Bisphenol A interferes with synaptic remodeling.

The potential adverse effects of Bisphenol A (BPA), a synthetic xenoestrogen, have long been debated. Although standard toxicology tests have revealed no harmful effects, recent research highlighted what was missed so far: BPA-induced alterations in the nervous system. Since 2004, our laboratory has been investigating one of the central effects of BPA, which is interference with gonadal steroid-induced synaptogenesis and the resulting loss of spine synapses. We have shown in both rats and nonhuman primates that BPA completely negates the ∼ 70-100% increase in the number of hippocampal and prefrontal spine synapses induced by both estrogens and androgens. Synaptic loss of this magnitude may have significant consequences, potentially causing cognitive decline, depression, and schizophrenia, to mention those that our laboratory has shown to be associated with synaptic loss. Finally, we discuss why children may particularly be vulnerable to BPA, which represents future direction of research in our laboratory.

Loss of asymmetric spine synapses in dorsolateral prefrontal cortex of cognitively impaired phencyclidine-treated monkeys.

Schizophrenia patients, long-term abusers of phencyclidine (PCP), and monkeys treated with PCP all exhibit enduring cognitive deficits. Evidence indicates that loss of prefrontal cortex spine synapses results in cognitive dysfunction, suggesting the presence of synaptic pathology in the monkey PCP model; however, there is no direct evidence of such changes. In this study we use the monkey PCP model of schizophrenia to investigate at the ultrastructural level whether remodelling of dorsolateral prefrontal cortex (DLPFC) asymmetric spine synapses occurs following PCP. Subchronic PCP treatment resulted in a decrease in the number of asymmetric spine synapses, which was greater in layer II/III than layer V of DLPFC, compared to vehicle-treated controls. This decrease may contribute to PCP-induced cognitive dysfunction in the non-human primate model and perhaps in schizophrenia. Thus, the synapse loss in the PCP model provides a novel target for the development of potential treatments of cognitive dysfunction in this model and in schizophrenia.

Bisphenol A prevents the synaptogenic response to estradiol in hippocampus and prefrontal cortex of ovariectomized nonhuman primates.

Exposure measurements from several countries indicate that humans are routinely exposed to low levels of bisphenol A (BPA), a synthetic xenoestrogen widely used in the production of polycarbonate plastics. There is considerable debate about whether this exposure represents an environmental risk, based on reports that BPA interferes with the development of many organs and that it may alter cognitive functions and mood. Consistent with these reports, we have previously demonstrated that BPA antagonizes spine synapse formation induced by estrogens and testosterone in limbic brain areas of gonadectomized female and male rats. An important limitation of these studies, however, is that they were based on rodent animal models, which may not be representative of the effects of human BPA exposure. To address this issue, we examined the influence of continuous BPA administration, at a daily dose equal to the current U.S. Environmental Protection Agency's reference safe daily limit, on estradiol-induced spine synapse formation in the hippocampus and prefrontal cortex of a nonhuman primate model. Our data indicate that even at this relatively low exposure level, BPA completely abolishes the synaptogenic response to estradiol. Because remodeling of spine synapses may play a critical role in cognition and mood, the ability of BPA to interfere with spine synapse formation has profound implications. This study is the first to demonstrate an adverse effect of BPA on the brain in a nonhuman primate model and further amplifies concerns about the widespread use of BPA in medical equipment, and in food preparation and storage.

Stress Resilience is Associated with Hippocampal Synaptoprotection in the Female Rat Learned Helplessness Paradigm.

The synaptogenic hypothesis of major depressive disorder implies that preventing the onset of depressive-like behavior also prevents the loss of hippocampal spine synapses. By applying the psychoactive drugs, diazepam and fluoxetine, we investigated whether blocking the development of helpless behavior by promoting stress resilience in the rat learned helplessness paradigm is associated with a synaptoprotective action in the hippocampus. Adult ovariectomized and intact female Sprague-Dawley rats (n = 297) were treated with either diazepam, fluoxetine, or vehicle, exposed to inescapable footshocks or sham stress, and tested in an active escape task to assess helpless behavior. Escape-evoked corticosterone secretion, as well as remodeling of hippocampal spine synapses at a timepoint representing the onset of escape testing were also analyzed. In ovariectomized females, treatment with diazepam prior to stress exposure prevented helpless behavior, blocked the loss of hippocampal spine synapses, and muted the corticosterone surge evoked by escape testing. Although fluoxetine stimulated escape performance and hippocampal synaptogenesis under non-stressed conditions, almost all responses to fluoxetine were abolished following exposure to inescapable stress. Only a much higher dose of fluoxetine was capable of partly reproducing the strong protective actions of diazepam. Importantly, these protective actions were retained in the presence of ovarian hormones. Our findings indicate that stress resilience is associated with the preservation of spine synapses in the hippocampus, raising the possibility that, besides synaptogenesis, hippocampal synaptoprotection is also implicated in antidepressant therapy.

Stress and remodeling of hippocampal spine synapses.

By nature's original design, stress is a protective mechanism, signaling danger to homeostasis and, as a result, stimulating the brain to initiate coping and adaptive responses, which ultimately increases the chances of survival. On the other hand, stress may become a danger to homeostasis itself, when it is so severe or so prolonged that it overpowers cellular reserves. One of the consequences of traumatic stress is loss of hippocampal spine synapses, which is the main topic of this chapter summarizing research findings from the last 10+ years. Loss of spine synapses is thought to be a neuronal defense mechanism against excitotoxic damage, so the subcellular mechanisms of synapse loss are reviewed in the context of glutamatergic insults. One of the main conceptual derivates of stress-induced synaptic alterations is the "synaptogenic" hypothesis of major depressive disorder. The synaptogenic hypothesis postulates an inverse correlation between the number of limbic, mainly prefrontal cortical and hippocampal, spine synapses and the severity of depressive behavior/symptoms. The synaptogenic hypothesis implies that synaptoprotective interventions, that are capable of countering the stress-induced loss of limbic spine synapses, are probably also capable of promoting stress resilience, which may provide the conceptual basis for a preventive approach in antidepressant therapy. Finally, we discuss why electron microscopic stereology is a reliable and highly accurate technique for the quantitative assessment of ultrastructural particulate objects, such as spines and synapses.

Bisphenol A prevents the synaptogenic response to testosterone in the brain of adult male rats.

Exposure measurement data from several developed countries indicate that human beings are widely exposed to low levels of the synthetic xenoestrogen, bisphenol A. We reported previously that bisphenol A, even at doses below the reference safe daily limit for human exposure, recommended by the U.S. Environmental Protection Agency, impairs the synaptogenic response to 17beta-estradiol in the hippocampus of ovariectomized rats. Recent experiments revealed that bisphenol A also interferes with androgen receptor-mediated transcriptional activities. Thus, to investigate whether bisphenol A impairs synaptogenesis in the medial prefrontal cortex (mPFC) and hippocampus of adult male rats, castrated and sham-operated animals were treated with different combinations of bisphenol A (300 microg/kg), testosterone propionate (1.5 mg/kg), and sesame oil vehicle. The brains were processed for electron microscopic stereology, and the number of asymmetric spine synapses in the mPFC and CA1 hippocampal area was estimated. In both regions analyzed, bisphenol A reduced the number of spine synapses in sham-operated, gonadally intact animals, which was accompanied by a compensatory increase in astroglia process density. In addition, bisphenol A prevented both the prefrontal and hippocampal synaptogenic response to testosterone supplementation in castrated males. These results demonstrate that bisphenol A interferes with the synaptogenic response to testosterone in the mPFC and hippocampus of adult male rats. Because the hippocampal synaptogenic action of androgens seems to be independent of androgen and estrogen receptors in males, the potential mechanisms that underlie these negative effects of bisphenol A remain the subject of further investigation.

Effects of androgens and estradiol on spine synapse formation in the prefrontal cortex of normal and testicular feminization mutant male rats.

Recent studies suggest that, in female monkeys and rats, estrogens elicit dendritic spine synapse formation in the prefrontal cortex, an area that, similar to the hippocampus, plays a critical role in cognition. However, whether gonadal hormones induce synaptic remodeling in the male prefrontal cortex remains unknown. Here we report that gonadectomy reduced, whereas administration of 5alpha-dihydrotestosterone or estradiol-benzoate to castrated male rats increased, the number of medial prefrontal cortical (mPFC) spine synapses, with estradiol-benzoate being less effective than 5alpha-dihydrotestosterone. To investigate whether the androgen receptor contributes to the mediation of these changes, we compared the response of testicular feminization mutant (Tfm) male rats to that of wild-type animals. The number of mPFC spine synapses in gonadally intact Tfm rats and 5alpha-dihydrotestosterone-treated castrated Tfm males was considerably reduced compared to intact wild-type animals, whereas the synaptogenic effect of estradiol-benzoate was surprisingly enhanced in Tfm rats. These data are consistent with the hypothesis that remodeling of spine synapses in the prefrontal cortex may contribute to the cognitive effect of gonadal steroids. Our findings in Tfm animals indicate that androgen receptors may mediate a large part of the synaptogenic action of androgens in the mPFC of adult males. However, because this effect of 5alpha-dihydrotestosterone is not completely lost in Tfm rats, additional mechanisms may also be involved.

Extrinsic afferent systems to the dentate gyrus.

The dentate gyrus is the first stage of the intrahippocampal, excitatory, trisynaptic loop, and a primary target of the majority of entorhinal afferents that terminate in a laminar fashion on granule cell dendrites and carry sensory information of multiple modalities about the external world. The electric activity of the trisynaptic pathway is controlled mainly by different types of local, GABAergic interneurons, and subcortical and commissural afferents. In this chapter we will outline the origin and postsynaptic targets in the dentate gyrus of chemically identified subcortical inputs. These systems are afferents originating from the medial septum/diagonal band of Broca GABAergic and cholinergic neurons, neurochemically distinct types of neurons located in the supramammillary area, serotonergic fibers from the median raphe, noradrenergic afferents from the pontine nucleus, locus ceruleus, dopamine axons originating in the ventral tegmental area, and the commissural projection system. Because of the physiological implications, these afferents are discussed in the context of the glutamatergic innervation of the dentate gyrus. One common feature of the extrinsic dentate afferent systems is that they originate from a relatively small number of neurons. However, the majority of these afferents are able to exert a powerful control over the electrical activity of the hippocampus. This strong influence is due to the fact that the majority of the extrinsic afferents terminate on a relatively small, but specific, populations of neurons that are able to control large areas of the hippocampal formation.

Sex steroids and the dentate gyrus.

In the late 1980s, the finding that the dentate gyrus contains more granule cells in the male than in the female of certain mouse strains provided the first indication that the dentate gyrus is a significant target for the effects of sex steroids during development. Gonadal hormones also play a crucial role in shaping the function and morphology of the adult brain. Besides reproduction-related processes, sex steroids participate in higher brain operations such as cognition and mood, in which the hippocampus is a critical mediator. Being part of the hippocampal formation, the dentate gyrus is naturally involved in these mechanisms and as such, this structure is also a critical target for the activational effects of sex steroids. These activational effects are the results of three major types of steroid-mediated actions. Sex steroids modulate the function of dentate neurons under normal conditions. In addition, recent research suggests that hormone-induced cellular plasticity may play a larger role than previously thought, particularly in the dentate gyrus. Specifically, the regulation of dentate gyrus neurogenesis and synaptic remodeling by sex steroids received increasing attention lately. Finally, the dentate gyrus is influenced by gonadal hormones in the context of cellular injury, and the work in this area demonstrates that gonadal hormones have neuroprotective potential. The expression of estrogen, progestin, and androgen receptors in the dentate gyrus suggests that sex steroids, which could be of gonadal origin and/or synthesized locally in the dentate gyrus, may act directly on dentate cells. In addition, gonadal hormones could also influence the dentate gyrus indirectly, by subcortical hormone-sensitive structures such as the cholinergic septohippocampal system. Importantly, these three sex steroid-related themes, functional effects in the normal dentate gyrus, mechanisms involving neurogenesis and synaptic remodeling, as well as neuroprotection, have substantial implications for understanding normal cognitive function, with clinical importance for epilepsy, Alzheimer's disease and mental disorders.

Stress induces equivalent remodeling of hippocampal spine synapses in a simulated postpartum environment and in a female rat model of major depression.

Stress and withdrawal of female reproductive hormones are known risk factors of postpartum depression. Although both of these factors are capable of powerfully modulating neuronal plasticity, there is no direct electron microscopic evidence of hippocampal spine synapse remodeling in postpartum depression. To address this issue, hormonal conditions of pregnancy and postpartum period were simulated in ovariectomized adult female Sprague-Dawley rats (n=76). The number of hippocampal spine synapses and the depressive behavior of rats in an active escape task were investigated in untreated control, hormone-withdrawn 'postpartum', simulated proestrus, and hormone-treated 'postpartum' animals. After 'postpartum' withdrawal of gonadal steroids, inescapable stress caused a loss of hippocampal spine synapses, which was related to poor escape performance in hormone-withdrawn 'postpartum' females. These responses were equivalent with the changes observed in untreated controls that is an established animal model of major depression. Maintaining proestrus levels of ovarian hormones during 'postpartum' stress exposure did not affect synaptic and behavioral responses to inescapable stress in simulated proestrus animals. By contrast, maintaining pregnancy levels of estradiol and progesterone during 'postpartum' stress exposure completely prevented the stress-induced loss of hippocampal spine synapses, which was associated with improved escape performance in hormone-treated 'postpartum' females. This protective effect appears to be mediated by a muted stress response as measured by serum corticosterone concentrations. In line with our emerging 'synaptogenic hypothesis' of depression, the loss of hippocampal spine synapses may be a novel perspective both in the pathomechanism and in the clinical management of postpartum affective illness.

Androgen effects on hippocampal CA1 spine synapse numbers are retained in Tfm male rats with defective androgen receptors.

The effects of estradiol benzoate (EB), dihydrotestosterone (DHT), or the antiandrogen hydroxyflutamide on CA1 pyramidal cell dendritic spine synapses were investigated in adult male rats. To elucidate the contribution of the androgen receptor to the hormone-induced increase in hippocampal CA1 synapses, wild-type males were compared with males expressing the Tfm mutation, which results in synthesis of defective androgen receptors. Orchidectomized rats were treated with EB (10 microg/rat.d), DHT (500 mug/rat.d), hydroxyflutamide (5 mg/rat.d), or the sesame oil vehicle sc daily for 2 d and examined using quantitative electron microscopic stereological techniques, 48 h after the second injection. In wild-type males, DHT and hydroxyflutamide both induced increases in the number of spine synapses in the CA1 stratum radiatum, whereas EB had no effect. DHT almost doubled the number of synaptic contacts observed, whereas hydroxyflutamide increased synapse density by approximately 50%, compared with the vehicle-injected controls. Surprisingly, in Tfm males, the effects of EB, DHT, and hydroxyflutamide were all indistinguishable from those observed in wild-type animals. These observations demonstrate that Tfm male rats resemble normal males in having no detectable hippocampal synaptic response to a dose of EB that is highly effective in females. Despite the reduction in androgen sensitivity as a result of the Tfm mutation, hippocampal synaptic responses to both DHT and a mixed androgen agonist/antagonist (hydroxyflutamide) remain intact in Tfm males. These data are consistent with previous results suggesting that androgen effects on hippocampal spine synapses may involve novel androgen response mechanisms.

Subchronic phencyclidine treatment decreases the number of dendritic spine synapses in the rat prefrontal cortex.

BACKGROUND: A growing body of evidence suggests the existence of synaptic pathology in schizophrenia. Here we used the phencyclidine schizophrenia model to directly investigate at the electron microscopic level whether structural synaptic alterations are present in these animals. METHODS: Adult male rats were treated according to our subchronic phencyclidine paradigm (5 mg/kg twice daily for 7 days, intraperitoneally). Following a one-week withdrawal period, the number of prefrontal cortical spine synapses was calculated using an unbiased electron microscopic stereological approach. The number of astroglia cells and the density of their processes was also analyzed following glial-fibrillary acidic protein immunohistochemistry. RESULTS: Subchronic phencyclidine treatment resulted in a 41.2% decrease in the number of prefrontal spine synapses when compared to controls. This was accompanied by a 58.8% increase in astroglia process density, without significant change in the number of astroglia cells. CONCLUSIONS: Our results demonstrate a severe reduction in the number of prefrontal spine synapses in an animal model of schizophrenia. This phenomenon may contribute to phencyclidine-induced cognitive dysfunction and decreased prefrontal cellular activity observed in this model.

Androgens increase spine synapse density in the CA1 hippocampal subfield of ovariectomized female rats.

The effects of androgen on the density of spine synapses on pyramidal neurons in the CA1 area of the hippocampus were studied in ovariectomized (OVX) adult female rats. Treatment of OVX rats with testosterone propionate (TP; 500 microg/d, s.c., 2 d) significantly increased spine synapse density (from 0.661 +/- 0.016 spine synapse/microm3 in OVX rats to 1.081 +/- 0.018 spine synapse/microm3 after TP treatment). A smaller, but still statistically significant, increase in synapse density (0.955 +/- 0.029 spine synapse/microm3) was observed in OVX animals after treatment with the nonaromatizable androgen dihydrotestosterone (DHT; 500 microg/d, s.c., 2 d). Administration of 1 mg of letrozole, a powerful nonsteroidal aromatase inhibitor, 1 hr before the steroid injections almost completely blocked the synaptic response to testosterone, resulting in a mean synapse density (0.723 +/- 0.003 spine synapse/microm3) only slightly higher than in OVX control rats. By contrast, the response to DHT was unaffected by letrozole pretreatment. These data suggest that androgen secretion during the female reproductive cycle may contribute to cyclical changes in hippocampal synaptic density. They also indicate that androgen treatment may be as effective as estrogen replacement in reversing the decline in hippocampal CA1 spine synapses that follows loss of ovarian function. Induction of hippocampal synapse formation by androgen is not mediated entirely via intracerebral estrogen biosynthesis, however, because aromatase-independent mechanisms also significantly affect CA1 spine synapse density.

Hypocretin/orexin innervation and excitation of identified septohippocampal cholinergic neurons.

Hypothalamic fibers containing the wake-promoting peptides, hypocretins (Hcrts) or orexins, provide a dense innervation to the medial septum-diagonal band of Broca (MSDB), a sleep-associated brain region that has been suggested to show intense axonal degeneration in canine narcoleptics. The MSDB, via its cholinergic and GABAergic projections to the hippocampus, controls the hippocampal theta rhythm and associated learning and memory functions. Neurons of the MSDB express very high levels of the Hcrt receptor 2, which is mutated in canine narcoleptics. In the present study, we investigated the electrophysiological effects of Hcrt peptides on septohippocampal cholinergic neurons that were identified in living brain slices of the MSDB using a selective fluorescent marker. Hcrt activation of septohippocampal cholinergic neurons was reversible, reproducible, and concentration dependent and mediated via a direct postsynaptic mechanism. Both Hcrt1 and Hcrt2 activated septohippocampal cholinergic neurons with similar EC(50) values. The Hcrt effect was dependent on external Na(+), reduced by external Ba(2+), and also reduced in recordings with CsCl-containing electrodes, suggesting a dual underlying ionic mechanism that involved inhibition of a K(+) current, presumably an inward rectifier, and a Na(+)-dependent component. The Na(+) component was dependent on internal Ca(2+), blocked by replacing external Na(+) with Li(+), and also blocked by bath-applied Ni(2+) and KB-R7943, suggesting involvement of the Na(+)-Ca(2+) exchanger. Using double-immunolabeling studies at light and ultrastructural levels, we also provide definitive evidence for a hypocretin innervation of cholinergic neurons. Thus Hcrt effects within the septum should increase hippocampal acetylcholine release and thereby promote hippocampal arousal.

The 17alpha and 17beta isomers of estradiol both induce rapid spine synapse formation in the CA1 hippocampal subfield of ovariectomized female rats.

Previous studies have demonstrated that estradiol-17beta and estradiol-17alpha both induce short-latency effects on spatial memory in rats, estradiol-17alpha being at least as potent as its 17beta isomer. To determine whether the mechanisms underlying these behavioral responses might include effects on hippocampal synaptic plasticity, CA1 pyramidal spine synapse density (PSSD) was measured in ovariectomized rats within the first few hours after s.c. estrogen injection. PSSD increased markedly (by 24%) 4.5 h after the administration of 45 microg/kg estradiol-17beta. The PSSD response was significantly greater (44% above control) 30 min after estradiol-17beta injection and was markedly dose dependent; a 3-fold lower estradiol-17beta dose (15 microg/kg) did not significantly affect CA1 PSSD at either 30 min or 4.5 h. Estradiol-17alpha was a more potent inducer of PSSD than estradiol-17beta. Dose-response analysis determined an ED50 for the effect of estradiol-17alpha on PSSD of 8.92 +/- 1.99 microg/kg, with a maximal response at 15 microg/kg. These results demonstrate that high doses of estradiol induce rapid changes in CA1 PSSD. CA1 spine synapse formation appears to be more sensitive to estradiol-17alpha than to estradiol-17beta, paralleling previous data on the effects of these two steroids on spatial memory. Rapid remodeling of hippocampal synaptic connections may thus contribute to the enhancement of spatial mnemonic processing observed within the first few hours after estrogen treatment. The potency of estradiol-17alpha suggests that hormone replacement therapy using this steroid might be useful clinically in ameliorating the impact of low endogenous estrogen production on the development and progression of neurodegenerative disorders involving the hippocampus.

The environmental estrogen bisphenol a inhibits estradiol-induced hippocampal synaptogenesis.

Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, as well as the BPA-containing plastics used in dental prostheses and sealants, considerable potential exists for human exposure to this compound. In this article we show that treatment of ovariectomized rats with BPA dose-dependently inhibits the estrogen-induced formation of dendritic spine synapses on pyramidal neurons in the CA1 area of the hippocampus. Significant inhibitory effects of BPA were observed at a dose of only 40 microg/kg, below the current U.S. Environmental Protection Agency reference daily limit for human exposure. Because synaptic remodeling has been postulated to contribute to the rapid effects of estrogen on hippocampus-dependent memory, these data suggest that environmental BPA exposure may interfere with the development and expression of normal sex differences in cognitive function, via inhibition of estrogen-dependent hippocampal synapse formation. It may also exacerbate the impairment of hippocampal function observed during normal aging, as endogenous estrogen production declines.

Effects of dehydroepiandrosterone and flutamide on hippocampal CA1 spine synapse density in male and female rats: implications for the role of androgens in maintenance of hippocampal structure.

The effects of androgens and the androgen antagonist, flutamide, on the density of dendritic spine synapses in the CA1 subfield of the hippocampus were studied in gonadectomized male and female rats. Treatment of orchidectomized male rats with dehydroepiandrosterone (DHEA; 2 d, 1 mg/d sc) increased the density of CA1 spine synapses observed 2 d later, by 106%, without significantly affecting ventral prostate weight. The hippocampal response to DHEA was unaffected by blockade of intracerebral estrogen biosynthesis using the aromatase inhibitor, letrozole. By contrast, flutamide alone (2 d; 5 mg/d, sc) increased CA1 spine synapse density by 66%, whereas in combination the effects of flutamide and DHEA were additive rather than inhibitory. Additive effects on CA1 synapse density were also observed in males using combinations of flutamide with 5alpha-dihydrotestosterone (2 d, 500 microg/d, sc). At the same doses, flutamide had no effect on prostate weight and completely blocked the effects on the prostate of treatment with 5alpha-dihydrotestosterone. Treatment of ovariectomized females with DHEA increased CA1 spine synapse density to a level similar to that observed in the male. As in males, flutamide in females increased CA1 spine synapse formation and further augmented the response to DHEA. These results demonstrate that flutamide and DHEA have positive effects on hippocampal CA1 spine synapse density in both sexes. They also suggest that conventional measures of androgen agonist or antagonist activity, exemplified by ventral prostate growth, may not be indicative of effects on hippocampal CA1 synaptogenesis.

Dehydroepiandrosterone increases hippocampal spine synapse density in ovariectomized female rats.

This study tests the hypothesis that dehydroepiandrosterone (DHEA) stimulates formation of hippocampal CA1 spine synapses in ovariectomized rats. Subcutaneous injections of DHEA (1 mg/d for 2 d) increased CA1 spine synapse density by more than 50% compared with vehicle-injected animals. The effect of DHEA on CA1 synapse density was abolished by pretreatment with the nonsteroidal aromatase inhibitor, letrozole. DHEA treatment, with or without letrozole, had no detectable uterotrophic effect. These observations are consistent with the hypothesis that DHEA treatment may be capable of reversing the decline in hippocampal spine synapse density observed after loss of ovarian steroid hormone secretion. The blockade of the synaptic response to DHEA by letrozole, despite the lack of a uterotrophic response to this steroid, suggests that the hippocampal response to DHEA may be mediated via aromatization in the brain.

Direct catecholaminergic-cholinergic interactions in the basal forebrain. III. Adrenergic innervation of choline acetyltransferase-containing neurons in the rat.

The central adrenergic neurons have been suggested to play a role in the regulation of arousal and in the neuronal control of the cardiovascular system. To provide morphological evidence that these functions could be mediated via the basal forebrain, we performed correlated light and electron microscopic double-immunolabeling experiments using antibodies against phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase, the synthesizing enzymes for adrenaline and acetylcholine, respectively. Most adrenergic/cholinergic appositions were located in the horizontal limb of diagonal band of Broca, within the substantia innominata, and in a narrow band bordering the substantia innominata and the globus pallidus. Quantitative analysis indicated that cholinergic neurons of the substantia innominata receive significantly higher numbers of adrenergic appositions than cholinergic cells in the rest of the basal forebrain. In the majority of cases, the ultrastructural analysis revealed axodendritic asymmetric synapses. By comparing the number and distribution of dopamine beta-hydroxylase (DBH)/cholinergic appositions, described earlier, with those of PNMT/cholinergic interactions in the basal forebrain, it can be concluded that a significant proportion of putative DBH/cholinergic contacts may represent adrenergic input. Our results support the hypothesis that the adrenergic/cholinergic link in the basal forebrain may represent a critical component of a central network coordinating autonomic regulation with cortical activation.

Estimation of the total number of hippocampal CA1 pyramidal neurons: new methodology applied to helpless rats.

We have recently reported that in the learned helplessness model of depression, the less hippocampal spine synapses rats have, the more helpless they become. It remains unclear, however, whether the observed synaptic changes are associated with the loss of CA1 pyramidal cells. Cell bodies in the CA1 pyramidal layer are very densely packed, making cell counting difficult in this hippocampal subregion. To address this issue, we developed a new approach that (1) yields excellent preservation of the three-dimensional tissue structure; (2) utilizes osmium tetroxide to unambiguously label nucleoli; and (3) facilitates and accelerates unbiased, reliable counting of densely packed cell bodies. Our method provides an improved tool for studies aiming to evaluate hippocampal atrophy and cell loss, the most characteristic features in many neurodegenerative diseases, such as Alzheimer's disease, temporal lobe epilepsy and ischemia, as well as in several psychiatric disorders. Using this new method, we demonstrated no significant changes in the number of CA1 pyramidal cells in the rat learned helplessness paradigm. In addition, volumes of the CA1 pyramidal cell layer and the entire CA1 subfield remained unchanged among treatment groups. We conclude that previously observed synaptic alterations in helpless rats are not associated with CA1 pyramidal cell loss. This finding suggests that behavioral outcome in the learned helplessness paradigm is related to plastic events at the synaptic level, rather than at the level of principal cells.