Masatoshi Takita

Transient Brain Ischaemia Provokes Ca2+, PIP2 and Calpain Responses Prior to Delayed Neuronal Death in Monkeys

To clarify the mechanism of postischaemic delayed cornu Ammonis (CA)‐1 neuronal death, we studied correlations among calpain activation and its subcellular localization, the immunoreactivity of phosphatidylinositol 4,5‐bisphosphate (PIP2) and Ca2+ mobilization in the monkey hippocampus by two independent experimental approaches: in vivo transient brain ischaemia and in vitro hypoxia‐hypoglycaemia of hippocampal acute slices. The CA‐1 sector undergoing 20 min of ischaemia in vivo showed microscopically a small number of neuronal deaths on day 1 and almost global neuronal loss on day 5 after ischaemia. Immediately after ischaemia, CA‐1 neurons ultrastructurally showed vacuolation and/or disruption of the lysosomes. Western blotting using antibodies against inactivated or activated μ‐calpain demonstrated μ‐calpain activation specifically in the CA‐1 sector immediately after ischaemia. This finding was confirmed in the perikarya of CA‐1 neurons by immunohistochemistry. CA‐1 neurons on day 1 showed sustained activation of μ‐calpain, and increased immunostaining for inactivated and activated forms of μ‐ and m‐calpains and for PIP2. Activated μ‐calpain and PIP2 were found to be localized at the vacuolated lysosomal membrane or endoplasmic reticulum and mitochondrial membrane respectively, by immunoelectron microscopy. Calcium imaging data using hippocampal acute slices showed that hypoxia‐hypoglycaemia in vitro provoked intense Ca2+ mobilization with increased PIP2 immunostaining specifically in CA‐1 neurons. These data suggest that transient brain ischaemia increases intracellular Ca2+ and PIP2 breakdown, which will activate calpain proteolytic activity. Therefore, we suggest that activated calpain at the lysosomal membrane, with the possible release of biodegrading enzyme, will cause postischaemic CA‐1 neuronal death.

Induction of stable long‐term depression in vivo in the hippocampal–prefrontal cortex pathway

We studied excitatory field potentials in the medial prefrontal cortex (mPFC, prelimbic area) to electrostimulation of the ventral hippocampus (CA1/subicular region) in the anaesthetized rat. Nine hundred stimulus trains (5 pulses at 250 Hz) applied at 1 Hz to the ventral hippocampus significantly and persistently depressed the amplitude and maximal slope (∼ 55% for each index) of the prelimbic field potentials, but did not change the latency of the maximal slope or peak negativity. Twelve stimulus trains (50 pulses at 250 Hz) applied subsequently at 0.1 Hz restored the depression back to control level, and this reversible depression was maintained for at least 13 h. Cumulative depressive effects on the prelimbic field potential amplitude and maximal slope were observed upon addition of stimulus trains in the hippocampus. An important implication of the results is that the direct pathway from the hippocampus to the mPFC in the rat retains long‐term depression (LTD) as a neuroplastic form in vivo. This form could cooperate with long‐term potentiation (LTP) and such a bi‐directional synaptic plasticity in the prefrontal cortex contributes to how cortical neural networks store information.

Specific role of the posterior dorsal hippocampus–prefrontal cortex in short-term working memory

Working memory in rats involves neural projections from the hippocampus (HP) to the prefrontal cortex (PFC), based on delayed task experiments in a radial‐arm maze, in which the time span of working memory is longer than seconds. To determine whether the HP–PFC pathway is involved in short‐term (on the order of seconds) working memory function, we lesioned the PFC and/or HP, and measured performance in an operant delayed alternation task. The posterior dorsal (pdHP) and ventral HP (vHP) were assessed separately. The bilateral PFC and bilateral pdHP ibotenate lesions produced significant working memory deficits, but the vHP lesion did not. Unilateral pdHP lesions combined with a PFC lesion in the opposite hemisphere reproduced the effects of bilaterally symmetrical lesions. By contrast, unilateral lesions of the pdHP combined with a PFC lesion in the same hemisphere had no effect on delayed alternation. These results indicate that the pdHP–PFC pathway is essential for working memory on the order of seconds in rats, and suggest that the pdHP and vHP pathways to the PFC play different behavioral roles.

Immunohistochemical analysis on the role of adenosine A1 receptors in epilepsy

Adenosine has an anticonvulsant effect in various models of epilepsy. This effect appears to be mediated through the activation of adenosine A1 receptors (A1Rs). We immunohistochemically investigated the changes of A1Rs expression in kainate-treated and hippocampus-kindled rats as chronic models of epilepsy. In the normal hippocampus, a predominant expression of A1Rs was detected in the CA2/CA3a field. The A1Rs immunoreactivity in this field began to decline drastically approximately 4 weeks after kainate treatment and remained minimal 8 weeks after treatment. In the hippocampus-kindled animals, A1Rs expression was minimal in the stimulated side but remained high in the nonstimulated side. The reduced expression of A1Rs in the CA2/CA3a field may be related to chronic epileptogenesis.

Differences between paired-pulse facilitation and long-term potentiation in the dorsal and ventral hippocampal CA1-prefrontal pathways of rats

We studied the interaction between paired-pulse facilitation (PPF) and long-term potentiation (LTP) in the hippocampo-prefrontal cortex (PFC, prelimbic area) pathway, stimulating the ventral or posterior dorsal CA1 region (vCA1 or pdCA1). In the vCA1-PFC, the group averaged PPF did not change after the LTP induction, and there was a negative correlation between the post-LTP PPF change and LTP magnitude. In contrast, the post-LTP PPF of the pdCA1-PFC appeared to decrease significantly, and the PPF change was independent of the LTP magnitude. We found that there were at least two mechanisms of PPF regulation following LTP induction in the pathway resulting from extensive CA1 projections into the prelimbic area. The results imply that the CA1-PFC pathway regulates the PFC PPF quantitatively in LTP-dependent and independent manners, which depend on the local properties of the CA1 regions.

Cooperativity between hippocampal–prefrontal short-term plasticity through associative long-term potentiation

The hippocampal-medial prefrontal cortex (mPFC) pathway provides highly convergent input to the mPFC in rats and shows two types of short-term plasticity in terms of paired-pulse facilitation (PPF) of the field potential under urethane anesthesia. We now report that stimulating either the dorsal or ventral subregions of the posterior hippocampus elicited PPF (by about 335 and 120%, respectively) of field potentials recorded in the mPFC at 100 ms interpulse interval. This PPF-like interaction occurred when projections were stimulated in the ventral-dorsal order (by about 200% of the single-pulsed response), but not vice versa. When weak long-term potentiation (LTP) of the dorsal projection was evoked simultaneously with strong LTP of the ventral projection, an associative effect was revealed (about +55%), although the magnitudes of LTP in each projection were not correlated. Even when the impermutable PPF-like facilitation was further enhanced (by about +120%), the enhancement was not correlated with either form of LTP, but exhibited the interaction of changes in the dorsal PPF, rather than in the heterotopic priming effect through the ventral projection. Moreover, this change was correlated with the associated LTP ratio of dorsal to ventral projection LTP (i.e., LTP associativity). Larger increases in LTP associativity correlated with greater impermutable PPF-like facilitation; in addition, there was hardly attenuation of the response to the dorsal projection by subsequent electrolytic lesions of the ventral subregion. These results indicate that the mPFC functionally integrates discrete sources of hippocampal information via cooperativity between short- and long-term plasticity.

Comparative induction of long-term depression between dorsal and ventral hippocampal CA1 in the anesthetized rat

We studied whether a protocol reported as in vivo prefrontal long-term depression (LTD) induction protocol, also induced LTD in the anesthetized rat hippocampal CA1, and whether differences in LTD induction existed between dorsal and ventral CA1, by low-frequency stimulation (LFS) (1 Hz, 900) or low-frequency burst stimulation (LFBS) (5-pulse burst at 4 ms interpulse intervals at 1 Hz, 900), hippocampo-prefrontal LTD induction protocol. Though LFS failed to induce stable LTD in dorsal or ventral CA1, LFBS reliably induced LTD in the ventral not dorsal CA1. This similarity between ventral hippocampal and hippocampo-prefrontal LTD induction thus implies their serial integration process, ventral CA3-CA1-prefrontal cortex pathway and observed dorsal and ventral differences involved in behavioral functions such as learning.

Compatibility of bidirectional synaptic plasticity on hippocampo-prefrontal cortex pathway in rats

The hippocampo-prefrontal cortex pathway reportedly expresses long-term potentiation (LTP) and depression (LTD) in anesthetized rats. We examined whether there were any effects governing the induction of LTD after prior induction of LTP, or vice versa. Induction in sequence of LTP and LTD resulted in significantly stable changes of about 140 and 70% of a common control for 1 h each. The reversed sequence, LTD and LTP, showed a mirror image of about 65 and 135% of control, which were not different from the respective changes in the first sequence (P>0.3 for each). The correlation coefficient between changes was significantly positive in the first sequence and weakly negative in the reverse. These results indicate that this pathway can express compatibility of bidirectional synaptic plasticity while historical changes remain covert.

Local properties of CA1 region in hippocampo-prefrontal synaptic plasticity in rats.

We studied paired-pulse facilitation and long-term potentiation/depression in anesthetized rats to determine whether the hippocampal CA1 region inhibits local differences in short-term and long-term synaptic plasticity in its projections to the prefrontal cortex. We compared projections with the PFC from the posterior dorsal and ventral hippocampal CA1 regions (pdCA1 and vCA1 respectively). The two pathways displayed similar properties. However, the PPF properties of the pdCA1, projections differed dramatically from those of the pdCA1 projections. The pdCA1 projections showed the opposite of facilitation (i.e. suppression) at 25–50 ms intervals and more pronounced facilitation at 100–400 ms intervals. These results suggest that there are functional differences between these pathways.

In vivo temporal property of GABAergic neural transmission in collateral feed-forward inhibition system of hippocampal–prefrontal pathway

Anatomical evidence suggests that rat CA1 hippocampal afferents collaterally innervate excitatory projecting pyramidal neurons and inhibitory interneurons, creating a disynaptic, feed-forward inhibition microcircuit in the medial prefrontal cortex (mPFC). We investigated the temporal relationship between the frequency of paired synaptic transmission and gamma-aminobutyric acid (GABA)ergic receptor-mediated modulation of the microcircuit in vivo under urethane anesthesia. Local perfusions of a GABAa antagonist (-)-bicuculline into the mPFC via microdialysis resulted in a statistically significant disinhibitory effect on intrinsic GABA action, increasing the first and second mPFC responses following hippocampal paired stimulation at interstimulus intervals of 100-200 ms, but not those at 25-50 ms. This (-)-bicuculline-induced disinhibition was compensated by the GABAa agonist muscimol, which itself did not attenuate the intrinsic oscillation of the local field potentials. The perfusion of a sub-minimal concentration of GABAb agonist (R)-baclofen slightly enhanced the synaptic transmission, regardless of the interstimulus interval. In addition to the tonic control by spontaneous fast-spiking GABAergic neurons, it is clear the sequential transmission of the hippocampal-mPFC pathway can phasically drive the collateral feed-forward inhibition system through activation of a GABAa receptor, bringing an active signal filter to the various types of impulse trains that enter the mPFC from the hippocampus in vivo.