Development and subunit composition of synaptic NMDA receptors in the amygdala: NR2B Synapses in the adult central amygdala

NMDA receptors are well known to play an important role in synaptic development and plasticity. Functional NMDA receptors are heteromultimers thought to contain two NR1 subunits and two or three NR2 subunits. In central neurons, NMDA receptors at immature glutamatergic synapses contain NR2B subunits and are largely replaced by NR2A subunits with development. At mature synapses, NMDA receptors are thought to be multimers that contain either NR1/NR2A or NR1/NR2A/NR2B subunits, whereas receptors that contain only NR1/NR2B subunits are extrasynaptic. Here, we have studied the properties of NMDA receptors at glutamatergic synapses in the lateral and central amygdala. We find that NMDA receptor-mediated synaptic currents in the central amygdala in both immature and mature synapses have slow kinetics and are substantially blocked by the NR2B-selective antagonists (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propano and ifenprodil, indicating that there is no developmental change in subunit composition. In contrast, at synapses on pyramidal neurons in the lateral amygdala, whereas NMDA EPSCs at immature synapses are slow and blocked by NR2B-selective antagonists, at mature synapses their kinetics are faster and markedly less sensitive to NR2B-selective antagonists, consistent with a change from NR2B to NR2A subunits. Using real-time PCR and Western blotting, we show that in adults the ratio of levels of NR2B to NR2A subunits is greater in the central amygdala than in the lateral amygdala. These results show that the subunit composition synaptic NMDA receptors in the lateral and central amygdala undergo distinct developmental changes.

Synapse formation on adult-born hippocampal neurons.

It is now widely accepted that adult neurogenesis plays a fundamental role in hippocampal function. Neurons born in the adult dentate gyrus of the hippocampus undergo a series of events before they fully integrate in the network and eventually become undistinguishable from neurons born during embryogenesis. Adult hippocampal neurogenesis is strongly regulated by neuronal activity and neurotransmitters, and the synaptic integration of adult-born neurons occurs in discrete steps, some of which are very different from perinatal synaptogenesis. Here, we review the current knowledge on the development of the synaptic input and output of neurons born in the adult hippocampus, from the stem/progenitor cell to the fully mature neuron. We also provide insight on the regulation of adult neurogenesis by some neurotransmitters and discuss some specificities of the integration of new neurons in an adult environment. The understanding of the mechanisms regulating the synaptic integration of adult-born neurons is not only crucial for our understanding of brain plasticity, but also provides a framework for the manipulation and monitoring of endogenous adult neurogenesis as well as grafted cells, for potential therapeutic applications.

Presynaptic long-term depression at a central glutamatergic synapse: a role for CaMKII

CaMKII is a calcium-activated kinase that is abundant in neurons and has been strongly implicated in memory and learning. Here we show that low-frequency stimulation of glutamatergic afferents in hippocampal slices from juvenile domestic chicks results in long-term depression of synaptic transmission. This reduction does not require activation of NMDA or metabotropic glutamate receptors and does not require a rise in postsynaptic calcium. However, buffering presynaptic calcium prevents the reduction of the excitatory postsynaptic potential or current that is induced by low-frequency stimulation. in addition, application of the calmodulin antagonist calmidazolium, or the specific CaMKII antagonist KN-93, completely blocks long-term depression. These findings demonstrate a newsy discovered form of long-term synaptic depression in the avian hippocampus.

Connexin 30 sets synaptic strength by controlling astroglial synapse invasion.

Astrocytes play active roles in brain physiology by dynamic interactions with neurons. Connexin 30, one of the two main astroglial gap-junction subunits, is thought to be involved in behavioral and basic cognitive processes. However, the underlying cellular and molecular mechanisms are unknown. We show here in mice that connexin 30 controls hippocampal excitatory synaptic transmission through modulation of astroglial glutamate transport, which directly alters synaptic glutamate levels. Unexpectedly, we found that connexin 30 regulated cell adhesion and migration and that connexin 30 modulation of glutamate transport, occurring independently of its channel function, was mediated by morphological changes controlling insertion of astroglial processes into synaptic clefts. By setting excitatory synaptic strength, connexin 30 plays an important role in long-term synaptic plasticity and in hippocampus-based contextual memory. Taken together, these results establish connexin 30 as a critical regulator of synaptic strength by controlling the synaptic location of astroglial processes.

Effects of Enriched Physical and Social Environments on Motor Performance, Associative Learning, and Hippocampal Neurogenesis in Mice.

We have studied the motor abilities and associative learning capabilities of adult mice placed in different enriched environments. Three-month-old animals were maintained for a month alone (AL), alone in a physically enriched environment (PHY), and, finally, in groups in the absence (SO) or presence (SOPHY) of an enriched environment. The animals' capabilities were subsequently checked in the rotarod test, and for classical and instrumental learning. The PHY and SOPHY groups presented better performances in the rotarod test and in the acquisition of the instrumental learning task. In contrast, no significant differences between groups were observed for classical eyeblink conditioning. The four groups presented similar increases in the strength of field EPSPs (fEPSPs) evoked at the hippocampal CA3-CA1 synapse across classical conditioning sessions, with no significant differences between groups. These trained animals were pulse-injected with bromodeoxyuridine (BrdU) to determine hippocampal neurogenesis. No significant differences were found in the number of NeuN/BrdU double-labeled neurons. We repeated the same BrdU study in one-month-old mice raised for an additional month in the above-mentioned four different environments. These animals were not submitted to rotarod or conditioned tests. Non-trained PHY and SOPHY groups presented more neurogenesis than the other two groups. Thus, neurogenesis seems to be related to physical enrichment at early ages, but not to learning acquisition in adult mice.

Transcription factor NF-kappaB is transported to the nucleus via cytoplasmic dynein/dynactin motor complex in hippocampal neurons

Background Long-term changes in synaptic plasticity require gene transcription, indicating that signals generated at the synapse must be transported to the nucleus. Synaptic activation of hippocampal neurons is known to trigger retrograde transport of transcription factor NF-κB. Transcription factors of the NF-κB family are widely expressed in the nervous system and regulate expression of several genes involved in neuroplasticity, cell survival, learning and memory. Principal Findings In this study, we examine the role of the dynein/dynactin motor complex in the cellular mechanism targeting and transporting activated NF-κB to the nucleus in response to synaptic stimulation. We demonstrate that overexpression of dynamitin, which is known to dissociate dynein from microtubules, and treatment with microtubule-disrupting drugs inhibits nuclear accumulation of NF-κB p65 and reduces NF-κB-dependent transcription activity. In this line, we show that p65 is associated with components of the dynein/dynactin complex in vivo and in vitro and that the nuclear localization sequence (NLS) within NF-κB p65 is essential for this binding. Conclusion This study shows the molecular mechanism for the retrograde transport of activated NF-κB from distant synaptic sites towards the nucleus.

Fine structure of hippocampal mossy fiber synapses following rapid high-pressure freezing

Synapses of hippocampal neurons play important roles in learning and memory processes and are involved in aberrant hippocampal function in temporal lobe epilepsy. Major neuronal types in the hippocampus as well as their input and output synapses are well known, but it has remained an open question to what extent conventional electron microscopy (EM) has provided us with the real appearance of synaptic fine structure under in vivo conditions. There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts. However, realistic fine-structural data of synapses are required for our understanding of the transmission process and for its simulation. Here, we used high-pressure freezing and cryosubstitution of hippocampal tissue that was not subjected to aldehyde fixation and dehydration in ethanol to monitor the fine structure of an identified synapse in the hippocampal CA3 region, that is, the synapse between granule cell axons, the mossy fibers, and the proximal dendrites of CA3 pyramidal neurons. Our results showed that high-pressure freezing nicely preserved ultrastructural detail of this particular synapse and allowed us to study rapid structural changes associated with synaptic plasticity.

A stereological study of synapse number in the epileptic human hippocampus

Hippocampal sclerosis is the most frequent pathology encountered in resected mesial temporal structures from patients with intractable temporal lobe epilepsy (TLE). Here, we have used stereological methods to compare the overall density of synapses and neurons between non-sclerotic and sclerotic hippocampal tissue obtained by surgical resection from patients with TLE. Specifically, we examined the possible changes in the subiculum and CA1, regions that seem to be critical for the development and/or maintenance of seizures in these patients. We found a remarkable decrease in synaptic and neuronal density in the sclerotic CA1, and while the subiculum from the sclerotic hippocampus did not display changes in synaptic density, the neuronal density was higher. Since the subiculum from the sclerotic hippocampus displays a significant increase in neuronal density, as well as a various other neurochemical changes, we propose that the apparently normal subiculum from the sclerotic hippocampus suffers profound alterations in neuronal circuits at both the molecular and synaptic level that are likely to be critical for the development or maintenance of seizure activity

Activity-dependent regulation of synaptic clustering in a hippocampal culture system

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-d-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.

Very short-term plasticity in hippocampal synapses

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2–10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (i) a form of synaptic depression manifested after a prior exocytotic event, (ii) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca2+ channels, and (iii) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.

Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Different modes of hippocampal plasticity in response to estrogen in young and aged female rats

Estrogen regulates hippocampal dendritic spine density and synapse number in an N-methyl-d-aspartate (NMDA) receptor-dependent manner, and these effects may be of particular importance in the context of age-related changes in endocrine status. We investigated estrogen's effects on axospinous synapse density and the synaptic distribution of the NMDA receptor subunit, NR1, within the context of aging. Although estrogen induced an increase in axospinous synapse density in young animals, it did not alter the synaptic representation of NR1, in that the amount of NR1 per synapse was equivalent across groups. Estrogen replacement in aged female rats failed to increase axospinous synapse density; however, estrogen up-regulated synaptic NR1 compared with aged animals with no estrogen. Therefore, the young and aged hippocampi react differently to estrogen replacement, with the aged animals unable to mount a plasticity response generating additional synapses, yet responsive to estrogen with respect to additional NMDA receptor content per synapse. These findings have important implications for estrogen replacement therapy in the context of aging.

Long-term potentiation at single fiber inputs to hippocampal CA1 pyramidal cells.

Despite extensive investigation, it remains unclear whether presynaptic and/or postsynaptic modifications are primarily responsible for the expression of long-term potentiation (LTP) in the CA1 region of the hippocampus. Here we address this issue by using techniques that maximize the likelihood of stimulating a single axon and thereby presumably a single synapse before and after the induction of LTP. Several basic properties of synaptic transmission were examined including the probability of neurotransmitter release (Pr), the quantal size (q), and the so-called potency, which is defined as the average size of the synaptic response when release of transmitter does occur. LTP was routinely associated with an increase in potency, whereas increases in Pr alone were not observed. LTP was also reliably induced when baseline Pr was high, indicating that synapses with high Pr can express LTP. These results suggest that the mechanism for the expression of LTP involves an increase in q and is difficult to explain by an increase in Pr alone.

Steel mutant mice are deficient in hippocampal learning but not long-term potentiation.

Mice carrying mutations in either the dominant white-spotting (W) or Steel (Sl) loci exhibit deficits in melanogenesis, gametogenesis, and hematopoiesis. W encodes the Kit receptor tyrosine kinase, while Sl encodes the Kit ligand, Steel factor, and the receptor-ligand pair are contiguously expressed at anatomical sites expected from the phenotypes of W and Sl mice. The c-kit and Steel genes are also both highly expressed in the adult murine hippocampus: Steel is expressed in dentate gyrus neurons whose mossy fiber axons synapse with the c-kit expressing CA3 pyramidal neurons. We report here that Sl/Sld mutant mice have a specific deficit in spatial learning. These mutant mice are also deficient in baseline synaptic transmission between the dentate gyrus and CA3 but show normal long-term potentiation in this pathway. These observations demonstrate a role for Steel factor/Kit signaling in the adult nervous system and suggest that a severe deficit in hippocampal-dependent learning need not be associated with reduced hippocampal long-term potentiation.

Long-term synaptic transformation of hippocampal CA1 gamma-aminobutyric acid synapses and the effect of anandamide.

Evidence is presented for a distinctive type of hippocampal synaptic modification [previously described for a molluscan gamma-aminobutyric acid (GABA) synapse after paired pre- and postsynaptic excitation]: transformation of GABA-mediated synaptic inhibition into synaptic excitation. This transformation persists with no further paired stimulation for 60 min or longer and is termed long-term transformation. Long-term transformation is shown to contribute to pairing-induced long-term potentiation but not to long-term potentiation induced by presynaptic stimulation alone. Further support for such mechanistic divergence is provided by pharmacologic effects on long-term transformation as well as these two forms of long-term potentiation by Cl- channel blockers, glutamate and GABA antagonists, as well as the endogenous cannabinoid ligand anandamide.