the mPFC in this type of learning, we investigated the participation of the mPFC in mnemonic processes both during and after daily conditioning using local microinfusion of the GABA(A) receptor agonist muscimol or the NMDA receptor antagonist APV into the rat mPFC. Muscimol infusions into the mPFC before daily conditioning significantly retarded CR acquisition and reduced CR expression if applied after sufficient learning. APV infusion also impaired acquisition of CRs, but not expression of well-learned CRs. When infusions were made immediately after daily conditioning, acquisition of the CR was partially impaired in both the muscimol and APV infusion groups. In contrast, rats that received muscimol infusions 3 h after daily conditioning exhibited improvement in their CR performance comparable to that of the control group. Both the pre- and post-conditioning infusion of muscimol had no effect on acquisition in the delay paradigm. These results suggest that the mPFC participates in both acquisition of a CR and the early stage of consolidation of memory in trace, but not delay eyeblink conditioning by NMDA receptor-mediated operations

Comparative study on neural oscillation in the procerebrum of the terrestrial slugs Incilaria bilineata and

Coherent oscillatory activities in procerebral neurones have been described in Limax maximus; however, the electrical properties of the procerebrum of other terrestrial molluscs are less well understood. We have examined oscillatory activity in the procerebrum of Incilaria bilineata and Limax marginatus. The local field potential measured in the procerebrum of I. bilineata showed repetitive peaks which had the opposite polarity from those measured in L. marginatus. Optical measurement of membrane potential using a potential-sensitive dye, di-4-ANEPPS, showed that the oscillations in I. bilineata occurred mainly in the internal mass while those in L. marginatus were located in the cell mass. An analysis of the waveform revealed that the depolarizing phase of the oscillations consists of both a slow and a rapid component in both species. The rapid component was most pronounced in the internal mass of I. bilineata but was prominent in the cell mass of L. marginatus. The superior tentacle nerve, which projects to the terminal mass, also showed oscillations in synchrony with those of the procerebrum. These results suggest that oscillations in procerebral interneurones are commonly generated in a region adjacent to the terminal mass and that these oscillations may affect the membrane potential of the neurones constituting the superior tentacle nerve

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

Optical recording analysis of olfactory response of the procerebral lobe in the slug brain.

We studied the oscillatory properties and the olfactory responses of the procerebral (PC) lobe of the cerebral ganglion in the terrestrial mollusc Limax marginatus. The PC lobe, a central olfactory organ in Limax, is a highly interconnected network of local interneurons that receives olfactory inputs from the inferior and superior tentacular noses. We used an optical recording technique with a voltage-sensitive dye to record the activity of the PC lobe from either the posterior or the dorsal surface. The recordings revealed that almost all PC interneurons showed spontaneous oscillatory activities that had been entrained with each other. Upon presentation to the nose of odors to which the slugs had been aversively conditioned, the basal level of the oscillation changed biphasically. In the early phase of the response, depolarization in the basal level of the oscillation occurred in one or more belt-shaped regions parallel to the dorsoventral axis. In the late phase of the response, hyperpolarization of basal potential level of the PC lobe oscillations occurred in a wider area. Such spatial and temporal modulation was not observed when the unpaired control odors were presented to the preparation, whereas the same preparations responded to the aversively conditioned stimuli. Thus, it was considered that the spatial and temporal response in the basal level of oscillation was specific to the aversively conditioned odors. Furthermore, the spatial pattern of the depolarization modulation in the early phase was repeatable in multiple trials performed using the same odor, although different odors produced different spatial patterns of the modulation. From these results, we conclude that in the PC lobe learned odors are represented as spatial and temporal activity patterns of oscillators that constitute a coherent network

N-methyl-D-aspartate receptors play important roles in acquisition and expression of the eyeblink conditioned response in glutamate receptor subunit delta2 mutant mice.

Classical eyeblink conditioning has been known to depend critically on the cerebellum. Apparently consistent with this, glutamate receptor subunit delta2 null mutant mice, which have serious morphological and functional deficiencies in the cerebellar cortex, are severely impaired in delay paradigm. However, these mutant mice successfully learn in trace paradigm, even in \u270-trace paradigm,\u27 in which the unconditioned stimulus starts just after the conditioned stimulus terminates. Our previous studies revealed that the hippocampus and the muscarinic acetylcholine receptors play crucial roles in 0-trace paradigm in glutamate receptor subunit delta2 null mutant mice unlike in wild-type mice, suggesting a large contribution of the forebrain to 0-trace conditioning in this type of mutant mice. In the present study, we investigated the role of N-methyl-D-aspartate receptors in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice. Mice were injected intraperitoneally with the noncompetitive N-methyl-d-aspartate receptor antagonist (+)MK-801 (0.1mg/kg) or saline, and conditioned with 350-ms tone conditioned stimulus followed by 100-ms periorbital shock unconditioned stimulus. Glutamate receptor subunit delta2 null mutant mice that received (+)MK-801 injection exhibited a severe impairment in acquisition of the conditioned response, compared with the saline-injected glutamate receptor subunit delta2 null mutant mice. In contrast, wild-type mice were not impaired in acquisition of 0-trace conditioned response by (+)MK-801 injection. After the injection solution was changed from (+)MK-801 to saline, glutamate receptor subunit delta2 null mutant mice showed a rapid and partial recovery of performance of the conditioned response. On the other hand, when the injection solution was changed from saline to (+)MK-801, glutamate receptor subunit delta2 null mutant mice showed a marked impairment in expression of the pre-acquired conditioned response, whereas impairment of the expression was small in wild-type mice. Injection of (+)MK-801 had no significant effects on spontaneous eyeblink frequency or startle eyeblink frequency to the tone conditioned stimulus in either glutamate receptor subunit delta2 null mutant mice or wild-type mice. These results suggest that N-methyl-D-aspartate receptors play critical roles both in acquisition and expression of the conditioned response in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice

The N-methyl-D-aspartate (NMDA)-type glutamate receptor GluRepsilon2 is important for delay and trace eyeblink conditioning in mice.

It has been proposed that the N-methyl-d-aspartate (NMDA)-type glutamate receptor (GluR) plays an important role in synaptic plasticity, learning, and memory. The four GluRepsilon (NR2) subunits, which constitute NMDA receptors with a GluRzeta (NR1) subunit, differ both in their expression patterns in the brain and in their functional properties. In order to specify the distinct participation of each of these subunits, we focused on the GluRepsilon2 subunits, which are expressed mainly in the forebrain. We investigated delay and trace eyeblink conditioning in GluRepsilon2 heterozygous mutant mice whose content of GluRepsilon2 protein was decreased to about half of that in wild-type mice. GluRepsilon2 mutant mice exhibited severe impairment of the attained level of conditioned response (CR) in the delay paradigm, for which the cerebellum is essential and modulation by the forebrain has been suggested. Moreover, GluRepsilon2 mutant mice showed no trend toward CR acquisition in the trace paradigm with a trace interval of 500 ms, in which the forebrain is critically involved in successful learning. On the other hand, the reduction of GluRepsilon2 proteins did not disturb any basic sensory and motor functions which might have explained the observed impairment. These results are different from those obtained with GluRepsilon1 null mutant mice, which attain a normal level of the CR but at a slower rate in the delay paradigm, and showed a severe impairment in the trace paradigm. Therefore, the NMDA receptor GluRepsilon2 plays a more critical role than the GluRepsilon1 subunit in classical eyeblink conditioning