Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

Glutamic acid decarboxylase 65 and 67 expression in the lateral septum is up-regulated in association with the postpartum period in mice.

The postpartum period in mammals undergoes a variety of physiological adaptations, including metabolic, behavioral and neuroendocrine alterations. GABA signaling has been strongly linked to various emotional states, stress responses and offspring protection. However, whether GABA signaling may change in the lateral septum (LS), a core brain region for regulating behavioral, emotional and stress responses in postpartum mice has not previously been examined. In this study, we tested whether the expression of two isoforms of glutamic acid decarboxylase (GAD), GAD65 (GAD2) and GAD67 (GAD1), the rate-limiting enzyme for GABA synthesis, exhibits altered expression in postpartum mice relative to nonmaternal, virgin mice. Using microdissected septal tissue from virgin and age-matched postpartum females, quantitative real-time PCR and Western blotting were carried out to assess GAD mRNA and protein expression, respectively. We found both protein and mRNA expression of GAD67 in the whole septum was up-regulated in postpartum mice. By contrast, no significant difference in the whole septum was observed in GAD65 expression. We then conducted a finer level of analysis using smaller microdissections and found GAD67 to be significantly increased in rostral LS, but not in caudal LS or medial septum (MS). Further, GAD65 mRNA expression in rostral LS, but not in caudal LS or MS was also significantly elevated in postpartum mice. These findings suggest that an increased GABA production in rostral LS of the postpartum mice via elevated GAD65 and GAD67 expression may contribute to multiple alterations in behavioral and emotional states, and responses to stress that occur during the postpartum period. Given that rostral LS contains GABA neurons that are projection neurons or local interneurons, it still needs to be determined whether the function of elevated GABA is for local or distant action or both.

Pyroglutamyl peptidase II inhibition enhances the analeptic effect of thyrotropin-releasing hormone in the rat medial septum.

Thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH(2)) has multiple, but transient, homeostatic functions in the brain. It is hydrolyzed in vitro by pyroglutamyl peptidase II (PPII), a narrow specificity ectoenzyme with a preferential localization in the brain, but evidence that PPII controls TRH communication in the brain in vivo is scarce. We therefore studied in male Wistar rats the distribution of PPII mRNA in the septum and the consequence of PPII inhibition on the analeptic effect of TRH injected into the medial septum. Twelve to 14% of cell profiles expressed PPII mRNA in the medial septum-diagonal band of Broca; in this region the specific activity of PPII was relatively high. Twenty to 35% of PPII mRNA-labeled profiles were positive for TRH-receptor 1 (TRH-R1) mRNA. The intramedial septum injection of TRH reduced, in a dose-dependent manner, the duration of ethanol-induced loss of righting reflex (LORR). Injection of the PPII inhibitor pGlu-Asn-Pro-7-amido-4-methylcoumarin into the medial septum enhanced the effect of TRH. The injection of a phosphinic TRH analog, a higher-affinity inhibitor of PPII, diminished the duration of LORR by itself. In contrast, the intraseptal injection of pGlu-Asp-Pro-NH(2), a peptide that did not inhibit PPII activity, or an inhibitor of prolyl oligopeptidase did not change the duration of LORR. We conclude that in the medial septum PPII activity may limit TRH action, presumably by reducing the concentration of TRH in the extracellular fluid around cells coexpressing PPII and TRH-R1.

Verifying of participation of nitric oxide in morphine place conditioning in the rat medial septum using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d).

BACKGROUND: Role of nitric oxide (NO) in morphine-induced conditioned place preference (CPP) has already been proposed in the rat medial septum (MS), but no molecular evidence has been provided to clear this fact. METHODS: Effects of intraseptal injections of L-arginine and/or NG-nitro-L-arginine methyl ester (L-NAME) on morphine place conditioning in Wistar rats were examined. Morphine (2.5-7.5 mg/kg) was injected s.c. using a three-day schedule of an unbiased place preference. All of the brain samples were examined histochemically by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), the main marker for NO activation. RESULTS: Morphine induced a significant CPP in the rats. Single injections of L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not induce CPP. In addition, co-administration of morphine (5.0 mg/kg) with L-arginine or L-NAME (0.3, 1.0 and 3.0 µg/rat) did not affect morphine response. However, administration of L-arginine (0.3, 1.0 and 3.0 µg/rat) prior to morphine conditioning testing enhanced the expression of morphine response. Moreover, pre-injection of L-NAME (0.3, 1.0 and 3.0 µg/rat) to L-arginine (0.3 µg/rat) did not reverse the response to the agent. The expression of NADPH-d was observed in the rat brain samples treated by L-arginine. A decreased expression of NADPH-d was also observed in rats pre-injected by L-NAME. CONCLUSION: This finding strongly suggests that NO system in the rat MS has an impact on the expression of morphine rewarding, and that the NO participates in place conditioning induced of morphine.

Extracellular signal-regulated kinase activation in the amygdala mediates elevated plus maze behavior during opioid withdrawal.

This study examined whether activation of extracellular signal-regulated kinase (ERK) contributes to the increased open-arm time observed in the elevated plus maze (EPM) during opioid withdrawal. We applied SL327, a selective ERK kinase (MEK) inhibitor, to specific limbic areas and examined the effect on EPM behaviors of controls and during naloxone-precipitated morphine withdrawal. We next confirmed that ERK activation increased in limbic areas of mice undergoing naloxone-precipitated morphine withdrawal. Direct injection of SL327 into the amygdala blocked the withdrawal-induced increase in open-arm time; however, injecting SL327 into the septum had no effect. Consistent with these results, both 0.2 and 2 mg/kg naloxone increased ERK activation in the central amygdala of morphine-dependent mice. In drug-naive mice, 2 mg/kg naloxone, but not 0.2 mg/kg, increased ERK activation in the central amygdala. During withdrawal, increased ERK activation was also observed in the lateral septum. In the locus coeruleus, a significant increase was observed only in morphine-dependent mice receiving 2 mg/kg, but not 0.2 mg/kg naloxone. In conclusion, ERK activation in limbic areas is likely involved in both the aversive properties of naloxone and in the affective/emotional symptoms of opioid withdrawal, including mediating EPM behaviors.

Nelumbo nucifera semen extract improves memory in rats with scopolamine-induced amnesia through the induction of choline acetyltransferase expression.

Nelumbo nucifera semen (NNS) is a traditional herb with anti-diarrheal, anti-ganacratia, and tranquilizer-like pharmacological activities. In this study, we examined the anti-amnesic effect of NNS on rats with scopolamine-induced amnesia. Passive avoidance tests, acetylcholinesterase (ACHE) activity, and choline acetyltransferase (CHAT) expression were used to evaluate the NNS anti-amnesic effects. The rats were divided into five groups: the normal group, scopolamine-treated group (1mg/kg; control), NNS (1g/kg) and scopolamine (1mg/kg) co-treatment group, and the ARICEPT (1mg/kg) and scopolamine (1mg/kg) co-treatment group (positive control). The rats were administered the compounds orally for 14 days. The latency time of passive avoidance significantly increased by 54% in the NNS-treated group compared to the scopolamine-treated group. The ACHE activity in the NNS-treated group significantly decreased to 7.35% than that of the control group. CHAT-positive neurons increased by 51.02% in the NNS group compared to the control group. These results suggest that NNS extract improves scopolamine-induced dementia by inhibiting ACHE activity and inducing CHAT expression.

Involvement of septal Cdk5 in the emergence of excessive anxiety induced by stress.

The aim of the present study was to evaluate whether the activation of Cdk5, a protein that has been suggested to participate in higher cognitive functions, is required for the onset of a sensitized anxiety-related behavior induced by stress. The exposure to restraint enhanced both Cdk5 expression in certain subareas of the septohippocampal system, principally in the lateral septum (LS) and septal Cdk5 kinase activity in rats. Behaviorally, restrained wild type mice showed a behavior indicative of enhanced anxiety in the elevated plus maze (EPM). In contrast, unstressed mice and stressed knockout mice, which lacked the p35 protein, the natural activator of Cdk5, displayed similar anxiety-like behavior in the EPM. Finally, the intra-LS infusion of olomoucine - a Cdk5 inhibitor - blocked the enhanced anxiety in the EPM induced by prior stress in rats. All these data provide evidence that septal Cdk5 is required in the emergence of a sensitized emotional process induced by stress.

[Effect of different needle-retained time of electroacupuncture on expression of nitric oxide synthase in the septum of the rat].

OBJECTIVE: To probe into the relationship of different needle-retained time with therapeutic effects. METHODS: Forty experimental rats were evenly randomized into 5 groups, electroacupuncture (EA) at "Zusanli" (ST 36) for 5 min, 15 min, 30 min and 60 min groups, who received EA for 5, 15, 30 and 60 min respectively, and a control group who did not receive EA. Nicotinamide adenosine dinucleotide phosphate diaphorase (NADPH-d) method and computer image analysis system were used to detect the meangray of nitric oxide synthase (NOS) positive neurons in the septum. RESULTS: NOS positive neurons in the septum did not change as the EA for 5 min group compared with the control group (P > 0.05); the number of heavily staining NOS positive neurons increased significantly (P < 0.05) and the meangray of the NOS positive neurons significantly decreased (P < 0.01) in some septal subnuclei (lateral septum, and medial septum, vertical diagnonal band) after EA for 15, 30, 60 min as compared with the control group, and the numbers and the meangray of the NOS positive neurons in the septum were not significantly difference among the 3 EA groups. CONCLUSION: NOS expression in the septum increases and keeps a same level from EA for 15 min to 60 min.

Glutamic acid decarboxylase isoforms are differentially distributed in the septal region of the rat.

The septal region of the brain consists of a heterogeneous population of GABAergic neurons that play an important role in the generation of hippocampal theta rhythms. While GABAergic neurons employ two isoforms of the enzyme glutamic acid decarboxylase (GAD) for the synthesis of GABA, distribution of GAD isoforms has not been investigated in the septum. Immunohistochemical techniques were used to investigate the expression of GAD enzymes in medial and lateral septum. GAD65 and GAD67 immunohistochemistry revealed dense fibers and punctuated immunoreactivity in septal regions. While few GAD65-positive neuronal somas were detected in medial septum, a significantly higher number of immunoreactive neurons were detected in lateral septum. GAD65- and GAD67-positive neurons in the lateral septum exhibit higher complexity of dendritic arborizations than in the medial septum where staining was mainly restricted to the soma. Presumptive axon terminals (puncta) showed abundant immunoreactivity predominantly for GAD65 isoforms in all septal regions. This suggests that septal GABAergic neurons differentially express GAD enzymes thereby potentially reflecting functional differences. Differences found between medial and lateral septal GABAergic neuronal populations are in agreement with the concept that medial and lateral septum are brain structures with highly different connectivity and function despite anatomical proximity.

Detection of nitric oxide synthase (NOS) immunoreactive neurons in the human septal area: a matter of method?

There is a remarkable discrepancy between biochemical and cell morphological findings with regard to the presence of NADPH diaphorase/neuronal nitric oxide synthase (NOS) in the primate septal area. Whereas considerable concentrations of neuronal nitric oxide synthase and high enzyme activities have been measured in postmortem human septal nuclei, histochemical studies were either unable to detect any nitric oxide synthase immunoreactivity in primate septal neurons, or found only a very few nitrergic neurons in this region. This study aimed to investigate the possible presence of nitrergic neurons in human the septal region in greater detail. After having studied a total of 16 postmortem human brains we conclude that the immunohistochemical demonstration of nitric oxide synthase in human septal neurons is largely dependent on the mode of tissue handling: in brain specimens which were fixed en-bloc with paraffin and embedded in paraplast, nitric oxide synthase immunoreactivity is barely detectable, whereas a satisfying immunostaining is obtained on free-floating frozen sections after an immersion-fixation with 4% paraformaldehyde and 0.5% glutaraldehyde, followed by sucrose protection of the specimens. We show herein that there are indeed nitric oxide synthase-containing neurons in the human septum, thus supporting results from previous biochemical studies.

Modulation of nerve growth factor and choline acetyltransferase expression in rat hippocampus after chronic exposure to haloperidol, risperidone, and olanzapine.

RATIONALE: Recently, we reported that compared to haloperidol, chronic exposure to either the risperidone (RISP) or olanzapine (OLZ) resulted in superior effects on spatial learning performance as well as the cholinergic neurons, although the mechanism for these effects was not addressed. OBJECTIVES: The objective of this study was to investigate one plausible mechanism whereby RISP and OLZ exert superior effects on cholinergic neurons, i.e. positive effects on nerve growth factor (NGF), which is known to regulate the brain cholinergic activity as well as cognitive function. Therefore, the effects of chronic exposure to HAL, RISP, or OLZ on the expression of NGF and choline acetyltransferase (ChAT) in the hippocampus (i.e. a brain area well known to be involved in cognitive function and known to receive major cholinergic projections from the medial septum) were compared. METHODS: Rats were treated with HAL (1 or 2 mg/kg per day), RISP (1.25 or 2.5 mg/kg per day), or OLZ (5 or 10 mg/kg per day) for 45 days in drinking water. NGF and ChAT were measured by immunohistochemistry and NGF protein was also measured by an enzyme-linked ImmunoSorbent assay. RESULTS: Compared to controls, HAL exposure resulted in a marked reduction in NGF immunoreactivity in the hippocampal dentate gyrus (DG), CA1 and CA3 areas. In contrast, RISP did not alter, while OLZ significantly increased levels of NGF. These changes in NGF levels corresponded well with changes in ChAT immunoreactivity in the hippocampus and the medial septum. CONCLUSIONS: These preclinical data, combined with previously published behavioral results, support the premise that OLZ and RISP, in contrast to HAL, preserve cholinergic pathways and cognitive function via superior effects on NGF expression and are thus therapeutically superior for extended use.

Cdk5: a novel role in learning and memory.

Learning and memory are processes by which organisms acquire, retain and retrieve information. They result in modifications of behavior in response to new or previously encountered stimuli thereby enabling adaptation to a permanently changing environment. Protein phosphorylation has long been known to play a key role in triggering synaptic changes underlying learning and memory. Although intracellular phosphorylation and dephosphorylation is orchestrated by a complex network of interactions between a number of protein kinases and phosphatases, significant advances in the understanding of neuronal mechanisms underlying learning and memory have been achieved by investigating the actions of individual molecules under defined experimental conditions, brain areas, neuronal cells and their subcellular compartments. On the basis of these approaches, the cyclic AMP protein kinase (PKA), protein kinase C (PKC) and extracellularly regulated protein kinases 1 and 2 (Erk-1/2) have been identified as the core signaling pathways in memory consolidation. Here we review recent findings demonstrating an important novel role for Cdk5 in learning and memory. We suggest that some of the well-characterized roles of Cdk5 during neurodevelopmental processes, such as interactions with distinct cytoplasmic and synaptic target molecules, may be also involved in synaptic plasticity underlying memory consolidation within the adult central nervous system.

Antidepressant effect of the calcium-activated tyrosine kinase Pyk2 in the lateral septum.

Accumulating evidence indicates that neural activity in the lateral septum (LS) influences the pathophysiology of depression and therapeutic effectiveness of antidepressant drugs. For example, the development of behavioral deficits in animal screens for antidepressant drug activity corresponds with a blunting of LS activity, whereas chronic treatment with antidepressants enhances cell firing in the LS; however, the molecular mechanisms underlying such behavioral functions of the LS have not been determined. The nonreceptor tyrosine kinase Pyk2 is highly expressed in the LS and plays important roles in regulating cellular excitability and synaptic plasticity, making it an attractive candidate for regulating the effects of stress and antidepressants on LS functioning and behavior. We provide evidence that stress decreases Pyk2 phosphorylation in the LS, whereas enhancing Pyk2 expression in LS neurons has an antidepressant effect behaviorally.Pyk2 messenger ribonucleic acid (mRNA) expression in the rat forebrain was detected by in situ hybridization, and a brief description of the distribution of Pyk2 mRNA in selected areas is presented. Levels of total Pyk2 protein and phosphorylated Pyk2 were subsequently measured in the LS and hippocampus following stress exposure, as were levels of extracellular stimuli-regulated kinase (Erk) and phospho-Erk. Herpes simplex virus (HSV)-mediated gene transfer was then used to enhance Pyk2 expression in the LS, and the effect this had on behavior in the learned helplessness model of depression was evaluated. High levels of Pyk2 mRNA were detected in a number of forebrain regions, including the hippocampus and LS. Following acute stress exposure, subjects showed a decrease in phosphorylated Pyk2 and Erk in the LS but not in the hippocampus. Total levels of Pyk2 and Erk remained unchanged following stress. In the learned helplessness paradigm, injection of HSV-Pyk2 into the LS prevented the active avoidance deficit caused by exposure to inescapable shock, indicative of an antidepressant effect. These results indicate that following acute stress, Pyk2 and Erk activity in the LS are decreased, whereas experimentally increasing Pyk2 activity in LS neurons reverses the behavioral deficits of acute, inescapable stress. These findings establish a role for the tyrosine kinase Pyk2 in the biochemical and behavioral responses to stress and suggest a possible role in the pathophysiology of depression, particularly notable considering Pyk2's role in promoting synaptic plasticity.

Regulation of contextual fear conditioning by baseline and inducible septo-hippocampal cyclin-dependent kinase 5.

In this work, we confirm the novel role of cyclin-dependent kinase (Cdk) 5 in associative learning by demonstrating that injection of the Cdk5 inhibitor butyrolactone I into the lateral septum or hippocampus profoundly impaired context-dependent fear conditioning of C57BL/6J mice. However, unlike the inducible up-regulation of Cdk5 and its regulator p35 observed in Balb/c mice, high baseline levels, which were not affected by fear conditioning, were found in C57BL/6J mice. Surprisingly, microinjections of butyrolactone I into the lateral septum or hippocampus significantly decreased baseline Cdk5 activity within the entire septo-hippocampal circuitry, suggesting a functional link between septal and hippocampal Cdk5 activity. Significantly higher levels of the transcription factor Sp4 in the septo-hippocampal system of C57BL/6J mice may account for the high baseline Cdk5/p35 production. On the other hand, the stronger cFos production observed in the lateral septum of fear conditioned Balb/c mice may be responsible, at least in part, for the inducible up-regulation of Cdk5 in this strain. These results suggest that the role of Cdk5 in memory consolidation is strain independent and functionally related to the septo-hippocampal circuitry. However, the molecular regulation of baseline and inducible Cdk5 protein might be different among individual mouse strains and possibly other species.

Drinking behavior elicited by central injection of angiotensin II: roles for protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

Prior studies utilizing neurons cultured from the hypothalamus and brain stem of newborn rats have demonstrated that ANG II-induced modulation of neuronal firing involves activation of both protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). The present studies were performed to determine whether these signaling molecules are also involved in physiological responses elicited by ANG II in the brain in vivo. Central injection of ANG II (10 ng/2 microl) into the lateral cerebroventricle (icv) of Sprague-Dawley rats increased water intake in a time-dependent manner. This ANG II-mediated dipsogenic response was attenuated by central injection of the PKC inhibitors chelerythrine chloride (0.5-50 microM, 2 microl) and Go-6976 (2.3 nM, 2 microl) and by the CaMKII inhibitor KN-93 (10 microM, 2 microl). Conversely, icv injection of chelerythrine chloride (50 microM, 2 microl) and KN-93 (10 microM, 2 microl) had no effect on the dipsogenic response elicited by central injection of carbachol (200 ng/2 microl). Furthermore, injection of ANG II (10 ng/2 microl) icv increases the activity of both PKC-alpha and CaMKII in rat septum and hypothalamus. These data suggest that signaling molecules involved in ANG II-induced responses in vitro are also relevant in physiological responses elicited by ANG II in the whole animal model.

Cholinergic deficits in the septal-hippocampal pathway of the SAM-P/8 senescence accelerated mouse.

Senescence accelerated prone mouse strains (SAM-P) and resistant strains (SAM-R) have proven useful in elucidating aspects of the aging process. The senescence accelerated mouse SAM-P/8 strain exhibits severe age-related learning and memory impairments well before the median age of survival. Disruption of the brain cholinergic system produces learning and memory impairments as severe as those seen in aging SAM-P/8 mice. Therefore, we compared the effects of aging on cholinergic parameters in the septal-hippocampal pathway, a region known to play a role in learning and memory, in SAM-P/8 mice and mice of the senescence resistant SAM-R/1 strain. Between 4 and 12 months of age we observed a 40-50% decrease in choline acetyltransferase (ChAT) activity in two of three subregions of the hippocampus in the SAM-P/8, but not the SAM-R/1 strain. Between 4 and 12 months, SAM-P/8 mice also showed a 40-50% decrease in ChAT activity in the septal region that was maximal by 8 months of age. By contrast, these age-related changes were not observed in the control SAM-R/1 mouse strain. The changes in ChAT in the SAMP/8 mouse strain were limited to the septal-hippocampal cholinergic pathway. There were no differences in ChAT activity in the nucleus basalis of Meynert, nor any of several neocortical areas to which it projects. To determine the neurochemical specificity of these alterations, the activity of glutamic acid decarboxylase (GAD), was also measured in the septum and hippocampus of SAM-P/8 mice. There were no age-related alterations in the hippocampus, but a significant 50% increase in GAD activity in the septal nucleus at 12 months of age. There were no age-related alterations in either nicotinic (3H-cytisine) or muscarinic (3H-QNB) cholinergic receptor binding in the cortex or hippocampus of SAM-P/8 mice. However, there were significant strain differences. At 2 months of age, 3H-QNB binding was higher in hippocampus of the SAM-R/1 than in SAM-P/8 mice. Similarly, 3H-cytisine binding in cortex of SAM-R/1 mice was higher at both 2 and 13 months than in SAM-P/8 mice. The results suggest that a compromised septal-hippocampal cholinergic pathway may contribute to the previously reported early onset of impaired learning and memory in the SAM-P/8 mouse strain.

Protective effect of the heparin-derived oligosaccharide C3, on AF64A-induced cholinergic lesion in rats.

Alzheimer's disease (AD) literature indicates that glycosaminoglycans (GAGs) may prevent proteoglycan-induced amyloid-beta (Abeta) aggregation, decrease Abeta-induced tau-2 immunoreactivity, and increase the axonal growth and arborization of hippocampal neurons. However, there is no information about the impact of GAGs on cholinergic lesions. Here, AF64A was administered stereotaxically into the lateral ventricles of rats, at doses that are selective for cholinotoxicity (1 and 2 nmol). The heparin-derived oligosaccharide (HDO), C3 (25mg/kg), was administered orally, once daily for 7 days before, and 7 days after AF64A administration. Choline acetyltransferase (ChAT) immunohistochemistry revealed that C3 administration reduced AF64A-induced cholinergic damage in the septum and cingulum bundle. Quantitative neuronal cell counts showed that C3 attenuated, by 60%, the decrease in cell number in the medial septum. Enzyme analysis showed that C3 also significantly restored ChAT (30%) and acetylcholinesterase (AChE) enzyme activity (45%), which had been diminished by AF64A. Our data suggest that, in addition to its effects of anti-Abeta aggregation, anti-Abeta-induced tau-2 immunoreactivity, and neurotrophic effects, C3 also effectively reduces AF64A-induced cholinergic damage; hence it may have potential therapeutic value in AD patients.

Tyrosine kinase activity of nerve growth factor and estrogen in embryonic septal neurons cultured from the rat.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in the septal nucleus of the brain. Nerve growth factor (NGF) and estrogen were studied to observe effects on tyrosine kinase activity in septal neurons. The time course of tyrosine kinase activation and number of cells in which tyrosine kinase was activated were measured. Tissue from embryonic day 16 rats was microdissected and the septal neurons obtained were treated with estrogen (10 microM) or NGF (100 ng/mL) at intervals of 1, 2, 3, 4, 5, or 10 min. Immunostaining for phosphotyrosine revealed that cells treated with NGF showed an increase in phosphotyrosine activity within 2-4 min followed by a decline to control levels of enzyme activity. Treatment with estrogen led to an increase in phosphotyrosine immunostaining within 2-3 min followed by a decline to control levels. This time course suggests a mechanism for estrogen activity other than the traditional method involving binding to nuclear receptors followed by protein synthesis.

Dissociation of memory and anxiety in a repeated elevated plus maze paradigm: forebrain cholinergic mechanisms.

The effect of intraseptal injection of the cholinergic immunotoxin 192-IgG-saporin on behavior in the elevated plus maze was investigated. A 5-min test-retest paradigm, with minute-by-minute analysis of the first session, was used to evaluate both anxiety and memory in this task. Biochemical analyses revealed a decrease in acetylcholinesterase (AChE) activity in the hippocampus (HPC), septum, and frontal cortex of animals injected with IgG-192 saporin (237.5 ng) when compared with controls. No statistical differences were found between groups in terms of behaviors associated with locomotor activity, conventional measures of anxiety, or ethological behaviors during either session 1 or 2. During test session 2 the controls exhibited decreased exploratory activity and increased indices of anxiety. In contrast, the saporin-treated rats did not exhibit these experience-dependent behavioral changes from session 1 to 2. The minute-by-minute analysis showed a significant decrease in exploratory as well in anxiety associated behaviors during the first session for the control group, but not for the saporin-treated group. These results suggest that the cholinergic innervation of the HPC, the frontal cortex, or both forebrain structures, modulate the initiation of exploratory activity which, results in the acquisition and retention of spatial information, but does not affect the expression of anxiety in the elevated plus-maze.

Postnatal development of opioid receptors modulating acetylcholine release in hippocampus and septum of the rat.

The postnatal development of presynaptic opioid receptors inhibiting the release of acetylcholine (ACh) was studied in rat brain hippocampus, medial septum (MS) and diagonal band of Broca (DB). To this end, the corresponding brain slices (350 microm thick) of rats of various postnatal ages (postnatal day 4 [P4] to P16, and adult) were preincubated with [(3)H]choline and stimulated twice for 2 min (S(1), S(2): at 3 Hz, 2 ms, 60 mA) during superfusion with physiological buffer containing hemicholinium-3. In parallel, the activity of choline acetyltransferase (ChAT) was determined in crude homogenates of the tissues as a marker for the development of cholinergic neurons. At any postnatal age, the electrically evoked overflow of tritium from slices preincubated with [(3)H]choline was highest in the DB, followed by the MS and the hippocampus. The evoked [(3)H]overflow increased with postnatal age, reached about 50% (MS, DB) or 30% (hippocampus) of the corresponding adult levels at P16 and correlated significantly with the corresponding ChAT activities. Presence of the preferential mu-opioid receptor agonist DAMGO during S(2) significantly inhibited the evoked overflow of tritium already at P4 in DB and MS, whereas in the hippocampus significant inhibitory effects were first observed at P8 only. Moreover, adult levels of inhibition due to DAMGO were reached at P16 in the DB and MS but not in the hippocampus. In septal areas, also the effect of the preferential delta-opioid receptor agonist DPDPE on the evoked [(3)H]overflow was studied: in contrast to DAMGO, however, significant inhibitory effects of DPDPE were first observed at P12 only. In conclusion, the postnatal development of presynaptic mu-opioid receptors on cholinergic neurons in the DB and MS starts earlier than in the hippocampus and precedes that of presynaptic delta-opioid receptors.