Behavioral dissociation of anterodorsal and posteroventral hippocampus by subseizure stimulation in mice

BALB/c mice were bilaterally implanted with bipolar electrodes either in anterodorsal (ADH) or posteroventral hippocampus (PVH) in order to compare the effects of postsession electrical stimulation on memory processes. For each experiment, 30 s after the end of the first session, the animals were stimulated during 80 s. For both hippocampal regions, the stimulation intensity was half of the afterdischarge threshold value. Control groups were naive, ADH and PVH implanted non-stimulated animals. Different appetitive and aversive tasks were used. Subseizure stimulation never created a deficit. Depending on the region of the hippocampus stimulated and on the learning task, a retention enhancement was eventually observed. These data are in agreement with the involvement of hippocampus in initial stages of memory consolidation. Further, the subseizure stimulation permitted a functional dissociation between the two hippocampal regions. Both regions seemed involved in the integration of information, but the anterodorsal part would be rather related to behavioral inhibition, while the posteroventral part would have the capacity to induce an arousal state allowing behavioral flexibility.     

Behavioral alterations in the pilocarpine model of temporal lobe epilepsy in mice

Psychiatric disorders frequently occur in patients with epilepsy, but the relationship between epilepsy and psychopathology is poorly understood. Frequent comorbidities in epilepsy patients comprise major depression, anxiety disorders, psychosis and cognitive dysfunction. Animal models of epilepsy, such as the pilocarpine model of acquired epilepsy, are useful to study the relationship between epilepsy and behavioral dysfunctions. However, despite the advantages of mice in studying the genetic underpinning of behavioral alterations in epilepsy, mice have only rarely been used to characterize behavioral correlates of epilepsy. This prompted us to study the behavioral and cognitive alterations developing in NMRI mice in the pilocarpine model of epilepsy, using an anxiety test battery as well as tests for depression, drug-induced psychosis, spatial memory, and motor functions. In order to ensure the occurrence of status epilepticus (SE) and decrease mortality, individual dosing of pilocarpine was performed by ramping up the dose until onset of SE. This protocol was used for studying the consequences of SE, i.e. hippocampal damage, incidence of epilepsy with spontaneous recurrent seizures, and behavioral alterations. SE was terminated by diazepam after either 60, 90 or 120 min. All mice that survived SE developed epilepsy, but the severity of hippocampal damage varied depending on SE length. In all anxiety tests, except the elevated plus maze test, epileptic mice exhibited significant increases of anxiety-related behavior. Surprisingly, a decrease in depression-like behavior was observed in the forced swimming and tail suspension tests. Furthermore, epileptic mice were less sensitive than controls to most of the behavioral effects induced by MK-801 (dizocilpine). Learning and memory were impaired in epileptic mice irrespective of SE duration. Thus, the pilocarpine-treated mice seem to reflect several of the behavioral and cognitive disturbances that are associated with epilepsy in humans. This makes these animals an ideal model to study the neurobiological mechanisms underlying the association between epilepsy and psychopathology.     

Memory enhancing actions of Asiasari radix extracts via activation of insulin receptor and extracellular signal regulated kinase (ERK) I/II in rat hippocampus

Brain insulin receptor and ERK I/II are suggested to play a role in memory formation. We designed a series of experiments to explore if Asiasari radix (AR) extracts could display memory enhancing actions possibly via the activation of insulin receptor and ERK I/II in mice and rats. Methanol extract of AR had significantly increased survival time in the NaNO(2) intoxication assay in mice. Methanol extract of Asiasari radix (fraction 1) and its subfractions, chloroform-soluble fraction (fraction 2) and chloroform-insoluble, methanol-soluble fraction (fraction 4) were further tested for memory formation. In eight-arm radial maze experiments, both reference memory errors and working memory errors were significantly decreased in mice by fractions 1, 2 and 4. In addition, these fractions were also effective in promoting memory in the passive avoidance test in mice and rats. To gain insight into the mechanism of memory enhancing effects by Asiasari radix extracts, the activities of hippocampal insulin receptors and ERK I/II were tested in mice and rats. Fraction 1 significantly stimulated tyrosine phosphorylation of the insulin receptor, whereas ERK I/II were stimulated by fractions 1, 2 and 4. These fractions also inhibited cholinesterase activities in rats. These results suggest that Asiasari radix extracts may exert memory enhancing effects via activation of insulin receptor and ERK I/II as well as decreasing cholinesterase activity.     

Changes in hippocampal connexin 36 mRNA and protein levels during epileptogenesis in the kindling model of epilepsy

Objective

Identification of key molecular changes occurring during epileptogenesis provides better understanding of epilepsy and helps to develop strategies to modify those changes and thus, block the epileptogenic process. Gap junctional communication is thought to be involved in epileptogenesis. This communication can be affected by changes in expression of gap junctional protein subunits called connexins (Cxs). One of the main brain regions involved in epileptogenesis is the hippocampus in which there is a network of gap junctional communication between different cell types.

Method

Cx36 and Cx43 expressions at both mRNA and protein level were measured in rat hippocampus during epileptogenesis in the kindling model of epilepsy.

Results

Cx36 expression at both mRNA and protein level was upregulated during acquisition of focal seizures but returned to basal level after acquisition of secondarily-generalized seizures. No change in Cx43 gene and protein expression was found during kindling epileptogenesis.

Conclusion

These results further point out the significance of Cx36 as a target to modify epileptogenic process and to develop antiepileptogenic treatments.     

Overexpression of wild-type human APP in mice causes cognitive deficits and pathological features unrelated to Aβ levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP_wt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ₄₂) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ₄₂ were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ-independent pathogenic pathways in Alzheimer's disease.     

Developmental changes of protein substrates of Ca / calmodulin-dependent protein kinase II in teh rat forebrain

We previously reported that the level of Ca²⁺ / calmodulin-dependent protein kinase II (CaM kinase II) α and β proteins increases with postnatal age. In the present study, we investigated the development changes in whole protein substrates of CaM kinase II as compared with those of cAMP-dependent protein kinase (A-kinase) in the rat forebrain. Protein substrates were phosphorylated with [γ-³³P]ATP, and analysed by two-dimensional gel electrophoresis. More than 50 substrates for CaM kinase II were found in the soluble and particulate fractions The phosphorylation level of more than 15 substrates increased in the particulate fraction during development. Similarly, that of more than 3 substrates increased in the soluble fraction. Some substrates for A-kinase also increased during development, although some decresed. These findings suggest that the expression of some substrates is regulated during development and that the phosphorylation reaction involves the regulation of neuronal development.     

Dynamic of neurochemical alterations in striatum,hippocampus and cortex after the 6-OHDA mesostriatal lesion

Immediate neurochemical alterations produced by 6-OHDA could explain the general toxic pattern in the central nervous system. However, no evidences describe the effects of 6-OHDA on early changes of neurotransmitters in rats’ striatum, cortex and hippocampus. In our study, unilateral 6-OHDA injection into medial forebrain bundle (MFB) was used in rats, then five neurotransmitters were analyzed at 3, 6, 12, 24, 48 and 72 h, respectively. Results showed that 6-OHDA injection caused a sharp decline of striatal dopamine (DA) levels in the first 12 h followed by a further reduction between 12 and 48 h. However, striatal levels of homovanillic acid (HVA) were stable in the first 12 h and showed a marked reduction between 12 and 24 h. Striatal levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) decreased linearly for 72 h, whereas levels of norepinephrine (NE) showed a slight reduction in the first 48 h, and returned back to normal afterwards. Striatal HVA/DA ratio increased significantly in the first 12 h, but 5-HIAA/5-HT ratio showed a sharp increase between 12 and 72 h. Besides, neurochemical alterations were also found in hippocampus and cortex, and the correlations of neurotransmitters were analyzed. Our study indicated that NE system had little influence in the early phase of 6-OHDA injection, moreover, early neurochemical alterations were involved with striatum, hippocampus and cortex.     

Calcium/calmodulin kinase II in the pedunculopontine tegmental nucleus modulates the initiation and maintenance of wakefulness

The pedunculopontine tegmentum nucleus (PPT) is critically involved in the regulation of wakefulness (W) and rapid eye movement (REM) sleep, but our understanding of the mechanisms of this regulation remains incomplete. The present study was designed to determine the role of PPT intracellular calcium/calmodulin kinase (CaMKII) signaling in the regulation of W and sleep. To achieve this aim, three different concentrations (0.5, 1.0, and 2.0 nmol) of the CaMKII activation inhibitor, KN-93, were microinjected bilaterally (100 nl/site) into the PPT of freely moving rats, and the effects on W, slow-wave sleep (SWS), REM sleep, and levels of phosphorylated CaMKII (pCaMKII) expression in the PPT were quantified. These effects, which were concentration-dependent and affected wake-sleep variables for 3 h, resulted in decreased W, due to reductions in the number and duration of W episodes; increased SWS and REM sleep, due to increases in episode duration; and decreased levels of pCaMKII expression in the PPT. Regression analyses revealed that PPT levels of pCaMKII were positively related with the total percentage of time spent in W (R(2) = 0.864; n = 28 rats; p < 0.001) and negatively related with the total percentage of time spent in sleep (R(2) = 0.863; p < 0.001). These data provide the first direct evidence that activation of intracellular CaMKII signaling in the PPT promotes W and suppresses sleep. These findings are relevant for designing a drug that could treat excessive sleepiness by promoting alertness.     

Contribution of cholinergic and gabaergic functions to memory processes in BALB/cANnCrlBR mice

Several lines of evidence indicate that glucose influences on memory depend on interactions between glucose, glucoregulation and hippocampal cholinergic function. We previously demonstrated that glucose and scopolamine differentially affected memory consolidation for an operant bar pressing task in two closely-related BALB/c mouse strains. Whereas glucose normally improves memory in several animal strains, memory consolidation was not effected by systemic glucose injections in BALB/cANnCrlBR mice. Moreover, these mice were relatively insensitive to the normally observed amnestic effects of scopolamine. We therefore sought to determine whether cholinergic mechanisms in the dorsal hippocampus were involved in such atypical drug effects on memory processing in that strain of mice. In Experiment 1, we examined whether post-training oxotremorine would also atypically influence memory consolidation for an appetitively reinforced operant bar pressing task following microinjection in the dorsal hippocampus. In Experiment 2, we examined the effects of intrahippocampal GABAA drugs on memory consolidation. The non-selective muscarinic agonist, oxotremorine, dose-dependently impaired memory and the GABAA antagonist, bicuculline, improved retention in BALB/cANnCrlBR mice. It was concluded that GABA-mediated influences on hippocampal pyramidal output in BALB/cANnCrlBR mice and other strains are similar; but the amnestic effects of oxotremorine from the dorsal hippocampus were opposite to facilitating effects normally observed in other animal strains. Results are discussed relative to possible altered septo-hippocampal cholinergic neurotransmission in BALB/cANnCrlBR mice.     

Differential effect of short-term REM sleep deprivation on NGF and BDNF protein levels in the rat brain

It is well known that REM sleep is associated with memory consolidation, especially, procedural skill learning. Neurotrophic factors are known to be involved in synaptic plasticity. We therefore investigated the effects of selective REM sleep deprivation (RSD) on NGF and BDNF proteins in the hippocampus, cerebellum and brainstem in the rat. NGF and BDNF were detected by an ELISA. Our findings show that 6 h RSD affected the NGF and BDNF protein levels in different manner. In the cerebellum and brainstem, BDNF was significantly decreased, while NGF was not changed. Conversely, in the hippocampus, NGF was significantly decreased while BDNF was not changed. This study indicates that REM sleep may be associated with the secretion of neurotrophic factors and thus contribute to the memory functions.     

Differential subcellular distribution of Ca2+/calmodulin-dependent protein kinase II isoforms in the striatum and NG108-15 cells

Four subunits of Ca2+/calmodulin-dependent protein kinase II (CaM KII) have several isoforms, which differ in the variable domain. We previously reported that all subunits were highly expressed in rat striatal neurons. To examine intracellular distributions of CaM KII subunits in the rat striatal neurons, we performed immunoblot analysis with antibodies specific to each subunit in cell extracts from the rat striatum after continuous sucrose density gradient fractionation. The alpha subunit, but not the beta, gamma, or delta subunits, was colocalized with synapsin I, and each subunit showed a distinct distribution pattern in the fractions. To examine further the intracellular distributions of CaM KII isoforms in the same subunit, we established NG108-15 cells stably expressing delta1, delta3, and delta4 isoforms and examined distributions of the delta and gamma isoforms in these cell lines after fractionation. Each of the overexpressed exogenous delta isoforms showed a distinct distribution pattern. The endogenous delta2 was colocalized with the overexpressed delta1, delta3, and delta4 isoforms. However, the endogenous gammaB/gammaC isoforms were not colocalized with the overexpressed delta isoforms. Furthermore, the endogenous delta1 was concentrated in the microsomal fraction from the rat striatum. With the results taken together, it is suggested that CaM KII forms oligomers between isoforms in the same subunit but not in different subunits. The variable domain of CaM KII isoforms might possibly be responsible for targeting to certain intracellular compartments.     

Immunohistochemical localization of Ca/calmodulin-dependent protein kinase II in the rat retina

Ca²⁺ / calmodulin-dependent protein kinase II (CaM kinase II) consisting of α and β isoforms is highly expressed in the central nervous system and is implicated in the regulation of various Ca²⁺-dependent physiological processes. We investigated the immunohistochemical distribution of the α and β isoforms of this enzyme in the rat retina, using highly specific monoclonal antibodies which recognize each isoform. Immunoblotting revealed that not only the α but also the β isoform of CaM kinase II were expressed in the retina. The immunohistochemical study showed that highly α-immunoreactive products were localized in amacrine cells in the inner nuclear layer and displaced amacrine cells and ganglion cells in the ganglion cell layer. In addition, two well-defined bands within the inner plexiform layer were densely stained with the anti-α antibody. By contrast, immunoreactivity against the anti-β antibody was very weak in the same neuronal components of the retina. β-Immunoreactive products were homogeneously distributed throughout the inner plexiform layer and no well-defined bands were detected in this layer. Glial cells such as Müller cells were immunoreactive neither to α nor β antibody. A possible co-existence of choline acetyl transferase (ChAT) within CaM kinase II α-immunopositive neurons was examined by evaluating adjacent sections stained with anti-CaM kinase II α antibody and anti-ChAT antibody, respectively. The distribution of CaM kinase II α immunoreactivity in the rat retina was remarkably similar to that of ChAT immunoreactivity. About 32% of total ChAT-immunopositive neurons in the inner nuclear layer contained the CaM II α isoform, whereas only an occasional co-existence of both enzymes was observed in the ganglion cell layer. The present immunoblot and immunohistochemical study elucidated that not only α but also β isoforms of CaM kinase II β were expressed and this enzyme may participate in the cholinergic system in the rat retina.     

Involvement of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse formalin pain model

In the present study, we investigated the role of phosphorylated calcium/calmodulin-dependent protein kinase II (pCaMK-II) and phosphorylated extracellular signal-regulated protein kinase (pERK) in nociceptive processing at the spinal and supraspinal levels in the formalin subcutaneous induced mouse pain model. In the immunoblot assay, subcutaneous (s.c.) injection with formalin increased the pERK and pCaMK-IIalpha level in the spinal cord, and an immunohistochemical study showed that the increase of pERK and pCaMK-IIalpha immunoreactivity mainly occurred in the laminae I and II areas of the spinal dorsal horn. At the supraspinal level, although pERK was not changed in the hippocampus induced by formalin s.c. injection, pCaMK-IIalpha was increased in the hippocampus and hypothalamus by s.c. formalin injection, and an increase of pCaMK-IIalpha immunoreactivity mainly occurred in the pyramidal cells and the stratum lucidum/radiatum layer of the CA3 region of hippocampus and paraventricular nucleus of the hypothalamus. Moreover, pERK immunoreactivity in the hypothalamic paraventricular nucleus was also increased. The second phase of nociceptive behavior induced by formalin administered either i.t. or intracerebroventricularly (i.c.v.) was attenuated by PD98059 (ERK inhibitor) as well as KN-93(a CaMK-II inhibitor). On the other hand, the first phase of nociceptive behavior induced by formalin s.c. injection was not affected by i.t. KN-93. Our results suggest that pERK and pCaMK-II located at both the spinal cord and supraspinal levels are an important regulator during the nociceptive processes induced by formalin administered s.c. respectively.     

Long-term potentiation persistence greater in C57BL/6 than DBA/2 mice: Predicted on basis of protein kinase C levels and learning performance

The gamma isoform of protein kinase C (γ-PKC) activity is elevated and learning is superior in the inbred C57BL/6 mouse when compared to the DBA/2 mouse strain. Given the proposed link between PKC and long-term potentiation (LTP) on the one hand and PKC and learning on the other, it was predicted that LTP persistence would be greater in C57BL/6 mouse. When suprathreshold levels of tetanic stimulation were used, similar persistent LTP was observed in both C57BL/6 and DBA/2 strains. However, when tetanus was at threshold, the response in DBA/2 mice decayed to baseline in 30 min. similar to short-term potentiation (STP). Using this same paradigm with C57BL/6 mice. LTP persisted for 4 h, the longest time tested. The time course of the results parallels those observed in rat when phorbol ester, a potent PKC activator, converts STP to LTP. The present findings thus confirm the predicted difference between the two mouse strains. Moreover, the present findings are consistent with a role for γ-PKC in LTP. Since such results call attention to the need for γ-PKC interventive procedures, the relative utility of current PKC inhibitors, null mutants and antisense methods are discussed.     

Behavioral effects of apamin, a selective inhibitor of the SK(Ca)-channel, in mice and rats

Apamin, a highly selective and potent peptide that blocks the SK_Ca-channels has been suggested to be a cognition enhancer. We tested apamin in the Morris water escape task, in shock motivated avoidance tasks, and in operant tasks in the Skinnerbox. We also used non-cognitive tests, such as the rat forced swimming test and cocaine-induced locomotor activity in the open field, and a test to assess the side effect profile. Mice and rats from different strains, and rats of different ages were used. The rat studies provided only weak support for the notion that apamin acts as a cognition enhancer. More convincing evidence was obtained from the mouse studies. Overt side effects of apamin were found at the dose of 0.3 mg kg⁻¹. This dose was close to, or even overlapped, the doses which improved cognition in mice. We conclude that apamin is a poor tool to assess the role of SK_Ca-channels in learning and memory processes.     

Developmental changes in the levels of Ca2+/calmodulin-dependent protein kinase II alpha and beta proteins in soluble and particulate fractions of the rat brain.

Developmental changes in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been immunochemically examined in the forebrain, cerebellum and brainstem of the rat using antibodies against its alpha or beta protein. The concentration of alpha and beta proteins varied markedly in different brain regions at increasing postnatal ages. In early postnatal brain, the concentration of the alpha and beta proteins was low, and a large increase was observed between postnatal days 10 and 30. The maximum expression of the alpha protein was in the order of 6.01, 2.33 and 0.168 micrograms/mg of forebrain, brainstem and cerebellum proteins respectively, in the soluble or particulate fraction. On the other hand, that of the beta protein was in the order of 1.81, 0.495 and 0.291 micrograms/mg of forebrain, cerebellum or brainstem protein. The ratio of alpha and beta proteins also differed in the soluble and particulate fractions. The maximum expression of the alpha protein was observed at day 30 in soluble and particulate fractions of forebrain, and at day 20 in those of the brainstem. The major alpha protein peak was observed on or after day 30 in particulate and soluble fractions from cerebellum, respectively. The maximum expression of the beta protein was observed at day 20 in soluble and particulate fractions of the forebrain as well as in soluble fraction of the cerebellum, and was observed at day 30 in the particulate fraction of cerebellum. The expression of the alpha and beta proteins roughly correlated with the CaM kinase II activity from forebrain and brainstem.