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Nerve gas organophosphates like sarin are likely to be used in urban terrorism, leading to widespread exposures of pregnant women and young children. Here, we established a model for sarin neurobehavioral teratogenicity in the developing chick so as to explore the consequences of apparently subtoxic sarin exposure and the mechanisms underlying synaptic and behavioral deficits. Chicken eggs were injected with sarin (2, 6 and 12 microg/kg) on incubation days 2 and 6, treatments that did not decrease hatching and did not evoke dysmorphology. After hatching the chicks were tested for filial imprinting and neurochemical markers known to be critical for imprinting. Imprinting was reduced at 2 and 6 microg/kg but not at the highest dose. Acetylcholinesterase and choline acetyltransferase were unaffected but sarin reduced the concentration of the high-affinity choline transporter, the rate-limiting factor in acetylcholine utilization. The concentration of PKC isoforms was assessed in the imprinting-related intermediate part of the medial hyperstriatum ventrale, the region most closely associated with cholinergic function in imprinting behavior. Sarin reduced the concentration of all isoforms (alpha, beta, gamma) with a similar, biphasic dose-response curve to that seen for behavioral performance, a relationship noted in previous work with organophosphate pesticides. Our results indicate that otherwise subtoxic exposures to sarin produce neurodevelopmental deficits; since we utilized a chick model, which is devoid of maternal confounds that are present in mammalian development, the adverse effects of sarin are mediated directly in the developing organism.

Increased levels of angiogenesis and neurogenesis possibly mediate the beneficial effects of physical activity on hippocampal plasticity. This study was designed to investigate the temporal dynamics of exercise-induced changes in hippocampal angiogenesis and cell proliferation. Mice were housed with a running wheel for 1, 3, or 10 days. Analysis of glucose transporter Glut1-positive vessel density showed a significant increase after 3 days of wheel running. Cell proliferation in the dentate gyrus showed a trend towards an increase after 3 days of running and was significantly elevated after 10 days of physical exercise. Ten days of wheel running resulted in a near-significant increase in the number of immature neurons, as determined by a doublecortin (DCX) staining. In the second part of the study, the persistence of the exercise-induced changes in angiogenesis and cell proliferation was determined. The running wheel was removed from the cage after 10 days of physical activity. Glut-1 positive vessel density and hippocampal cell proliferation were determined 1 and 6 days after removal of the wheel. Both parameters had returned to baseline 24 h after cessation of physical activity. The near-significant increase in the number of DCX-positive immature neurons persisted for at least 6 days, indicating that new neurons formed during the period of increased physical activity had survived. Together these experiments show that the hippocampus displays a remarkable angiogenic and neurogenic plasticity and rapidly responds to changes in physical activity., (Copyright 2008 Wiley-Liss, Inc.)

Stressful experiences, especially when prolonged and severe are associated with psychopathology and impaired neuronal plasticity. Among other effects on the brain, stress has been shown to negatively regulate hippocampal neurogenesis, and this effect is considered to be exerted via glucocorticoids. Here, we sought to determine the temporal dynamics of changes in hippocampal neurogenesis after acute and chronic exposure to foot-shock stress. Rats subjected to a foot-shock procedure showed strong activation of the hypothalamic-pituitary-adrenal (HPA) axis, even after exposure to daily stress for 3 weeks. Despite a robust release of corticosterone, acute foot-shock stress did not affect the rate of hippocampal cell proliferation. In contrast, exposure to foot-shock stress daily for 3 weeks led to reduced cell proliferation 2 hours after the stress procedure. Interestingly, this stress-induced effect did not persist and was no longer detected 24 hours later. Also, while chronic foot-shock stress had no impact on survival of hippocampal cells that were born before the stress procedure, it led to a decreased number of doublecortin-positive granule neurons that were born during the chronic stress period. Thus, whereas a strong activation of the HPA axis during acute foot-shock stress is not sufficient to reduce hippocampal cell proliferation, repeated exposure to stressful stimuli for prolonged period of time ultimately results in dysregulated neurogenesis. In sum, this study supports the notion that chronic stress may lead to cumulative changes in the brain that are not seen after acute stress. Such changes may indicate compromised brain plasticity and increased vulnerability to neuropathology.

Recently, activated microglia have been shown to be involved in the regulation of several aspects of neurogenesis under certain experimental conditions both in vitro and in vivo. A neurogenesis supportive microglia phenotype has been suggested to arise from the interaction of microglia with homing encephalitogenic T cells. However, a unified hypothesis regarding the exact nature of microglia activity that is supportive of neurogenesis is yet missing from the field. Our aim was to investigate the connection between microglia activity and adult hippocampal neurogenesis under physiological conditions. To address this question we compared the level of microglia activation in the hippocampus of mice, which had access to a running wheel for 10 days and that of sedentary controls. Surprisingly, despite elevated levels of proliferation of neural precursors and survival of newborn neurons in the dentate gyrus microglia remained in a "resting" state morphologically, antigenically, and at the transcriptional level. Moreover, neither T cells nor MHCII expressing microglia were present in the hippocampal brain parenchyma. Though microglia in the dentate gyrus of the runners proliferated at a higher level than in the sedentary controls, this difference was also present in non-neurogenic sites. Therefore, our findings suggest that classical signs of microglia activation and microglia activation arising from interaction with T cells in particular are not a prerequisite for the activity-induced increase in adult hippocampal neurogenesis in C57Bl/6 mice. Thus, our results draw attention on the species and model differences that might exist regarding the regulation of adult hippocampal neurogenesis., ((c) 2008 Wiley-Liss, Inc.)

Besides a beneficial cardiovascular effect, it was recently suggested that statins can also exert neuroprotective actions. In a previous study, we provided in vitro evidence that lovastatin treatment abates excitotoxic cell death in primary cortical neurons. Here, we investigated the neuroprotective effect of lovastatin in an in vivo mouse model. We found that administration of lovastatin (20 mg/kg) significantly protects cholinergic neurons and their cortical projections against N-methyl-D-aspartate (60 nmol)-induced cell death in the magnocellular nucleus basalis, a neuronal cell group that is characteristically affected in Alzheimer's disease. Furthermore, lovastatin-mediated neuroprotection was shown to be dependent on protein kinase B (PKB)/Akt signaling since treatment with the PKB/Akt inhibitor LY294002 blocked the lovastatin-induced neuroprotective effect. The loss of cholinergic neurons after the lesion in the magnocellular nucleus basalis resulted in memory impairment as tested in a passive avoidance paradigm. This was reverted by pre-lesion lovastatin treatment. From these studies we conclude that treatment with lovastatin may provide protection against neuronal injury in excitotoxic conditions associated with neurodegenerative diseases including Alzheimer's disease.

The suprachiasmatic nuclei of the hypothalamus (SCN) are the master circadian clock in mammals. Transcriptional activity in this master clock has a marker in the immediate-early gene c-Fos. Within the SCN, distinct differences in c-Fos in the ventrolateral and the dorsomedial SCN have been reported for rodent species such as rats, mice, and hamsters. We studied C-FOS expression in the common vole (Microtus arvalis) SCN under LD 12:12 h and under constant dim light conditions. In the vole dorsomedial SCN, rhythmic C-FOS expression was seen in LD with a clear peak in the middle of the light period. Under constant dim light, we report constitutive, non-rhythmic expression of C-FOS in the dorsomedial SCN. This pattern is consistent with the circadian organization of behavioral activity, which is weak in voles and may be lost under constant dim-light conditions. In the ventrolateral SCN, we observed a rise in C-FOS expression under LD conditions prior to lights-on, followed by peak expression at lights-on. Another peak was seen at lights-off. In an additional experiment, we subjected animals to LD 16:8 to test the hypothesis that the dawn and dusk peaks in ventrolateral C-FOS expression change phase along with the photoperiod. The peak in C-FOS expression did not shift with the time of lights on, but remained at the same external time 6. The results are consistent with the interpretation that in the vole, c-Fos expression reports transcriptional activity associated more likely with an internal, gating process than with an external effect of light.

Both genetic and pharmacological studies demonstrated that contextual fear conditioning is critically regulated by cyclic AMP-dependent protein kinase (PKA). Since PKA is a broad range protein kinase, a mechanism for confining its activity is required. It has been shown that intracellular spatial compartmentalization of PKA signaling is mediated by A-kinase anchoring proteins (AKAPs). Here, we investigated the role of PKA anchoring to AKAPs in different stages of the memory process (acquisition, consolidation, retrieval and extinction) using contextual fear conditioning, a hippocampus-dependent learning task. Mice were injected intracerebroventricularly or intrahippocampally with the membrane permeable PKA anchoring disrupting peptides St-Ht31 or St-superAKAP-IS at different time points during the memory process. Blocking PKA anchoring to AKAPs resulted in an impairment of fear memory consolidation. Moreover, disrupted PKA anchoring promoted contextual fear extinction in the mouse hippocampus. We conclude that the temporal and spatial compartmentalization of hippocampal PKA signaling pathways, as achieved by anchoring of PKA to AKAPs, is specifically instrumental in long-term contextual fear memory consolidation and extinction, but not in acquisition and retrieval.

Endogenous biological clocks allow organisms to anticipate daily environmental cycles. The ability to achieve time-place associations is key to the survival and reproductive success of animals. The ability to link the location of a stimulus (usually food) with time of day has been coined time-place learning, but its circadian nature was only shown in honeybees and birds. So far, an unambiguous circadian time-place-learning paradigm for mammals is lacking. We studied whether expression of the clock gene Cryptochrome (Cry), crucial for circadian timing, is a prerequisite for time-place learning. Time-place learning in mice was achieved by developing a novel paradigm in which food reward at specific times of day was counterbalanced by the penalty of receiving a mild footshock. Mice lacking the core clock genes Cry1 and Cry2 (Cry double knockout mice; Cry1(-/-)Cry2(-/-)) learned to avoid unpleasant sensory experiences (mild footshock) and could locate a food reward in a spatial learning task (place preference). These mice failed, however, to learn time-place associations. This specific learning and memory deficit shows that a Cry-gene dependent circadian timing system underlies the utilization of time of day information. These results reveal a new functional role of the mammalian circadian timing system.

It is unclear whether protein phosphatases, which counteract the actions of protein kinases, play a beneficial role in the formation and extinction of previously acquired fear memories. In this study, we investigated the role of the calcium/calmodulin dependent phosphatase 2B, also known as calcineurin (CaN) in the formation of contextual fear memory and extinction of previously acquired contextual fear. We used a temporally regulated transgenic approach, that allowed us to selectively inhibit neuronal CaN activity in the forebrain either during conditioning or only during extinction training leaving the conditioning undisturbed. Reducing CaN activity through the expression of a CaN inhibitor facilitated contextual fear conditioning, while it impaired the extinction of previously formed contextual fear memory. These findings give the first genetic evidence that neuronal CaN plays an opposite role in the formation of contextual fear memories and the extinction of previously formed contextual fear memories.

In common voles (Microtus arvalis), natural variation in locomotor behavior can be exploited to study the mechanism of pacemaker control over circadian timing of behavior. Here we studied daily patterns in numbers of neuropeptide immunoreactive suprachiasmatic nucleus neurons in rhythmic, weakly rhythmic, and non-rhythmic voles. Circadian rhythmic voles showed circadian variation in numbers of vasoactive intestinal polypeptide and vasopressin immunoreactive suprachiasmatic nucleus neurons with a peak at zeitgeber time 0. In contrast, voles with weak or no circadian rhythmicity exhibited similar fluctuations for vasoactive intestinal polypeptide, but a continuous, non-rhythmic high profile for vasopressin. Vole suprachiasmatic nucleus neurons do not produce somatostatin or substance P. We conclude that the vasopressin system in the common vole suprachiasmatic nucleus acts as a principal correlate with expression of circadian behavior, in contrast to vasoactive intestinal polypeptide, somatostatin, and substance P. We also conclude that high levels of vasopressin immunoreactivity in the non-rhythmic vole suprachiasmatic nucleus is in line with previously demonstrated hampered release, probably resulting in vasopressin accumulation in the suprachiasmatic nucleus. Vasopressin could be a candidate in mediating output of the vole circadian clock, leading to circadian expression of locomotor behavior.

A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that specifically target cAMP-dependent protein kinase (PKA) to unique subcellular compartments and thus, provide high specificity to PKA signaling. For example, the AKAP79/150 family tethers PKA, PKC and PP2B to neuronal membranes and postsynaptic densities and plays an important role in synaptic function. Several studies suggested that AKAP79/150 anchored PKA contributes to mechanisms associated with synaptic plasticity and memory processes, but the precise role of AKAPs in these processes is still unknown. In this study we established the mouse brain distribution of AKAP150 using two well-characterized AKAP150 antibodies. Using Western blotting and immunohistochemistry we showed that AKAP150 is widely distributed throughout the mouse brain. The highest AKAP150 expression levels were observed in striatum, cerebral cortex and several other forebrain regions (e.g. olfactory tubercle), relatively high expression was found in hippocampus and olfactory bulb and low/no expression in cerebellum, hypothalamus, thalamus and brain stem. Although there were some minor differences in mouse AKAP150 brain distribution compared to the distribution in rat brain, our data suggested that rodents have a characteristic AKAP150 brain distribution pattern. In general we observed that AKAP150 is strongly expressed in mouse brain regions involved in learning and memory. These data support its suggested role in synaptic plasticity and memory processes.

A-kinase anchoring protein 150 (AKAP150) is a multi-enzyme signaling complex that coordinates the action of PKA, PKC, and PP2B at neuronal membranes and synapses. We measured levels of AKAP150 protein in the hippocampus 6h after training mice in a contextual fear conditioning paradigm. In contextual fear conditioning mice learn to associate a context with a footshock presentation. Mice were divided in four experimental groups with different training protocols: naive, no footshock exposure, immediate footshock exposure, and footshock 3min after exposure to the context. We found that AKAP150 protein levels were increased upon exposing mice to the novel context independent of the training protocol. However, when the animals were habituated to the experimental context, only mice that learned to associate the context with the footshock showed an upregulation of AKAP150. We suggest that upregulated levels of AKAP150 contribute to processing the exposure to a novel context and associative learning.

Recent theories on the function of arousals from torpor in hibernating mammals focus on the repair of the central nervous system from damage accumulating during prolonged hypothermia. In this framework, we investigated the synaptic ultrastructure in Layer 2 of the frontal cortex from hibernating European ground squirrels (Spermophilus citellus) sacrificed at four different phases in the torpor-arousal cycle. Using electron microscopy, we quantified synapse number and morphometric data on asymmetric axospinous synapses. Length, width, and surface area of postsynaptic densities (PSDs), and the synaptic apposition length of the analyzed synapse were measured. Five groups of animals were compared during entrance into torpor (Torpor Early, TE, n = 6), late torpor (Torpor Late, TL, n = 5), beginning of euthermic arousal episodes (Arousal Early, AE, n = 5), late in the euthermic arousal episode (Arousal Late, AL, n = 5), and during continuous euthermy in spring (EU, n = 6). The results showed that during torpor and at the beginning of arousals the PSD length and synaptic apposition length are significantly increased compared to synapses during late arousal and in spring conditions. In contrast, the width and surface area of the PSDs are decreased in torpor. At the beginning of an arousal the width of the PSD increases and gains maximum value in late arousals (AL), returning to spring (EU) values. No differences were found in total number of synapses during the torpor-arousal cycle. The results indicate reversible changes in ultrastructure of (asymmetric axospinous) synapses in the frontal cortex, which may be critical for the maintenance of cortical neuronal networks and for protection against potential deleterious effects of prolonged hypothermic phases of hibernation.

Enhanced physical activity is associated with improvements in cognitive function in rodents as well as in humans. The authors examined in detail which aspects of learning and memory are influenced by exercise, using a spatial Y-maze test combined with a 14-day exercise paradigm at different stages of learning. The authors show that 14 days of wheel running promotes memory acquisition, memory retention, and reversal learning. The exercise paradigm that was employed also significantly increased the number of maturing neurons, suggesting that an increase in neurogenesis underlies the positive effects of exercise on Y-maze performance. Finally, the authors show that memory acquisition in itself does not have a major impact on the number of immature neurons. However, memory retention testing and reversal learning both cause a significant reduction in the number of doublecortin and Ser133- phosphorylated pCREB-positive cells, indicating that a decrease in neurogenesis might be a prerequisite for optimal memory retrieval., ((c) 2007 APA, all rights reserved)

It is suggested that the hippocampus functions as a comparator by making a comparison between the internal representation and actual sensory information from the environment (for instance, comparing a previously learned location of a food reward with an actual novel location of a food reward in a Y-maze). However, it remains unclear to what extent the various hippocampal regions contribute to this comparator function. One of the proteins known to be crucially involved in the formation of hippocampus-dependent long-term memory is the adenosine 3',5' cyclic monophosphate dependent protein kinase (PKA). Here, we examined region-specific changes in immunoreactivity (ir) of the regulatory IIalpha,beta subunits of PKA (PKA RIIalpha,beta-ir) in the hippocampus during various stages of spatial learning in a Y-maze reference task. Thereafter, we compared changes in hippocampal PKA RIIalpha,beta-ir induced by training and reversal training in which the food reward was relocated to the previously unrewarded arm. We show that: (1) There was a clear correlation between behavioral performance and elevated PKA RIIalpha,beta-ir during the acquisition phase of both training and reversal training in area CA3 and dentate gyrus (DG), (2) PKA RIIalpha,beta-ir was similarly enhanced in area CA1 during the acquisition phase of reversal training, but did not correlate with behavioral performance, (3) PKA RIIalpha,beta-ir did not change during training or reversal training in the subiculum (SUB), (4) No changes in PKA RIIalpha,beta protein levels were found using Western blotting, and (5) AMPA receptor phosphorylation at serine 845 (S845p; the PKA site on the glutamate receptor 1 subunit (GluR1)), was enhanced selectively during the acquisition phase of reversal training. These findings reveal that training and reversal training induce region-specific changes in hippocampal PKA RIIalpha,beta-ir and suggest a differential involvement of hippocampal subregions in match-mismatch detection in case of Y-maze reference learning. Alterations in AMPA receptor regulation at the S845 site seems specifically related to the novelty detector function of the hippocampus important for match-mismatch detection.

The regulation and function of the calcium-dependent phosphatase calcineurin (CaN, protein phosphatase 2B) in learning and memory remain unclear, although recent work indicates that CaN may play a differential role in training and reversal training. To gain more insight into the involvement of CaN in these two types of learning, hippocampal CaN activity, protein levels, and expression patterns were studied in mice subjected to a reference memory version of the Y-maze task. We show that (1) training but not habituation induces a decrease in cytosolic CaN activity, (2) the recovery of cytosolic CaN activity is reversal training specific and does not reflect normal restoration of basal levels unrelated to subsequent learning, (3) cytosolic protein levels for the catalytic subunit of CaN (CaNA) are decreased at the early phase of training, but not at the early phase of reversal training, (4) CaNA immunoreactivity in the dorsal hippocampus is enhanced in the CA1 and CA3 area (but not in the dentate gyrus [DG] or subiculum [SUB]) only during reversal training. These findings indicate that memory formation is accompanied by reduced CaN activity, whereas adapting to changes in a familiar environment is accompanied by restored CaN activity. Moreover, reversal training selectively affects hippocampal CA3 and CA1 regions, suggesting a specific function of these hippocampal subregions in reversal learning.

Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of long-term memories for emotionally arousing experiences but not that for less arousing or neutral information. However, previous studies have not determined the basis of such arousal-induced selectivity. Here we report the finding that endogenous noradrenergic activation of the basolateral complex of the amygdala (BLA) induced by emotional arousal is essential in enabling glucocorticoid memory enhancement. Corticosterone administered immediately after object recognition training enhanced 24-h memory of naïve male rats but not that of rats previously habituated to the training context in order to reduce novelty-induced emotional arousal. The beta-adrenoceptor antagonist propranolol administered either systemically or into the BLA blocked the corticosterone-induced memory enhancement. Further, in habituated rats, corticosterone activated BLA neurons, as assessed by phosphorylated cAMP response element binding (pCREB) immunoreactivity levels, and enhanced memory only when norepinephrine release was stimulated by administration of the alpha(2)-adrenoceptor antagonist yohimbine. These findings strongly suggest that synergistic actions of glucocorticoids and emotional arousal-induced noradrenergic activation of the BLA constitute a neural mechanism by which glucocorticoids may selectively enhance memory consolidation for emotionally arousing experiences.

The present study investigated whether proliferation of hippocampal progenitors is subject to circadian modulation. Mice were perfused using 3h intervals throughout the light-dark cycle and brains were stained for Ki-67. Since Ki-67 is not expressed during the G0 phase of the cell cycle, we expected a decline in Ki-67 expression at the moment cells synchronously exit the cell cycle. However, despite the fact that various hippocampal factors fluctuate across the day, the number of dividing cells remained constant. In a second experiment, we studied whether disturbance of normal sleep affected the stable rate in cell proliferation. Our data show that 12h of sleep deprivation during the light phase did not influence proliferating cell number. A third experiment investigated whether physical activity, a condition known to enhance hippocampal cell proliferation, caused an elevation of the steady baseline number of proliferating progenitors, or a peak directly following the active phase of the animals. Mice were housed with a running wheel for 9 days. On the last day, animals were sacrificed either directly before or directly after the active phase. Exercise significantly promoted cell proliferation and this effect appeared to be strongest directly after the active period and to disappear during the resting phase. Our data suggest that hippocampal cell proliferation is not synchronized under basal conditions and is unchanged by sleep deprivation. However, running affected cell proliferation differentially at two times of day. These data demonstrate that the steady rate in cell proliferation is not indispensable, but can be changed by behavioral activity.

Mounting evidence suggests that sleep loss negatively affects learning and memory processes through disruption of hippocampal function. In the present study, we examined whether sleep loss alters the generation, differentiation, and survival of new cells in the dentate gyrus. Rats were sleep restricted by keeping them awake in slowly rotating drums for 1 day or repeatedly for 20 h/day over a period of 8 days. In addition to home cage controls, we included forced activity controls which, compared to sleep restricted rats, walked at double speed for half the time. These animals thus walked the same distance but had sufficient time to sleep. The results show that a single day of sleep deprivation significantly reduced hippocampal cell proliferation in the hilus of the dentate gyrus as measured by immunostaining for the proliferation marker Ki-67. Repeated partial sleep deprivation reduced cell proliferation in both the hilus and the subgranular zone. However, the latter was also found after chronic forced activity, and may not have been specific for sleep loss. To study neuronal survival, rats received a single intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) 5 days before the experiment. The number of surviving, BrdU-positive cells was not affected by sleep restriction. Also, the differentiation of BrdU-positive new cells into NeuN-positive neuronal and GFAP-positive glial phenotypes was not significantly altered by sleep loss. In conclusion, since new cells in the hilus mostly differentiate into glia, our findings indicate that sleep loss may reduce hippocampal gliogenesis.

Arg8-vasopressin (AVP), a circadian clock-controlled gene product, is released from the hypothalamic suprachiasmatic nuclei (SCN) in mice in a circadian fashion. Previously reported differences in two mouse lines, initially selected for thermoregulatory nest-building behavior (building small nests (S-mice) or big nests (B-mice)) with different circadian organization of behavior and in number of SCN-AVP immunoreactive neurons, were further investigated. We confirmed and expanded the finding that S-mice exhibited constant high levels of SCN-AVP content with no apparent circadian rhythmicity, whereas B-mice had lower numbers of AVP positive cells which varied with time of day. We found that AVP mRNA expression levels at midnight and midday were similar in both lines, as established by in situ hybridization. When AVP transport and release were blocked by colchicine, SCN-AVP immunoreactivity was similar in both lines. This suggests that differences in SCN-AVP content depend on transport or release. Organotypic SCN cultures of B-mice showed more AVP release per neuron than cultures of S-mice. These results reveal that on a mechanistic level the mouse lines differed in transport and/or release of AVP in the SCN, rather than differential regulation of AVP gene transcription or number of AVP immunoreactive neurons.

Neural progenitors in the subgranular zone of the hippocampal formation form a continuously proliferating cell population, generating new granule neurons throughout adult life. Between 10 days and 1 month after their formation, many of the newly generated cells die. The present study investigated whether a partial lesion of one of the main nuclei projecting to the hippocampus, the medial septum (MS), affects survival and differentiation of cells during this critical period. Rats were injected with BrdU and 5 days later excitotoxic lesion of the MS was applied by infusion of either 30 or 60 nmol of N-methyl-D-aspartate (NMDA). One week after the lesion, quantification of immunopositive cells revealed that the number of GABAergic cells was significantly reduced in both lesioned groups, whereas a decline in cholinergic cell number was observed only after injection of 60 nmol of NMDA. The partial septohippocampal denervation significantly reduced hippocampal neurogenesis. Survival of newly generated neurons was decreased by approximately 40%. The MS lesion did not affect proliferation of hippocampal progenitors. The present study points out the importance of a functional septohippocampal pathway for the regulation of hippocampal neurogenesis and highlights the potential role of GABA as a mediator in this phenomenon.

Daily rhythms in behavior and physiology are under control of the suprachiasmatic nucleus (SCN), the main mammalian circadian pacemaker located in the hypothalamus. The SCN communicates with the rest of the brain via various output systems. The aim of the present study was to determine the neuroanatomical and temporal relationship between two output systems, arginine-vasopressin (AVP) and transforming growth factor alpha (TGFalpha), in the mouse SCN. TGFalpha-positive cells were found throughout the SCN, but more abundantly in the core than the shell area, while AVP was predominantly found in the shell. Fluorescent double labeling revealed a total lack of co-expression for the two proteins in SCN cells. The circadian profile, studied by way of optical density in immunostaining at 3 h intervals, showed peak values for AVP shortly after the LD transitions. Immunoreactivity for TGFalpha was highly variable, especially at time points before the LD transitions. In addition, strong lateralization in TGFalpha immunostaining in the SCN was found in some individuals. Daily fluctuations in the paraventricular nucleus were absent for TGFalpha, and only weakly present for AVP. The main conclusion derived from this study is that these two output systems of the biological clock are anatomically separated with different daily profiles in expression.

In the current study, the authors investigated whether Morris water maze learning induces alterations in hippocampal neurogenesis or neural cell adhesion molecule (NCAM) polysialylation in the dentate gyrus. Two frequently used rat strains, Wistar and Sprague-Dawley, were trained in the spatial or the nonspatial version of the water maze. Both training paradigms did not have an effect on survival of newly formed cells that were labeled 7-9 days prior to the training or on progenitor proliferation in the subgranular zone. However, the granule cell layer of the spatially trained rats contained significantly more positive cells of the polysialylated form of the NCAM. These data demonstrate that Morris water maze learning causes plastic change in the dentate gyrus without affecting hippocampal neurogenesis., ((c) 2005 APA)

Hippocampal granule neurons that are newly formed during adulthood might be involved in learning and memory processes. Experimental data suggest that only hippocampus-dependent learning tasks stimulate neurogenesis. To further address this issue, the effects of active shock avoidance (ASA) learning on hippocampal progenitor proliferation and survival of newly formed cells were investigated. ASA training, although considered as hippocampus-independent, is known to induce several neurobiological alterations in the hippocampus. Adult Wistar rats were trained in a shuttle box using a 1-day or 4-day paradigm and brains were analyzed for the mitotic marker Ki-67. Effects on survival of newly generated cells were examined by immunocytochemistry for 5-bromo-2-deoxyuridine (BrdU), which was injected 1 week before the training. Neither proliferation nor survival was affected by the ASA learning task. Because elevated glucocorticoid levels have a negative impact on hippocampal neurogenesis, blood samples were taken throughout the 4-day training paradigm. Both trained animals and control rats that were only placed in the shuttle box without receiving foot shocks showed a similar rise in corticosterone, enabling us to exclusively investigate the effects of ASA learning on neurogenesis without differential interference of stress between groups. On the other hand, the finding that ASA induced elevations in plasma corticosterone, but did not influence proliferation or survival of newly formed cells, indicates that this type of stress does not affect neurogenesis. The present study shows that, in line with the existing data on other hippocampus-independent learning tasks, ASA training has no effect on hippocampal neurogenesis.

The chemical warfare blistering agent, sulfur mustard (SM), is a powerful mutagen and carcinogen. Due to its similarity to the related chemotherapy agents nitrogen mustard (mechlorethamine), it is expected to act as a developmental neurotoxicant. The present study was designed to establish a chick model for the mechanisms of SM on neurobehavioral teratogenicity, free of confounds related to mammalian maternal effects. Chicken eggs were injected with SM at a dose range of 0.0017-17.0 microg/kg of egg, which is below the threshold for dysmorphology, on incubation days (ID) 2 and 7, and then tests were conducted posthatching. Exposure to SM elicited significant deficits in the intermedial part of the hyperstriatum ventrale (IMHV)-related imprinting behavior. Parallel decreases were found in the level of membrane PKCgamma in the IMHV, while eliciting no net change in cytosolic PKCgamma. The chick, thus, provides a suitable model for the rapid evaluation of SM behavioral teratogenicity and elucidation of the mechanisms underlying behavioral anomalies. The results obtained, using a model that controls for confounding maternal effects, may be replicated in the mammalian model and provide the groundwork for studies designed to offset or reverse the SM-induced neurobehavioral defects in both avian and mammals.

The suprachiasmatic nucleus (SCN) is engaged in modulation of memory retention after (fear) conditioning, but it is unknown which pathways and neurotransmitter system(s) play a role in this action. Here we examine immunocytochemically whether muscarinic acetylcholine receptors (mAChRs), mediating cholinergic signal transduction in the SCN, are involved. For this purpose, mAChR immunoreactivity (mAChR-ir) was studied in the SCN after various stages of passive shock avoidance (PSA) and active shock avoidance (ASA) training and, for ASA, at various posttraining time points. mAChR-ir was significantly enhanced in SCN neurons as a result of the training procedure, and the number of mAChR-positive glial cells in the SCN increased significantly. The increase in mAChR-ir as a result of PSA and ASA training was not due to fear conditioning or the number of correct avoidances (in case of ASA training) but rather to behavioral arousal as a consequence of (brief) exposure to a novel environment (the test apparatus). This finding was confirmed by a cage-change experiment in which the rats were allowed to stay in a novel cage for 15 min or 24 hr. Only the brief exposure to the fresh cage triggered alterations for SCN mAChRs 24 hr later. These results shed new light on a possible function of the cholinergic system in the SCN mediated by mAChRs in relation to modulation of memory processes and demonstrate that behavioral arousal during (the habituation stage of) a learning task is sufficient to alter the mAChR system in the SCN.

A wide variety of otherwise unrelated neuroteratogens elicit a common set of behavioral defects centering around cholinergic contributions to cognitive function. We utilized the developing chick to overcome confounds related to maternal effects and compared the actions of nicotine, chlorpyrifos, and heroin on cholinergic signaling in the intermedial part of the hyperstriatum ventrale (IMHV), which controls imprinting behavior. Chicken eggs were injected with nicotine (10 mg/kg of egg), chlorpyrifos (10 mg/kg of egg), or heroin (20 mg/kg of egg; all doses below the threshold for dysmorphology) on incubation days (ID) 0 and 5, and then tests were conducted posthatching. All three compounds elicited significant deficits in imprinting behavior. We also found defects in cholinergic synaptic signaling specifically involving the muscarinic receptor-mediated membrane translocation of protein kinase C (PKC)-gamma and in the basal levels of both PKCgamma and PKCbetaII, the two isoforms known to be relevant to behavioral performance. In contrast, there were no alterations in the response of PKCalpha, an isoform that does not contribute to the behavior, nor were cytosolic levels of any of the isoforms affected. Taken together with similar results obtained in rodents, our findings suggest that disparate neuroteratogens all involve signaling defects centering on the ability of cholinergic receptors to elicit PKCgamma translocation/activation and that this effect is direct, i.e., not mediated by maternal confounds. The chick thus provides a suitable model for the rapid screening of neuroteratogens and elucidation of the mechanisms underlying behavioral anomalies.

The immunocytochemical and subcellular localization of the Ca(2+)-dependent protein kinase C (cPKC) isoforms (PKCalpha, beta1, beta2, and gamma) was examined in rabbit hippocampus of young (3 months of age; n = 11) and aging (36 months of age; n = 14) subjects. Detailed immunocytochemical analyses revealed a significant increase in PKCbeta1, beta2, and gamma immunoreactivity in principal cell bodies and associated dendrites, and interneurons of the hilar region in the aging rabbits. The number of PKCalpha- and gamma-positive interneurons in the aging stratum oriens declined significantly. PKCalpha was least affected in principal cells, showing an increase in immunostaining in granule cells only. Weakly PKC-positive principal cells intermingled between densely stained ones were seen in parts of the hippocampus in most of the aging rabbits, showing that the degree of aging-related alterations in PKC-immunoreactivity varies between neurons. Changes in PKC expression in the molecular and subgranular layer of the aging dentate gyrus suggested a reorganization of PKC-positive afferents to this region. Western blot analysis revealed a significant loss of PKC in the pellet fraction for all isoforms, and a tendency for increased levels of cytosolic PKC. However, no significant changes were found in total PKC content for any PKC isoform. A concurrent dramatic loss of the PKC anchoring protein receptor for activated C kinase (RACK1) in the pellet fraction was shown by Western blotting. These findings suggest that the loss of RACK1 contributes to the dysregulation of the PKC system in the aging rabbit hippocampus. The enhanced PKC-immunoreactivity might relate to reduced protein-protein interactions of PKC with the anchoring protein RACK1 leading to increased access of the antibodies to the antigenic site. In conclusion, the results suggest that memory deficits in aging rabbits are (in part) caused by dysregulation of subcellular PKC localization in hippocampal neurons., (Copyright 2004 Wiley-Liss, Inc.)

Neurofibrillary pathology [paired helical filaments (PHFs)] formed by the microtubule-associated protein tau in a hyperphosphorylated form is a major hallmark of Alzheimer's disease and related disorders. The process of tau phosphorylation, thought to be of critical importance for PHF formation, and its potential link to neurodegeneration, however, is not understood very well, mostly because of the lack of a physiological in vivo model of PHF-like tau phosphorylation. Here we describe the formation of highly phosphorylated tau, containing a number of PHF-like epitopes in torpor during hibernation. PHF-like phosphorylation of tau was not associated with fibril formation and was fully reversible after arousal. Distribution of PHF-like tau followed a consistent pattern, being most intense in the entorhinal cortex, hippocampus, and isocortical areas. Within the hippocampus, a particularly high labeling was seen in CA3 pyramidal cells. Somewhat lesser reactivity was present in CA1 neurons while dentate gyrus granule cells were not reactive. Formation of PHF-like tau in CA3 neurons was paralleled by the regression of synaptic contacts of the mossy fiber system terminating on CA3 apical dendrites. Mossy fiber afferentation was re-established during arousal, concomitantly with the decrease of PHF-like tau in CA3 neurons. These findings implicate an essential link between neuronal plasticity and PHF-like phosphorylation of tau. The repeated formation and degradation of PHF-like tau might, thus, represent a physiological mechanism not necessarily associated with pathological effects. Hibernation will, therefore, be a valuable model to study the regulation of PHF-like tau-phosphorylation and its cell biological sequelae under physiological in vivo conditions.

Natural hypothermia during hibernation results in physiological and behavioral deficits. These changes may be traced at the level of hippocampal signal transduction. We investigated synaptophysin immunoreactivity (SYN-ir) in the hippocampus after short and long periods of hypothermia and short and long periods of euthermy in hibernating ground squirrels. SYN-ir in the stratum lucidum of the hippocampus was transiently reduced during natural hypothermia. Natural hypothermia thus reduces synaptic efficacy. This may play a role in the reduced neuronal connectivity of CA3 pyramidal cell dendrites observed in hibernating ground squirrels.

Active shock avoidance was used to explore the impact of behavioural stimulation on the neurochemistry of the suprachiasmatic nucleus. We have found previously that the expression of muscarinic acetylcholine receptors in the suprachiasmatic nucleus of young rats was significantly enhanced 24 hours after fear conditioning. Here, we investigated whether this observation is age-dependent. We used 26 month-old Wistar rats with a deteriorated circadian system, and compared them with young rats (4 months of age) with an intact circadian system. Vasopressin, representing a major output system of the suprachiasmatic nucleus, was studied in addition to muscarinic receptors. Young rats showed a significant increase in immunostaining for muscarinic acetylcholine receptors 24 h after training, corroborating earlier observations. Aged rats did not show such an increase. In contrast, aged rats did show an increase in vasopressin immunoreactivity 24 h after fear conditioning, both at the level of content and cell number, while young rats did not reveal a significant rise. Thus, it seems that these two neurochemical systems in the suprachiasmatic nucleus are regulated independently. The results further demonstrate that the circadian pacemaker is influenced by fear conditioning in an age-dependent manner.

The suprachiasmatic nucleus (SCN) is the locus of the main pacemaker for circadian behavioral rhythms. In common voles, variation in circadian behavioral rhythmicity correlates with vasopressin (AVP) immunoreactive cells in the SCN. Here we studied the immunostaining of four AVP linked Ca(2+)-dependent protein kinase C (PKC) isoforms (PKCalpha, PKCbeta1, PKCbeta2, and PKCgamma) at the beginning of the light period, and conclude that PKCalpha is highly expressed in the vole SCN compared to the other isozymes. Voles, characterized as strongly circadian rhythmic showed circadian variation in numbers of PKCalpha immunoreactive SCN neurons, while voles with weak or no circadian rhythmicity did not reveal such a circadian profile. PKCalpha immunoreactivity in acute SCN slices that were treated with a physiological dose of AVP was significantly lowered when compared with control slices. The intracellular messenger PKCalpha may reflect variation in locomotor behavior via the AVP system in the vole SCN. This system could play a key role in the vole SCN by mediating output of its circadian clock.

Decreased immunoreactivity has been reported for several neuropeptides in the aged suprachiasmatic nucleus (SCN). We compared somatostatin (SS) and substance P (SP) immunoreactivity (ir) in aged (20-26 months) and young (6 months) Wistar rats. The old rat SCN revealed a significant increase in SSir (2.6-fold) and SPir. The results show that not all SCN-neuropeptidergic systems decline with age, and suggest a specific age-related role for SS in the SCN., (Copyright 2002 Elsevier Science B.V.)

Despite the prominent role of the Syrian hamster (Mesocricetus auratus) in studies of circadian rhythms, there are no data available on the temporal dynamics of the neuropeptide vasopressin (AVP), a major output system of the suprachiasmatic nucleus (SCN). We studied the hamster SCN-AVP system in vivo across the light period and in vitro using long-term organotypic SCN cultures. Additionally, we compared wild-type and tau mutant hamsters with an endogenous circadian period of approximately 24 h and approximately 20 h, respectively. The in vivo study revealed no differences in the number of SCN-AVP neurons between the two genotypes of hamsters studied at three time points across the light period of the circadian cycle. A significantly higher level of AVP-immunoreactivity, however, was found in the SCN of wild-type compared to tau mutant hamsters at the beginning and in the middle of the light period, but not at the end of the light period. SCN-AVP cell number and immunostaining decreased significantly across the light period in wild-type hamsters, but not in tau mutants. The in vitro study revealed a significantly higher rate of AVP release per 24 h from the tau mutant SCN compared to the wild-type SCN. Robust circadian oscillations in AVP release were not found in either type of hamster. These results may suggest that the SCN-AVP system of hamsters, irrespective of genotype, is relatively weak compared to other species. Moreover, the tau mutation seems to influence the SCN-AVP system by enhancing the rate of AVP release and by reducing AVP content and its daily fluctuation.

Hypothermia is commonly used to restrict organ damage during preservation of tissue, but does not offer complete protection. Organ damage after reperfusion/rewarming is amongst others caused by an impairment of vascular properties, particularly endothelium-dependent vasodilatation. We hypothesized that hibernating small animals, which frequently cycle through periods of deep cooling (torpor) and full rewarming (arousal), employ specific mechanisms to preserve vascular function after cooling and reperfusion. Therefore we measured contraction of aortic tissue of hibernating European ground squirrels after 24 h and 7 days of torpor, arousal (1.5 h) and in non-hibernating animals. To assess the role of nitric oxide (NO), experiments were performed in the absence and presence of the NO-synthesis inhibitor, L-NMMA (10(-4) M). Maximum contraction to phenylephrine and angiotensin II was doubled in 7-days torpid animals without a shift in EC50, compared to the other 3 groups. Maximum contraction to KCl was doubled in 7-days torpid animals compared to the arousal group and non-hibernating animals. Relaxation to acetylcholine (ACh) and sodium nitrite in phenylephrine precontracted rings did not differ between groups. In the presence of L-NMMA, the maximum of concentration-response curves for all three vasoconstrictors was increased by about 30% in the arousal group, but unaffected in other groups. L-NMMA completely inhibited ACh-induced relaxation in 24-h torpid animals and non-hibernating animals, but only partially in 7-days torpid animals and in the arousal group. From this we conclude that vascular adaptation proceeds during torpor. Further, increased contractility of aortic tissue during long torpor returns to normal within 1.5 hours of arousal, which is associated with an increased basal NO synthesis. In addition, involvement of NO in agonist-mediated relaxation differs between the various stages of hibernation.Thus, hibernating animals have effectively developed mechanisms to preserve vascular function after cooling and rewarming.

European ground squirrels (Spermophilus citellus) in outside enclosures show suppressed circadian rhythmicity in body temperature patterns during the first days of euthermia after hibernation. This may reflect either gradual reappearance of circadian rhythmicity following suppressed functioning of the circadian system during hibernation, or it may reflect transient days during re-entrainment of the circadian system which, during hibernation, has drifted out of phase with the environmental light-dark cycle. Here we report that animals kept under continuous dim light conditions also showed absence of circadian rhythmicity in activity and body temperature in the first 5-15 days after hibernation. After post-hibernation arrhythmicity, spontaneous circadian rhythms re-appeared gradually and increased daily body temperature range. Numbers of arginine-vasopressin immunoreactive neurons in the suprachiasmatic nuclei correlated positively with individual circadian rhythmicity and increased gradually over time after hibernation. Furthermore, circadian rhythmicity was enhanced rather than suppressed after exposure to a light-dark cycle but not after a single 1-h light pulse (1,700 lux). The results support the view that the functioning of the circadian system in the European ground squirrel is suppressed during hibernation at low temperatures and that it requires several days of euthermia to resume its summer function.

Estradiol exerts beneficial effects on neurodegenerative disorders associated with the decline of cognitive performance. The present study was designed to further investigate the effect of 17beta-estradiol on learning and memory, and to evaluate its neuroprotective action on cholinergic cells of the nucleus basalis magnocellularis, a neural substrate of cognitive performance. Female rats were ovariectomized at an age of 6 months. Three weeks later they received injections of either a mid-physiological dose of 17beta-estradiol or vehicle (oil), every other day for 2 weeks. The effect of estradiol on cognitive performance was tested in two associative learning paradigms. In the two-way active shock avoidance task estradiol-replaced animals learned significantly faster, while in the passive shock avoidance test no differences were observed between the experimental groups. Subsequent unilateral infusion of N-methyl-D-aspartate in the nucleus basalis magnocellularis resulted in a significant loss of cholinergic neurons concomitant with the loss of their fibers invading the somatosensory cortex. Estradiol treatment did not affect the total number of choline-acetyltransferase-immunoreactive neurons and their coexpression of the p75 low-affinity neurotrophin receptor either contralateral or ipsilateral to the lesion. In contrast, cholinergic fiber densities in estradiol-treated animals were greater both in the contralateral and ipsilateral somatosensory cortices as was detected by quantitative choline-acetyltransferase and vesicular acetylcholine transporter immunocytochemistry. However, estradiol treatment did not affect the lesion-induced relative percentage loss of cholinergic fibers. A significant decline of synaptophysin immunoreactivity paralleled the cholinergic damage in the somatosensory cortex of oil-treated animals, whereas an almost complete preservation of synaptic density was determined in estradiol-treated rats. Our results indicate that estradiol treatment enhances the cortical cholinergic innervation but has no rescuing effect on cholinergic nerve cells in the basal forebrain against excitotoxic damage. Nevertheless, estradiol may restore or maintain synaptic density in the cerebral cortex following cholinergic fiber loss. This estradiol effect may outweigh the lack of cellular protection on cholinergic cells at the functional level.

The functional significance of the suprachiasmatic nucleus (SCN) in circadian rhythm control of mammals has been well documented. The role of protein phosphorylation mediated by protein kinase C (PKC), however, is not well known. We report the immunocytochemical localization of three Ca(2+)-dependent PKC isoforms (alpha, betaI, betaII) within the SCN of selected house mouse lines that differ in behavioral circadian rhythm parameters. Optical density measurements revealed that the adult mice selected for low levels of nest-building behavior (small nest-builders) had more than threefold higher PKCbetaI immunostaining in the SCN than the mice selected for high levels of nest-building behavior (big nest-builders). A similar twofold difference between the adult small and big nest-builders was observed for the number of PKCbetaI-containing cells in the SCN. The non-selected control lines were intermediate. Ten-day-old pups revealed similar differences in PKCbetaI immunostaining in the SCN between the small and big nest-builders. PKCalpha and PKCbetaII immunostaining in the SCN was not different among the lines. PKCbetaI immunostaining was not different among the selected lines in the lateroanterior hypothalamic nucleus (LA) and the cornu ammonis field 1 (CA1) of the dorsal hippocampus and confirms the specificity of the difference in PKCbetaI immunostaining in the SCN among the selected lines. The significance of these findings is discussed in the context of differences among the lines in arginine-vasopressin (AVP) and light-induced Fos expression in the SCN, behavioral phase-delay responses to 15-min light pulses in constant darkness, and measures of the strength of the circadian activity rhythm expressed.

The formation of new synapses has been suggested to underlie learning and memory. However, previous work from this laboratory has demonstrated that hippocampus-dependent associative learning does not induce a net gain in the total number of hippocampal synapses and, hence, a net synaptogenesis. The aim of the present work was to determine whether associative learning involves a specific synaptogenesis confined to the formation of multiple-synapse boutons (MSBs) that synapse with more than one dendritic spine. We used the behavioral paradigm of trace eyeblink conditioning, which is a hippocampus-dependent form of associative learning. Conditioned rabbits were given daily 80-trial sessions to a criterion of 80% conditioned responses in a session. During each trial, the conditioned stimulus (tone) and the unconditioned stimulus (corneal airpuff) were presented with an intervening trace interval of 500 msec. Brain tissue was taken for morphological analyses 24 hr after the last session. Unbiased stereological methods were used for obtaining estimates of the total number of MSBs in the stratum radiatum of hippocampal subfield CA1. The results showed that the total number of MSBs was significantly increased in conditioned rabbits as compared with pseudoconditioned or unstimulated controls. This conditioning-induced change, which occurs without a net synaptogenesis, reflects a specific synaptogenesis resulting in MSB formation. Models of the latter process are proposed. The models postulate that it requires spine motility and may involve the relocation of existing spines from nonactivated boutons or the outgrowth of newly formed spines for specific synaptogenesis with single-synapse boutons activated by the conditioning stimulation.

We studied vasopressin (AVP) release patterns from organotypic suprachiasmatic nucleus (SCN) cultures obtained from circadian rhythmic and non-rhythmic voles. All eight SCN cultures made from non-rhythmic voles did not produce any circadian pattern in AVP release, while five out of six SCN cultures of rhythmic voles produced significant (circadian) peak values. The total amount of AVP released was 2-fold higher in SCN cultures from rhythmic vole. These data confirm our previously formulated AVP release deficit hypothesis for nonrhythmic voles, and suggest that AVP in the vole SCN plays an important role in mediating output of its circadian clock, regulating circadian organization of locomotor behavior.

Songbirds (Oscines) learn their songs from a tutor. It is not known where in the brain the memories of these learned sounds are stored. Recent evidence suggests that song perception in songbirds involves neuronal activation in brain regions that have not traditionally been implicated in the control of song production or song learning, notably the caudal part of the neostriatum (NCM) and of the hyperstriatum ventrale. Zebra finch males (Taeniopygia guttata castanotis) were reared without their father and exposed to a tape-recorded song during the sensitive period for song learning. When, as adults, they were reexposed to the tutor song, the males showed increased expression of the protein products of the immediate early genes egr-1 (ZENK) and c-fos in the NCM and caudal hyperstriatum ventrale, but not in the conventional "song-control nuclei." The strength of the immediate early gene response (which is a reflection of neuronal activation) in the NCM correlated significantly and positively with the number of song elements that the birds had copied from the tutor song. These results show localized neural activation in response to tutor song exposure that correlates with the strength of song learning.

The intrinsic properties of the suprachiasmatic nucleus (SCN), the site of the main circadian pacemaker in mammals, have recently been studied in vitro by means of organotypic slice culturing. So far, only neonatal rats and mice have been used for such developmental and functional analyses of the isolated pacemaker. Here, the authors present a comparative developmental study of the SCN of voles, rats, and hamsters in organotypic slice cultures. In contrast to strictly circadian organization of behavior in rats and hamsters, common voles (Microtus arvalis) are characterized by large variability in the strength of circadian organization of behavior. It is not known to what extent this variability is reflected in the intrinsic features of the SCN. Cultures were prepared from rat, hamster, and vole pups (6 to 9 days old) for the purpose of species comparison. In addition, the authors studied the relation between age and development in cultures from pup (7 to 10 days old), juvenile (15 to 16 days old), and young adult (1 to 2 months old) voles. In contrast to the situation in rat and hamster, the most striking feature in neonatal voles is the variability in shape of the final, fully developed culture and its poor resemblance with the in vivo SCN. The SCN of adult voles, however, could be cultured successfully while retaining its morphological organization seen in situ. Phase-contrast microscopy and immunocytochemical staining for vasopressin and glial fibrillary acidic protein revealed that cultures of pup and juvenile voles still have potential for neurogenesis and morphological reorganization. Young voles, therefore, can serve as a model to study the developmental establishment of a functional circadian pacemaker, while adult voles allow the study of intrinsic pacemaker properties in relation to previously recorded behavior of the donor and aging-related pacemaker dysfunction.

The aim of this study was to determine whether hippocampus-dependent associative learning involves changes in the number and/or structure of hippocampal synapses. A behavioral paradigm of trace eyeblink conditioning was used. Young adult rabbits were given daily 80 trial sessions to a criterion of 80% conditioned responses in a session. During each trial, the conditioned (tone) and unconditioned (corneal airpuff) stimuli were presented with a stimulus-free or trace interval of 500 msec. Control rabbits were pseudoconditioned by equal numbers of random presentations of the same stimuli. Brain tissue was taken for morphological analyses 24 hours after the last session. Synapses were examined in the stratum radiatum of hippocampal subfield CA1. Unbiased stereological methods were used to obtain estimates of the total number of synapses in this layer as well as the area of the postsynaptic density. The data showed that the total numbers of all synaptic contacts and various morphological subtypes of synapses did not change in conditioned animals. The area of the postsynaptic density, however, was significantly increased after conditioning in axospinous nonperforated synapses. This structural alteration may reflect an addition of signal transduction proteins (such as receptors and ion channels) and the transformation of postsynaptically silent synapses into functional ones. The findings of the present study indicate that cellular mechanisms of hippocampus-dependent associative learning include the remodeling of existing hippocampal synapses. Further studies examining various time points along the learning curve are necessary to clarify the issue of whether these mechanisms also involve the formation of additional synaptic contacts.

In the present study plastic neural responses to N-methyl-D-aspartate-induced excitotoxic lesions and the neuroprotective effects of the L-type voltage-dependent Ca(2+) channel antagonist nimodipine were investigated in the rat magnocellular nucleus basalis. Assessment of spontaneous behaviour in the elevated plus maze and small open-field paradigms on day 5 and day 14 post-surgery indicated anxiety and persistent hypoactivity of N-methyl-D-aspartate-lesioned rats, as compared with sham-operated controls. Nimodipine administration significantly alleviated the behavioural deficits. Quantitative histochemical analysis of acetylcholinesterase-positive fibre innervation of the somatosensory cortex and determination of the numbers of choline-acetyltransferase-positive proximal fibre branches of cholinergic projection neurons in the magnocellular nucleus basalis demonstrated a severe cholinergic deficit as a consequence of the excitotoxic lesion 14 days post-surgery. Nimodipine pre-treatment significantly attenuated the loss of cortical cholinergic innervation and preserved the functional integrity of cholinergic projection neurons in the magnocellular nucleus basalis. Double-labelling immunocytochemistry demonstrated increased amyloid precursor protein expression in shrinking and presumably apoptotic choline-acetyltransferase-positive neurons, whereas surviving cholinergic nerve cells were devoid of excessive amyloid precursor protein immunoreactivity. Moreover, as a consequence of N-methyl-D-aspartate infusion, rim-like accumulation of amyloid precursor protein-positive astrocytes was visualized in a penumbra-like zone of the excitotoxic injury. Furthermore, abundant sprouting of serotonergic projection fibres invading the damaged magnocellular nucleus basalis subdivision was demonstrated. Pharmacological blockade by the Ca(2+) antagonist nimodipine significantly attenuated both neuronal and glial amyloid precursor protein immunoreactivity and serotonergic fibre sprouting following N-methyl-D-aspartate infusion. The present data characterize plastic endogenous glial and neuronal responses in the magnocellular nucleus basalis model of acute excitotoxic brain damage. The increased amyloid precursor protein expression may indicate effective means of intrinsic neuroprotection, as secreted amyloid precursor protein isoforms are suggested to play a role in neuronal rescue following excitotoxic injury. From a pharmacological point of view, extensive sprouting of serotonergic projections in the damaged magnocellular nucleus basalis may also counteract N-methyl-D-aspartate excitotoxicity via serotonin-induced inhibition of Ca(2+) currents and membrane hyperpolarization. Hence, lesion-induced changes in spontaneous animal behaviour, such as anxiety and novelty-induced hypoactivity, may well be attributed to the considerable re-distribution of serotonergic projections in the basal forebrain. In conclusion, our present data emphasize a role of neuron-glia and neurotransmitter-system interactions in functional recovery after acute excitotoxic brain injury, and the efficacy of L-type Ca(2+) channel blockade by the selective 1,4-dihydropyridine antagonist nimodipine.

This study is the first to demonstrate organotypic culturing of adult suprachiasmatic nuclei (SCN). This approach was used to obtain organotypic SCN cultures from adult vole brain with a previously determined state of behavioral circadian rhythmicity. We examined vasopressin (AVP) immunoreactivity in these organotypic slice cultures. AVP is one of the major neuropeptides produced by the SCN, the main mammalian circadian pacemaker. AVP immunoreactivity in the SCN of adult common voles in vivo has been shown to correlate with the variability in expression of circadian wheel-running behavior. Here, cultures prepared from circadian rhythmic and nonrhythmic voles were processed immunocytochemically for AVP. Whereas in all cultures AVP could be observed, AVP immunoreactivity differed considerably between vole SCN cultures. SCN cultures from rhythmic voles contained significantly lower numbers of AVP immunoreactive (AVPir) cells per surface area than cultures from nonrhythmic voles. The correlation between timing of behavior and AVP immunoreactivity in vitro is similar to the correlation found earlier in vivo. Apparently, such correlation depends on intrinsic AVP regulation mechanisms of SCN tissue, and not on neural or hormonal input from the environment, as present in intact brain.

Immunocytochemical mapping studies employing the extensively used monoclonal anti-muscarinic acetylcholine receptor (mAChR) antibody M35 are reviewed. We focus on three neuronal muscarinic cholinoceptive substrates, which are target regions of the cholinergic basal forebrain system intimately involved in cognitive functions: the hippocampus; neocortex; and amygdala. The distribution and neurochemistry of mAChR-immunoreactive cells as well as behaviorally induced alterations in mAChR-immunoreactivity (ir) are described in detail. M35+ neurons are viewed as cells actively engaged in neuronal functions in which the cholinergic system is typically involved. Phosphorylation and subsequent internalization of muscarinic receptors determine the immunocytochemical outcome, and hence M35 as a tool to visualize muscarinic receptors is less suitable for detection of the entire pool of mAChRs in the central nervous system (CNS). Instead, M35 is sensitive to and capable of detecting alterations in the physiological condition of muscarinic receptors. Therefore, M35 is an excellent tool to localize alterations in cellular cholinoceptivity in the CNS. M35-ir is not only determined by acetylcholine (ACh), but by any substance that changes the phosphorylation/internalization state of the mAChR. An important consequence of this proposition is that other neurotransmitters than ACh (especially glutamate) can regulate M35-ir and the cholinoceptive state of a neuron, and hence the functional properties of a neuron. One of the primary objectives of this review is to provide a synthesis of our data and literature data on mAChR-ir. We propose a hypothesis for the role of muscarinic receptors in learning and memory in terms of modulation between learning and recall states of brain areas at the postsynaptic level as studied by way of immunocytochemistry employing the monoclonal antibody M35.

In the present study we report the effect of corticosterone in the regulation of hippocampal muscarinic acetylcholine receptor immunoreactivity (mAChR-ir) expression in rats. Adrenalectomy (ADX) or a single injection of a mineralocorticoid antagonist RU-28318 (1.0 mg/100 g body weight (b.w.)) in adrenally intact rats 24 h prior to sacrifice revealed an increased mAChR-ir in hippocampal CA1 and CA3 areas. Corticosterone replacement (100 microg/100 g b.w.) prevented the increase in mAChR-ir of ADX animals. However, glucocorticoid receptor antagonist (RU38486) treatment in adrenally intact rats failed to affect the mAChR immunolabeling. These results point to a modulation of muscarinic receptors by corticosterone that is predominantly mediated by the mineralocorticoid receptor.

Aging leads to a decrease in circadian organization of behavior. Whether this general observation is related to the finding that in older subjects the arginine-vasopressin (AVP) system in the suprachiasmatic nucleus (SCN) has deteriorated is an unsolved question. Here we assessed circadian organization of running wheel behavior and numbers of AVP cells in the SCN of old voles (n=12, 11. 5 months of age) and compared the results with data from young voles (n=16, 4.5 months of age). A third of the young voles, but three-quarter of the old voles lost circadian rhythmicity. Analysis of daily onset to onset periodicity of running wheel activity at the age of 5 and 10 months in individual voles revealed a significant loss of precision of circadian rhythmicity at the higher age. The number of AVP cells in the SCN of old voles decreased substantially, over 78% compared to young voles in general. AVP cell numbers, however, cannot be directly correlated with the state of rhythmicity in old voles; in one of the three circadian rhythmic old voles the SCN contained the least AVP cells. This study does not support the idea of a causal relationship between aging induced reduction in AVP cells in the SCN and the presence of circadian rhythmicity in behavior., (Copyright 1999 Elsevier Science B.V.)

This study describes the translocation of the brain specific protein kinase C gamma isoenzyme (PKCgamma) in the hippocampus during food rewarded spatial learning. The holeboard test was used for spatial orientation, and immunoblot analysis was used for assessment of PKCgamma in cytosolic, membrane-inserted and membrane-associated fractions. Membrane-associated PKCgamma was increased during early acquisition of spatial learning, but not in a later phase of training. This transient and apparently temporary intracellular PKCgamma translocation was only observed in the posterior but not in the anterior hippocampus, and was only detected within 10 min after termination of the learning trial. This study supports the idea that PKCgamma is significantly involved in the biochemical events underlying learning and memory, notably during the period of novel information processing. The results further promote the hypothesis that the hippocampus is specifically involved in temporal information processing, which requires the engagement of PKCgamma.

Vasopressin (AVP) is a major neuropeptide in the suprachiasmatic nucleus, the mammalian hypothalamic circadian pacemaker. Protein kinase Calpha is a putatively coupled intracellular messenger. Mean numbers of AVP- and protein kinase Calpha-immunoreactive neurons were determined in the suprachiasmatic nucleus of common voles, entrained to a 12:12 h light-dark (LD) cycle, at the beginning of the light period (zeitgeber time zero) and 6 h later (zeitgeber time six). At zeitgeber time zero, mean numbers of AVP- and protein kinase Calpha- immunoreactive neurons were 2194 and 9897, respectively. Both numbers decreased significantly with about 40% at zeitgeber time six. This concurrent decrease was most pronounced in the dorsomedial aspect of the suprachiasmatic nucleus. These findings are consistent with the findings of a peak of AVP release in rats during the early light phase.