Estradiol increases delayed, N-methyl-D-aspartate receptor-mediated excitation in the hippocampal CA1 region.

Hippocampal functions, e.g. synaptic plasticity and hippocampal-dependent behavior, are influenced by the circulating levels of ovarian steroids in adult, female rats. The mechanisms underlying this estradiol-dependent modulation, however, are poorly understood. One possibility is that estradiol alters N-methyl-D-aspartate (NMDA)-receptor functioning in the hippocampus. Here, using the in vitro hippocampal slice preparation, we evaluate estradiol-dependent changes in the NMDA receptor- and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated components of excitatory postsynaptic potentials (EPSPs) evoked in CA1 by Schaffer collateral test stimulation. Using established experimental conditions [J Neurosci 17 (1997) 1848], we replicate the observation that estradiol pretreatment of ovariectomized rats increases a pharmacologically isolated NMDA receptor-mediated EPSP evoked by Schaffer collateral stimulation. However, using different conditions that optimize study of this evoked response, the estradiol-dependent increase in the monosynaptic NMDA receptor-mediated EPSP is eliminated. Low-intensity test stimulation of the Schaffer collaterals in this optimized medium reveals a novel, late NMDA receptor-mediated EPSP in CA1 from estradiol-pretreated rats. The mechanism(s) underlying this estradiol-dependent increase in a late, NMDA receptor-mediated EPSP is not known, but enhanced CA1-CA1 excitatory circuitry and glutamate spillover could contribute to this response. We conclude that estradiol pretreatment enhances NMDA receptor function in the female hippocampus by increasing not the monosynaptic, but rather a late NMDA receptor-mediated response. Variations in the magnitude of this late response may well contribute to ovarian steroid-dependent modulation of hippocampal synaptic plasticity.

Estradiol modulates long-term synaptic depression in female rat hippocampus.

Fluctuating estradiol levels in the adult, female rat modify the anatomical and functional organization of the hippocampal CA1 region. When systemic levels of estradiol are low, e.g., on estrus or in ovariectomized (OVX) rats, long-term synaptic potentiation is difficult to induce in vivo. However, little is known about the role of this ovarian hormone in long-term synaptic depression. Using multiple conditioning paradigms, we assess the magnitude of long-term depression (LTD) at CA3-CA1 synapses in vitro from adult, ovariectomized rats as a function of systemic estradiol replacement. In hippocampal slices from control OVX rats with low levels of estradiol, a low-frequency (2 Hz), asynchronous conditioning stimulation protocol does not produce LTD at 1 h postconditioning. However, this same protocol induces robust LTD in slices from estradiol-treated OVX rats. When the conditioning frequency is increased to 4 Hz, slices from both groups of rats show robust LTD in vitro. At an even higher conditioning frequency (10 Hz), the 2-Hz-based observations are reversed; no consistent changes in synaptic transmission are observed in slices from estradiol-treated OVX rats, but those from control rats (OVX + oil) show robust LTD. Thus estradiol reduces the frequency threshold for LTD induction at the CA3-CA1 synapses. Further, regardless of the conditioning frequency employed, where robust LTD is seen, its induction depends on normally functioning N-methyl-D-aspartate (NMDA) receptors during conditioning. The shift in conditioning frequency needed to elicit LTD is consistent with a decrease in NMDA receptor activation with decreasing estradiol levels.

N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning.

The present study demonstrates that impairments of spatial learning and hippocampal long-term potentiation in rats chronically exposed to lead are associated with changes in gene and protein expression of N-methyl-D-aspartate receptor subunits. Rats exposed to 750 and 1500 ppm lead acetate were found to exhibit deficits in acquisition of a water maze spatial learning task. Furthermore, lead-exposed rats show dose-dependent reductions in the maintenance of in vivo hippocampal long-term potentiation induced in entorhinal cortex-dentate gyrus synapses. We found an unexpected, but significant (P<0.05), correlation between spatial learning and long-term potentiation when control and lead-exposed rats were analysed as a single, combined population. Dentate gyrus NR1 subunit messenger RNA was reduced 18% and 28% by exposure to 750 and 1500 ppm lead acetate, respectively. NR2A subunit messenger RNA was reduced 18% but only in the dentate gyrus of rats exposed to 1500 ppm lead acetate. No significant changes in dentate NR2B messenger RNA expression were measured in either of the lead-exposed groups. NR1 subunit protein was reduced 24% and 58% in hippocampal homogenates from rats exposed to 750 and 1500 ppm lead acetate. In contrast, no changes in NR2A or NR2B subunit protein were observed in the same hippocampal homogenates. These data show that reductions of specific N-methyl-D-aspartate receptor subunits are associated with deficits of both hippocampal long-term potentiation and spatial learning, induced in rats by chronic exposure to environmentally relevant levels of lead. These findings strongly suggest that the effects of lead on N-methyl-D-aspartate receptors may be the mechanistic basis for lead-induced deficits in cognitive function.

Estradiol enhances the induction of homosynaptic long-term depression in the CA1 region of the adult, ovariectomized rat.

An ovarian steroid-dependent cycle of synaptogenesis and synapse shedding occurs naturally in the hippocampus of the adult female rat. The newly formed axospinous synapses in CA1 may differ functionally from extant axospinous synapses, e.g., in terms of their modifiability. Here we assess whether estradiol alters the induction of homosynaptic long-term depression of the Schaffer collateral-CA1 synapses in vitro. Sprague-Dawley rats were bilaterally ovariectomized and, beginning 6-8 days later, received a series of injections of either 17beta-estradiol or sesame oil sc. Field potentials were recorded in hippocampal slices. In estradiol-treated animals, asynchronous, low-frequency stimulation led to significant long-term depression of the activated synapses in CA1 s. radiatum and no change of the inactive synapses in s. oriens. In contrast, this conditioning stimulation did not significantly alter any CA1 responses in oil-treated control animals. Subsequent high-frequency conditioning stimulation significantly potentiated the activated s. radiatum synapses in both estradiol- and oil-treated animals. Thus, given the stimulation conditions used here, estradiol enables the induction of homosynaptic long-term depression at the CA3-CA1 synapses in adult females.

Free postsynaptic densities in the hippocampus of the female rat.

The number of synapses in the adult, female hippocampal CA1 region fluctuates naturally across the estrous cycle in an ovarian steroid-dependent manner. This phasic variation in synapse number occurs without identifiable degenerating synapses. Ultrastructural correlates of the dynamic aspect of this synapse loss and synapse formation thus remain undescribed. During early development, one hallmark of synaptogenesis is the presence of free postsynaptic densities (PSDs). Here we report that the incidence of free PSDs in CA1 fluctuates across the rat estrous cycle. The number of free PSDs is greatest on the afternoon of proestrus and is significantly decreased on the afternoon of estrus, 24 h later. We hypothesize that these free PSDs reflect synapse turnover in the adult CA1 region.

Homosynaptic long-term depression of CA3-CA3 synapses in the in vivo hippocampus.

We tested the hypothesis that homosynaptic long-term depression (LTD) can be induced at the CA3-CA3 synapses in the adult, in vivo hippocampus while the CA3-CA1 synapses remain unchanged. Low-frequency conditioning stimulation of the contralateral fimbria significantly depressed the CA3 population response but did not change the simultaneously recorded CA3 response to angular bundle test stimulation. Similarly, in another group of animals, low-frequency conditioning stimulation of the contralateral fimbria depressed the CA3 synaptic response and left the collateral CA1 synaptic response unchanged. Among the possible explanations for this differential induction of homosynaptic LTD at the CA3-CA3 and CA3-CA1 synapses are differential control of intracellular calcium, differing levels of inhibition in these two regions, and the recency of 'natural' long-term potentiation in the two regions.

Enhanced expression of AMPA receptor protein at perforated axospinous synapses.

We performed a quantitative electron microscopic analysis of the middle third of the molecular layer in the dentate gyrus of rat, using material processed with postembedding gold labeling for the glutamate receptor subunits GluR1, GluR2/3, or NMDAR1. Perforated axospinous synapses were at least twice as likely as non-perforated ones to express detectable levels of AMPA receptor subunits, whereas no significant differences in NMDA receptor expression were observed. These data imply that perforated synapses may be especially potent, and are consistent with the hypothesis that insertion of AMPA receptor protein into the postsynaptic membrane of previously silent synapses contributes to long-term potentiation.

Perforant path activation modulates the induction of long-term potentiation of the schaffer collateral--hippocampal CA1 response: theoretical and experimental analyses.

In one computational model of hippocampal function, the entorhinal cortical input to CA1 is hypothesized to play a key role in the ability of CA1 to decode CA3 recodings. Here, we develop a modification of this CA1 decoder hypothesis that is applicable to several computational theories of hippocampal function, and then we electrophysiologically investigate one assumption of this new hypothesis. First, using biologically realistic estimates, we calculate that CA3-induced CA1 excitation is too high and that inhibition plausibly plays a role in this CA1 decoder model. Thus motivated, we turn to a physiological demonstration to substantiate the plausibility of the proposed mechanism. Using the rat hippocampal slice, we examine an interlaminar interaction between the distal perforant path input to hippocampal CA1 stratum moleculare and the more proximal Schaffer collateral input to stratum radiatum. Perforant path activation provides sufficient inhibition to block homosynaptic long-term potentiation elicited by a suitably strong stratum radiatum input. For this interlaminar interaction to be most effective, perforant path activation must both precede and follow Schaffer collateral activation. Perforant path-evoked inhibition in CA1 can thus serve as a viable mechanism in the learned decoder theory of hippocampal CA1.

Ovarian steroidal control of connectivity in the female hippocampus: an overview of recent experimental findings and speculations on its functional consequences.

Experimental evidence accumulated over the past 5 years clearly indicates that ovarian steroids regulate the number of synapses in the rat hippocampal CA1 region. When estradiol levels are high such as during proestrus and ovulation, the number of synapses is high; when estradiol levels are low such as during estrus, the number of synapses is low. Here we address three questions that are frequently raised by these phasic fluctuations in synapse number in a brain region to which cognitive functions are classically attributed. First, what neuronal signals might produce the changes in synapse number? Second, how are the hippocampal functions of memory encoding and cognitive mapping affected by fluctuating levels of ovarian steroids? Third, for mammals in general, what might be the ecological/cognitive significance of such changes? In this last section, we integrate some of the relevant human and rodent cognitive/behavioral literature and propose a hypothesis. Namely, by altering its quantitative connectivity, the female hippocampus is optimized for different cognitive/behavioral functions when the female is sexually receptive and ovarian steroid levels are high rather than when she is not receptive and steroid levels are low. The hippocampus thus shifts its optimal computational functions across the estrous/menstrual cycle.

Astrocytic volume fluctuates in the hippocampal CA1 region across the estrous cycle.

The number of dendritic spine synapses in the hippocampal CA1 stratum radiatum fluctuates across the rat estrous cycle, being high on proestrus and low on estrus [20]. We hypothesized that the volume occupied by astrocytic processes changes in a complementary manner. The volume fraction of astrocytic processes was determined stereologically in CA1 s. radiatum and s. lacunosum-moleculare of cycling female rats. Consistent with our hypothesis, the volume fraction was significantly lower on the afternoon of proestrus than on the afternoon of estrus in both laminae.

Ultrastructural identification of entorhinal cortical synapses in CA1 stratum lacunosum-moleculare of the rat.

The goal of the present study was to identify and characterize, at the electron microscopic level, the synapses formed by entorhinal cortical (EC) axons in the hippocampal CA1 stratum lacunosum-moleculare of the adult rat. Phaseolus vulgaris leucoagglutinin was ionotophoresed at various loci throughout the mediolateral and dorsoventral extent of the EC to label EC-CA1 synapses. Virtually all labeled synapses were asymmetric and axospinous. EC axons did not preferentially synapse with any particular type of dendritic spine; rather, EC axons formed synapses with the range of dendritic spine morphologies observed in CA1 s. lacunosum-moleculare. Spines with either perforated or nonperforated postsynaptic densities were contacted by EC axons. Occasionally both a labeled and an unlabeled axon synapsed on a single dendritic spine head. The data are discussed in relation to the morphology of other afferent systems synapsing in s. lacunosum-moleculare and to the physiology of the EC-CA1 system.

Rapid development of somatic spines in stratum granulosum of the adult hippocampus in vitro.

Somatic spines on granule cells have been observed occasionally in the adult rat dentate gyrus in vivo. Here we evaluate the appearance and formation of somatic spines in s. granulosum from adult rat hippocampal slices immediately after slice preparation, 45 min, 3 h, and 5 h later. Initially somatic spines are extremely rare but, after 3 h in vitro, they are readily apparent. Some of these somatic spines form asymmetric synapses that have a spherical vesicle-containing presynaptic bouton and a postsynaptic density. Other somatic spines lack a postsynaptic density and may also lack an apposed presynaptic bouton (free somatic spines) as observed in single thin sections. Both the number of these free somatic spines and the number of somatic spine synapses increase in a time-dependent manner. No somatic spines were observed on CA1 pyramidal cells in the same hippocampal slices. Electrophysiological observations indicate that the formation of somatic spine synapses on granule cells in the hippocampal slice occurs without any apparent granule cell activation. The trigger event for this very rapid synaptogenesis in the adult dentate gyrus remains to be determined.

Somatic ribosomal changes induced by long-term potentiation of the perforant path-hippocampal CA1 synapses.

The present study quantified ribosomes, as an ultrastructural marker of neuronal protein synthesis, following long-term potentiation (LTP) in the hippocampal CA1 region in vitro. Sixty min after LTP-inducing, high-frequency stimulation of the perforant path, the total number of ribosomes, the number of polysomes, and the number of membrane-bound ribosomes increased significantly. These increases are a postsynaptic morphological correlate consistent with enhanced protein synthesis following the induction of LTP in the perforant path-CA1 system.

NMDA receptor antagonists block the induction of long-term depression in the hippocampal dentate gyrus of the anesthetized rat.

The present study tested the effect of two non-competitive NMDA receptor antagonists, ketamine and phencyclidine, on the induction of long-term depression (LTD) in the dentate gyrus of urethane-anesthetized rats. Both drugs blocked the induction of LTD as well as long-term potentiation (LTP). NMDA receptor activation thus seems to be required for the induction of both LTD and LTP in the dentate gyrus. High-intensity conditioning stimulation did not overcome the phencyclidine block of LTD. Strong, but brief, postsynaptic depolarization is apparently not the only event needed to trigger LTD.

Morphological correlates of long-term potentiation imply the modification of existing synapses, not synaptogenesis, in the hippocampal dentate gyrus.

This report evaluates two morphological markers of synaptogenesis following the induction of long-term potentiation (LTP) in the dentate gyrus of the anesthetized rat. These two morphological features, polyribosomes and multiple synaptic contacts, are known to increase in number with synaptogenesis in the mature hippocampus. The analysis focused on the middle third of the dentate molecular layer. As shown previously, this is the region of primary synaptic activation in our electrophysiological protocol and the region of localized morphological changes with LTP. Here the incidence of a polyribosome at the base of a dendritic spine declined 57% with LTP. In addition, the number of multiple synaptic contacts decreased 18% there with LTP. Both decreases were more pronounced immediately following conditioning stimulation than at later intervals. Because both morphological features decrease with LTP but increase with synaptogenesis, the data do not support the hypothesis that new synapses form with LTP. Instead, the data add further support to the view that the strengthening of existing excitatory synapses underlies LTP.

Associative synaptic potentiation and depression: quantification of dissociable modifications in the hippocampal dentate gyrus favors a particular class of synaptic modification equations.

This report further characterizes associative long-term synaptic modification of the ipsilateral and contralateral synapses formed by the bilateral entorhinal cortical (EC) projection to the dentate gyrus (DG). The experimental model is the anesthetized hooded rat. The quantitative results qualify this system as a model for studying the rules of associative synaptic modification formulated in terms of individual synapses. Bilateral DG microelectrodes recorded both ipsilateral and contralateral EC-DG responses before and after brief, high-frequency EC conditioning stimulation. The weak contralateral pathway received high-frequency conditioning before, during, or after similar conditioning of the strong, converging ipsilateral pathway. Statistical analyses revealed two types of significant, dissociated synaptic modifications, which depend on the relationship of the ipsilateral and contralateral afferents. First, contralateral EC-DG responses potentiated or depressed when the converging ipsilateral responses concurrently either potentiated or remained unchanged. Second, contralateral EC-DG responses potentiated, depressed, or showed no change when the collateral ipsilateral responses concurrently either potentiated or remained unchanged. Correlation and contingency table analyses indicated that changes in the contralateral synaptic responses are not well predicted by changes at either neighboring synapses of the converging ipsilateral pathway or at synapses of the collateral ipsilateral pathway. The contingencies of associated pre- and postsynaptic activation determined by the conditioning paradigm, however, accurately predicted the altered synaptic responses of both ipsilateral and contralateral EC-DG pathways. The results imply that associative synaptic modification in the EC-DG system is specific to individual synapses and requires both appropriate presynaptic and postsynaptic activation. Because this system provides suitable controls for nonspecific effects of conditioning stimulation and because modification of neighboring synapses is dissociable, the EC-DG system can be used to study further those rules of activity-dependent associative modification that are formulated in terms of individual synapses. The discussion briefly considers published rules of synaptic modification, pointing out several rules that are not consistent with the experimental observations and one that agrees with the present results.

Synaptic interface surface area increases with long-term potentiation in the hippocampal dentate gyrus.

The present study continues our attempt to understand the ultrastructural changes that accompany and may underlie long-term potentiation (LTP). This report describes changes with LTP in the surface area of the pre- and postsynaptic membrane apposition at the synapses formed by entorhinal cortical (EC) axons with granule cell dendritic spines of the dentate gyrus (DG). The electrophysiology and electron microscopy of the DGs from each animal followed conventional procedures. The trace length of the pre- and postsynaptic apposition was measured for identified asymmetric synapses in the dentate molecular layer. The total apposed membrane surface area per unit volume (Sv) was then computed for 4 categories of synaptic profiles for each third of the molecular layer. Statistical analysis of the Sv data used multivariate analyses of variance. Across the entire molecular layer, total apposed Sv does not change significantly with LTP. However, in the activated portion of the molecular layer, total apposed Sv increases significantly, reflecting a significant increase in the apposed Sv for the concave spine profiles there. For these spine profiles, the increased apposed Sv is due to the increased membrane area both at the postsynaptic density and beyond. The average apposed surface area per individual synapse also increases markedly with LTP. The present data support the hypothesis of coordinated pre- and postsynaptic anatomical changes with LTP in the EC-DG system.

Changes in the numerical density of synaptic contacts with long-term potentiation in the hippocampal dentate gyrus.

Long-term potentiation (LTP) in the rat dentate gyrus is a multifaceted phenomenon, including synaptic potentiation; simultaneous synaptic depression at neighboring, unconditioned synapses; and a change in the amount of cell firing produced by a specified amount of synaptic current (see Levy and Desmond: In G. Buzsaki and C. Vanderwolf (eds): Electrical Activity of The Archicortex. Budapest: Akademiai Kiado, pp. 359-373, '85b). This study presents long-term anatomical modifications that seem related to excitatory synaptic modification. These anatomical alterations appear early and persist for at least 60 minutes following conditioning stimulation. Each animal received test pulse stimulation delivered alternately to the angular bundles before and after brief, unilateral high-frequency conditioning stimulation that is typical of many LTP paradigms. Anatomical preparation followed standard procedures. Double-blind scoring procedures quantified the number of asymmetric synapses in the dentate molecular layer. These counts were converted to the number of synapses per unit volume using stereological corrections that combined geometrically derived theory and modest serial sectioning. Multivariate analysis of variance evaluated the statistical significance of changes in synapse density. Across all three groups of animals, conditioning stimulation does not significantly change the density of synaptic contacts across the entire molecular layer. There is a trend for a decreased density of synaptic contacts in the middle molecular layer, the region activated by the conditioning stimulation. Here the density of concave spine profiles increases significantly in all three groups of animals with conditioning stimulation. This increase accompanies significant decreases in the density of nonconcave, simple and ellipsoid, spine profiles. No significant changes in the density of shaft synapses occur with LTP-inducing conditioning stimulation. These data suggest that the concave spine profiles are a correlate of LTP-inducing stimulation and may be the potentiated synapses. We hypothesize that with synaptic potentiation there occurs an interconversion of spine synapses such that some nonconcave spine profiles become concave spine profiles. Such an interconversion apparently begins shortly after the conditioning stimulation and persists for at least 60 minutes.

Changes in the postsynaptic density with long-term potentiation in the dentate gyrus.

The present study documents alterations in the size of the postsynaptic density (PSD) of synapses formed by entorhinal afferents with granule cell dendritic spines with long-term potentiation (LTP). These changes appear early and persist for at least 60 minutes after LTP-inducing conditioning stimulation. Each animal received test and conditioning stimulation typical of LTP paradigms. Electron microscopic preparation of the dentate gyri from each animal followed conventional procedures. PSD trace lengths of identified asymmetric synaptic profiles were measured. The total PSD length for four categories of synaptic profiles was determined for each third of the molecular layer. PSD surface area per unit volume of tissue (SV) was then computed from these data. Statistical analysis of the SV data used multivariate analysis of variance. PSD surface area per synapse was also estimated. Total PSD surface area per unit volume does not change significantly throughout the entire molecular layer with LTP-inducing conditioning stimulation. However, in the activated portion of the molecular layer, total PSD surface area per unit volume tends to increase with conditioning stimulation. In the middle third of the molecular layer, total PSD surface area per unit volume associated with the concave spine profiles increases significantly while there is a statistically significant decrease in total PSD SV associated with the nonconcave spine profiles. The PSD surface area per synapse also increases markedly. Since it seems that there is an interconversion of spine synapses from nonconcave to concave with LTP (Desmond and Levy: J. Comp. Neurol. In press, '86a), these data suggest that potentiated synapses have larger responses because, in part, they have larger neurotransmitter receptive regions.