Synaptic Enhancement Research Articles

Endogenous Opioids as Physiological Antidepressants: Complementary Role of Delta Receptors and Dopamine

Major depressive disorder was shown to be associated with a reduction in response to rewarding stimuli in the dopaminergic mesolimbic pathway in a recent neuroimaging study (Forbes et al, 2009). This neuronal network is modulated by opioids at the level of dopamine (DA) neurons and afferent structures, typically by activation of mu- and delta-opioid receptors (MORs and DORs, respectively) enhancing reward- and motivation-related processes. Therefore, a deficit in endogenous opioids, mainly enkephalins (ENKs), in the nucleus accumbens and ventral tegmental area, may lead to a decrease in the neurobiological control of mood states and reward. To develop fast-acting therapeutics for severe depression, particularly in adolescents, MORs and DORs have been investigated for potential antidepressant activity, using: (i) exogenous agonists and antagonists for both receptors and (ii) ENKs protected from their inactivating enzymes by dual inhibitors such as RB101 (N-(R,S)-2-benzyl-3-((S)(2-amino-4-methyl-thio)-butyldithio)-1-oxopropyl)-1-phenylalanine benzyl ester) (Noble and Roques, 2007), which induced a synaptic enhancement of phasically released ENKs. In animal models, exogenous delta agonists such as SNC80 ((+)-4-(aR)-a-((2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl)-(3-methoxyphenyl)methyl)-N,N-diethylbenzamide induce highly significant antidepressant effects whereas mu agonists do not. This is associated with an increase in brain-derived neurotrophic factor mRNA (Jutkiewicz, 2006), which has been suggested to have a role in antidepressant effects, as well as an indirect increase in DA efflux into the striatum (Bosse et al, 2008). As frequently observed with some delta agonists, SNC80 causes seizures. This is not observed with RB101, which is effective in almost all animal models of depression, eg, decreases conditioned suppression of mobility in mice, immobility in the forced swim test in rats (Jutkiewicz, 2006) and in mice (Nieto et al, 2005) and escape failures in the learned helplessness test (Noble and Roques, 2007). The effects of RB101 are observed after a single administration and no tolerance is observed during chronic use (Cordonnier et al, 2005). Moreover, stimulation of opioid receptors by RB101 protected ENKs does not induce the undesirable side effects of morphine (Noble and Roques, 2007). The selective DOR antagonist naltrindole or DA antagonists reverse antidepressant effects of RB101 and SNC80, demonstrating that the effects are mediated by DORs, and involve the DA-dependent mesolimbic pathway. Consistently, DOR KO mice present depressive-like behaviors, which are reversed by antidepressant drugs, whereas MOR KO mice are unaffected (Filliol et al, 2000; Nieto et al, 2005). The key role of interconnected endogenous opioid and DAergic systems in mood control is demonstrated by the facilitation of antidepressant-like effects of RB101 after deafferentation of the DAergic mesolimbic pathway (Cordonnier et al, 2005), which increases the concentrations of PENK and ENKs. Taken together, these results suggest a phasic control of the DAergic mesolimbic pathway by ENKs, which may be impaired in depressive-like syndromes, and clinical studies are starting to investigate this endogenous opioid-DA interaction in human depression (Kennedy et al, 2006; Scott et al, 2008). The development of (i) dual orally active ENK inhibitors with strong analgesic properties and immediate antidepressant effects (Noble and Roques, 2007) and (ii) delta agonists devoid of side effects may lead to significant improvements in the treatment of depression and mood disorders.

Coactivation of thalamic and cortical pathways induces input timing–dependent plasticity in amygdala

Long-term synaptic enhancements in cortical and thalamic auditory inputs to the lateral nucleus of the amygdala (LAn) mediate encoding of conditioned fear memory. It remained unknown, however, whether the convergent auditory conditioned stimulus (CSa) pathways may interact with each other producing changes in their synaptic function. Here we show that continuous paired stimulation of thalamic and cortical auditory inputs to the LAn with the interstimulus delay approximately mimicking a temporal pattern of their activation in behaving animals during auditory fear conditioning results in persistent potentiation of synaptic transmission in cortico-amygdala pathway in rat brain slices. This novel form of input timing-dependent plasticity (ITDP) in cortical input depends on InsP3-sensitive Ca2+ release from the internal stores and postsynaptic Ca2+ influx through calcium-permeable kainate receptors during its induction. ITDP in the auditory projections to the LAn, determined by characteristics of presynaptic activity patterns, may contribute to the encoding of the complex CSa.

Counteracting roles of metabotropic glutamate receptor subtypes 1 and 5 in regulation of pain-related spatial and temporal synaptic plasticity in rat entorhinal–hippocampal pathways

It was previously found that persistent inflammatory pain state resulted in enhancement of synaptic connections and efficacy in direct entorhinal–hippocampal (EC-HIP) pathways. In the current study, the roles of two subtypes of group I metabotropic glutamate receptors in the above processes were evaluated. Similarly, pain-related spatial and temporal synaptic enhancement model was stably achieved by the multi-electrode array (8 × 8) recordings in the hippocampal slices of rats pre-treated with intraplantar (i.pl.) bee venom (BV) injection. I.pl. saline injection was used as control. Inhibition of mGluR1 by a selective antagonist 7-hydroxyiminocyclopropan [b] chromen-1α-carboxylic acid ethyl ester (CPCCOEt) resulted in a dramatic increase in synaptic connections in the hippocampal slices of rats treated by BV, but not by saline. However, inhibition of mGluR5 by a selective antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) produced no spatial change from either of the two groups. Temporally, the BV-enhanced LTP could be further incremented by antagonism of mGluR1 with CPCCOEt perfusion when plateau LTP was well established. However, the BV-enhanced LTP was significantly suppressed by antagonism of mGluR5 with MPEP. Neither of the two drugs affected magnitude of LTP in rats treated by i.pl. saline. Taken together with our previous results, it is suggested that mGluR1 be involved in tonic inhibition of EC-HIP synaptic enhancement, while mGluR5 be involved in maintenance of persistent inflammatory pain-associated EC-HIP synaptic enhancement that is largely based upon activation of ionic glutamate receptors.

Compartmentalized versus Global Synaptic Plasticity on Dendrites Controlled by Experience

Synapses in the brain are continuously modified by experience, but the mechanisms are poorly understood. In vitro and theoretical studies suggest threshold-lowering interactions between nearby synapses that favor clustering of synaptic plasticity within a dendritic branch. Here, a fluorescently tagged AMPA receptor-based optical approach was developed permitting detection of single-synapse plasticity in mouse cortex. Sensory experience preferentially produced synaptic potentiation onto nearby dendritic synapses. Such clustering was significantly reduced by expression of a phospho-mutant AMPA receptor that is insensitive to threshold-lowering modulation for plasticity-driven synaptic incorporation. In contrast to experience, sensory deprivation caused homeostatic synaptic enhancement globally on dendrites. Clustered synaptic potentiation produced by experience could bind behaviorally relevant information onto dendritic subcompartments; global synaptic upscaling by deprivation could equally sensitize all dendritic regions for future synaptic input.

Mechanisms underlying rule learning-induced enhancement of excitatory and inhibitory synaptic transmission

Training rats to perform rapidly and efficiently in an olfactory discrimination task results in robust enhancement of excitatory and inhibitory synaptic connectivity in the rat piriform cortex, which is maintained for days after training. To explore the mechanisms by which such synaptic enhancement occurs, we recorded spontaneous miniature excitatory and inhibitory synaptic events in identified piriform cortex neurons from odor-trained, pseudo-trained, and naive rats. We show that olfactory discrimination learning induces profound enhancement in the averaged amplitude of AMPA receptor-mediated miniature synaptic events in piriform cortex pyramidal neurons. Such physiological modifications are apparent at least 4 days after learning completion and outlast learning-induced modifications in the number of spines on these neurons. Also, the averaged amplitude of GABA(A) receptor-mediated miniature inhibitory synaptic events was significantly enhanced following odor discrimination training. For both excitatory and inhibitory transmission, an increase in miniature postsynaptic current amplitude was evident in most of the recorded neurons; however, some neurons showed an exceptionally great increase in the amplitude of miniature events. For both excitatory and inhibitory transmission, the frequency of spontaneous synaptic events was not modified after learning. These results suggest that olfactory discrimination learning-induced enhancement of synaptic transmission in cortical neurons is mediated by postsynaptic modulation of AMPA receptor-dependent currents and balanced by long-lasting modulation of postsynaptic GABA(A) receptor-mediated currents.

NADPH oxidase-mediated Rac1 GTP activity is necessary for nongenomic actions of the mineralocorticoid receptor in the CA1 region of the rat hippocampus

Mineralocorticoid receptors (MRs) in the central nervous system play important roles in spatial memory, fear memory, salt sensitivity, and hypertension. Corticosterone binds to MRs to induce presynaptic vesicle release and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor aggregation, which are necessary for induction of long-term potentiation under psychological stress. On the other hand, cognitive dysfunction is an important problem clinically in patients with hypertension, diabetes, and cerebral infarction, and all of these conditions are associated with an increase in reactive oxygen species (ROS) generation. Oxidative stress has been shown to modify the genomic actions of MRs in the peripheral organs; however, there have been no reports until now about the relation between the nongenomic actions of MRs and ROS in the central nervous system. In this study, we investigated the relationship between ROS and the nongenomic actions of MR. We examined the nongenomic actions of MR by measuring the slope of the field excitatory postsynaptic potentials and found that ROS induced an additive increase of these potentials, which was accompanied by Rac1 GTP activation and ERK1/2 phosphorylation. An NADPH oxidase inhibitor, apocynin, blocked the nongenomic actions of MRs. A Rac1 inhibitor, NSC23766, was also found to block synaptic enhancement and ERK1/2 phosphorylation induced by NADPH and corticosterone. We concluded that NADPH oxidase activity and Rac1 GTP activity are indispensable for the nongenomic actions of MRs and that Rac1 GTP activation induces ERK1/2 phosphorylation in the brain.

Modification of network activity induced by synaptic enhancement in cultured rathippocampal neurons

Modification of network activity induced by synaptic enhancement in cultured rathippocampal neurons

Expression and distribution of GluR1 subunit of glutamate receptor during development of long-lasting synaptic enhancement coupled with synaptogenesis

Expression and distribution of GluR1 subunit of glutamate receptor during development of long-lasting synaptic enhancement coupled with synaptogenesis

A mechanism emerges for the critical period hypothesis for estrogen treatment

During the past few decades, a significant body of literature has emerged from the basic science community demonstrating neuroprotective effects and synaptic enhancements induced by estrogen in brain regions that mediate memory and cognition, such as the hippocampus (1). A parallel but less consistent literature emerged from clinical studies suggesting that estrogen treatment (ET) is protective against cognitive decline in surgically and naturally menopausal women (2, 3). However, the Women's Health Initiative (WHI) studies published in 2003 not only failed to support such positive effects, but, in contrast, reported an increased risk of cognitive decline and dementia with hormone therapy (HT) (4, 5). Although the WHI results generated great concern in the clinical research and basic science communities, they also galvanized both groups to address conflicting reports on benefits vs. risks of HT and inconsistencies between the preclinical and clinical findings (6). Recent analyses of the clinical literature (2, 3, 7) have supported a hypothesis that was initially proposed by Gibbs based on animal studies (8); ET or HT has to be administered soon after estrogen depletion as a result of a “window of opportunity” during which estrogen could exert positive effects. Currently, there is strong support in both the basic science and clinical research communities for what is now referred to as the critical period hypothesis, which posits that ET or HT must be administered relatively soon after ovarian estrogen is depleted to be neuroprotective and exert positive effects on brain circuitry and cognitive function (7). Although there are extensive data supporting the critical period hypothesis, the cellular mechanism(s) underlying the decreased response to estrogen with time are poorly understood. The report by Brann and colleagues in PNAS (9) offers compelling evidence for a mechanism underlying the critical period that centers on sustaining sufficient levels of estrogen receptor (ER)-α. In addition, Brann and colleagues link the same mechanism to underlying events that occur with natural aging, which further extends the translational relevance of their findings (9).

Assessing the roles of presynaptic ryanodine receptors and adenosine receptors in caffeine-induced enhancement of hippocampal mossy fiber transmission

Caffeine robustly enhances transmitter release from the hippocampal mossy fiber terminals, although it remains uncertain whether calcium mobilization through presynaptic ryanodine receptors mediates this enhancement. In this study, we adopted a selective adenosine A1 blocker to assess relative contribution of A1 receptors and ryanodine receptors in caffeine-induced synaptic enhancement. Application of caffeine further enhanced transmission at the hippocampal mossy fiber synapse even after full blockade of adenosine A1 receptors. This result suggests that caffeine enhances mossy fiber synaptic transmission by two distinct presynaptic mechanisms, i.e., removal of A1 receptor-mediated tonic inhibition and ryanodine receptor-mediated calcium release from intracellular stores.

Local establishment of repetitive long-term potentiation-induced synaptic enhancement in cultured hippocampal slices with divided input pathways

Long-term potentiation (LTP) in the rodent hippocampus is a popular model for synaptic plasticity, which is considered the cellular basis for brain memory. Because most LTP analysis involves acutely prepared brain slices, however, the longevity of single LTP has not been well documented. Using stable hippocampal slice cultures for long-term examination, we previously found that single LTP disappeared within 1 day. In contrast, repeated induction of LTP led to the development of a distinct type of plasticity that lasted for more than 3 weeks and was accompanied by the formation of new synapses. Naming this novel plastic phenomenon repetitive LTP-induced synaptic enhancement (RISE), we proposed it as a model for the cellular processes involved in long-term memory formation. However, because in those experiments LTP was induced pharmacologically in the whole slice, it is not known whether RISE has input-pathway specificity, an essential property for memory. In this study, we divided the input pathway of CA1 pyramidal neurons by a knife cut and induced LTP three times, the third by tetanic stimulation in one of the divided pathways to express RISE specifically. Voltage-sensitive dye imaging and Golgi-staining performed 2 weeks after the three LTP inductions revealed both enhanced synaptic strength and increased dendritic spine density confined to the tetanized region. These results demonstrate that RISE is a feasible cellular model for long-term memory.

Acute and chronic effects of hypercalcaemia on cortical excitability as studied by 5 Hz repetitive transcranial magnetic stimulation

We designed the present study to disclose changes in cortical excitability in humans with hypercalcaemia, by delivering repetitive transcranial magnetic stimulation (rTMS) over the primary motor area (M1). In 22 patients with chronic hypercalcaemia related to primary hyperparathyroidism and 22 age-matched healthy subjects 5 Hz-rTMS was delivered at rest and during a sustained voluntary contraction of the target muscle. Changes in the resting motor threshold (RMT), motor evoked potential (MEP) amplitudes and cortical silent period (CSP) duration were measured and compared in patients and healthy controls. Two of the 22 patients were re-tested after parathyroidectomy when serum calcium had normalized. In a subgroup of healthy subjects, changes in the rTMS parameters were tested before and after acute hypercalcaemia. No significant difference between healthy normocalcaemic subjects and chronic hypercalcaemic patients was found in the RMT values and MEP amplitude and CSP duration evoked by the first stimulus of the trains. During the course of 5 Hz-rTMS trains, MEP size increased significantly less in patients with chronic hypercalcaemia than in healthy subjects, whereas the CSP duration lengthened to a similar extent in both groups. In the two patients studied after parathyroidectomy, rTMS elicited a normal MEP amplitude facilitation. Our findings indicate that acute hypercalcaemia significantly decreased the MEP amplitude facilitation. Given that 5 Hz-rTMS modulates cortical excitability through mechanisms resembling short-term synaptic enhancement, the reduction of MEP amplitude facilitation by hypercalcaemia may be related to Ca2+-dependent changes in synaptic plasticity.

Receptor saturation controls short-term synaptic plasticity at corticothalamic synapses

Glutamatergic synapses of layer 6 corticothalamic (CT) neurons form a major excitatory input onto thalamic relay cells, allowing neocortex to continuously control sensory information processing in thalamic circuits. CT synapses display both short- and long-term forms of use-dependent synaptic enhancement, mediated at least in part by increases in the probability of transmitter release. At some synapses, such increases in release probability are accompanied by a higher degree of multivesicular release (MVR) and larger glutamate transients at individual release sites, resulting in the saturation of postsynaptic receptors. The extent to which MVR and postsynaptic saturation interact and control short-term plasticity at CT synapses is not known. Here we examined two distinct presynaptic forms of short-term enhancement, facilitation and augmentation, at CT synapses contacting relay neurons in the ventrobasal nucleus of the mouse thalamus. We found that, in the presence of the low-affinity antagonist γ-D-glutamylglycine, to relieve postsynaptic DL-α-amino-3-hydroxy-5-methylisox azole-propionic acid (AMPA) receptor saturation, the magnitude of facilitation and augmentation increased. Whereas receptor saturation was prominent for both AMPA and N-methyl-D-aspartate receptors, desensitization of AMPA receptors did not significantly alter short-term plasticity. Our results suggest that at CT synapses the activity-dependent increase in synaptic strength is controlled by postsynaptic receptor saturation.

Excitatory synaptic activity is associated with a rapid structural plasticity of inhibitory synapses on hippocampal CA1 pyramidal cells

Synaptic activity, such as long-term potentiation (LTP), has been shown to induce morphological plasticity of excitatory synapses on dendritic spines through the spine head and postsynaptic density (PSD) enlargement and reorganization. Much less, however, is known about activity-induced morphological modifications of inhibitory synapses. Using an in vitro model of rat organotypic hippocampal slice cultures and electron microscopy, we studied activity-related morphological changes of somatic inhibitory inputs triggered by a brief oxygen–glucose deprivation (OGD) episode, a condition associated with a synaptic enhancement referred to as anoxic LTP and a structural remodeling of excitatory synapses. Three-dimensional reconstruction of inhibitory axo-somatic synapses at different times before and after brief OGD revealed important morphological changes. The PSD area significantly and markedly increased at synapses with large and complex PSDs, but not at synapses with simple, macular PSDs. Activity-related changes of PSD size and presynaptic bouton volume developed in a strongly correlated manner. Analyses of single and serial sections further showed that the density of inhibitory synaptic contacts on the cell soma did not change within 1 h after OGD. In contrast, the proportion of the cell surface covered with inhibitory PSDs, as well as the complexity of these PSDs significantly increased, with less macular PSDs and more complex, segmented shapes. Together, these data reveal a rapid activity-related restructuring of somatic inhibitory synapses characterized by an enlargement and increased complexity of inhibitory PSDs, providing a new mechanism for a quick adjustment of the excitatory–inhibitory balance. This article is part of a Special Issue entitled ‘Synaptic Plasticity & Interneurons’.

Dose-dependent effects of morphine on hippocampal seizure

Opiates have complex effects on seizure thresholds as these substances have both anti and proconvulsive actions in the mammalian brain. A reduction of inhibitory synaptic activity or enhancement of excitatory synaptic activity would be expected to trigger a seizure. This study is designed to determine how morphine and naloxone affect seizures induced by a low Mg2+ perfusate in the whole, intact hippocampus of mouse in vitro. In this research, C57BL/ 6 mice (P11-17) were used. Intact hippocampal structures were carefully dissected and incubated for at least 1 hour prior to recording. The tissues were perfused by low Mg2+ (0.2 mM)-ACSF at a rate of 3 ml/min. Morphine sulfate was used in various doses (10, 30, 100, 200 µM) as an opioid receptor agonist, while naloxone (5 and 10 µM) was utilized as an antagonist of these receptors. Various concentrations of morphine sulfate showed variable effects on whole hippocampus activity: Morphine (30 and 100 µM) noticeably elongated the length of epileptiform activity, while decreasing the frequency of the tonic stage of seizures. Morphine (200 µM) attenuated the hippocampal seizure activities. Low concentrations (5 µM) of naloxone did not show any significant effect on hippocampal activity, but higher concentrations of naloxone (10 µM) depressed both interictal and ictal like events. When combining perfusion with morphine (10, 30, 100, and 200) and naloxone (10 µM), both interictal and ictal-like events were depressed. Our results indicate that morphine in therapeutic doses of 30 µM may enhance seizure activity and its use in epileptic patients should be considered with caution. Conversely, naloxone has anticonvulsant actions.

The Muscarinic Long-Term Enhancement of NMDA and AMPA Receptor-Mediated Transmission at Schaffer Collateral Synapses Develop through Different Intracellular Mechanisms

We had described a muscarinic-mediated long-term synaptic enhancement at Schaffer collateral synapses caused by the insertion of AMPARs in spines of rat hippocampal CA1 pyramidal neurons that requires Ca(2+) release from IP3-sensitive stores (Fernández de Sevilla et al., 2008). We now show that this AMPA-mediated LTP(IP3) is precisely matched by an amplification of NMDAR-mediated transmission. The enhanced AMPAR transmission involves SNARE protein activity and CaMKII activation. The amplification of NMDA transmission requires combined CaMKII, PKC, and SRC kinase activity without detectable surface incorporation of NMDARs, suggesting that changes in receptor properties mediate this process. The enhanced AMPAR- and NMDAR-mediated transmission markedly reduce the induction threshold of "Hebbian" LTP. We conclude that both modes of glutamatergic synaptic potentiation may play a critical functional role in the regulation of the learning machinery of the brain by adding flexibility to the demands of the hippocampal network.

Analysis of gene expression changes associated with long‐lasting synaptic enhancement in hippocampal slice cultures after repetitive exposures to glutamate

We have previously shown that repetitive exposures to glutamate (100 muM, 3 min, three times at 24-hr intervals) induced a long-lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (for repetitive LTP-induced synaptic enhancement). To investigate the molecular mechanisms underlying RISE, we first analyzed the time course of gene expression changes between 4 hr and 12 days after repetitive stimulation using an original oligonucleotide microarray: "synaptoarray." The results demonstrated that changes in the expression of synapse-related genes were induced in two time phases, an early phase of 24-96 hr and a late phase of 6-12 days after the third stimulation. Comprehensive screening at 48 hr after the third stimulation using commercially available high-density microarrays provided candidate genes responsible for RISE. From real-time PCR analysis of these and related genes, two categories of genes were identified, 1) genes previously reported to be induced by physiological as well as epileptic activity (bdnf, grm5, rgs2, syt4, ania4/carp/dclk) and 2) genes involved in cofilin-based regulation of actin filament dynamics (ywhaz, ssh1l, pak4, limk1, cfl). In the first category, synaptotagmin 4 showed a third stimulation-specific up-regulation also at the protein level. Five genes in the second category were coordinately up-regulated by the second stimulation, resulting in a decrease in cofilin phosphorylation and an enhancement of actin filament dynamics. In contrast, after the third stimulation, they were differentially regulated to increase cofilin phosphorylation and enhance actin polymerization, which may be a key step leading to the establishment of RISE.

A Novel Postsynaptic Mechanism for Heterosynaptic Sharing of Short-Term Plasticity

Postsynaptic release of Ca(2+) from intracellular stores is an important means of cellular signaling that mediates numerous forms of synaptic plasticity. Previous studies have identified a postsynaptic intracellular Ca(2+) requirement for a form of short-term plasticity, post-tetanic potentiation (PTP) at sensory neuron (SN)-motor neuron synapses in Aplysia. Here, we show that postsynaptic IP(3)-mediated Ca(2+) release in response to a presynaptic tetanus in an SN that induces PTP can confer transient plasticity onto a neighboring SN synapse receiving subthreshold activation. This heterosynaptic sharing of plasticity represents a dynamic, short-term synaptic enhancement of synaptic inputs onto a common postsynaptic target. Heterosynaptic sharing is blocked by postsynaptic disruption of Ca(2+)- and IP(3)-mediated signaling, and, conversely, it is mimicked by postsynaptic injection of nonhydrolyzable IP(3), and by photolysis of caged IP(3) in the MN. The molecular mechanism for heterosynaptic sharing involves metabotropic glutamate receptors and Homer-dependent interactions, indicating that Homer can facilitate the integration of Ca(2+)-dependent plasticity at neighboring postsynaptic sites and provides a postsynaptic mechanism for the spread of plasticity induced by presynaptic activation. Our results support a model in which postsynaptic summation of IP(3) signals from suprathreshold and subthreshold inputs results in molecular coincidence detection that gives rise to a novel form of heterosynaptic plasticity.

Expression of GAP-43 during development and after monocular enucleation in the rat superior colliculus

The retinotectal projection of rodents presents a precise retinotopic organization that develops, from diffuse connections, from the day of birth to post-natal day 10. Previous data had demonstrated that these projections undergo reorganization after retinal lesions, nerve crush and monocular enucleation. The axonal growth seems to be directly related to growth-associated protein-43 (GAP-43) expression, a protein predominantly located in growth cones, which is regulated throughout development. GAP-43 is presented both under non-phosphorylated and phosphorylated (pGAP-43) forms. The phosphorylated form, has been associated to axon growth via polymerization of F-actin, and synaptic enhancement through neurotransmitter release facilitation. Herein we investigated the spatio-temporal expression of GAP-43 in the rat superior colliculus during normal development and after monocular enucleation in different stages of development. Lister Hooded rats ranging from post-natal day 0 to 70 were used for ontogeny studies. Another group of animals were submitted to monocular enucleation at post-natal day 10 (PND10) or PND21. After different survival-times, the animals were sacrificed and the brains processed for either immunohistochemistry or western blotting analysis. Our data show that GAP-43 is expressed in retinotectal axons in early stages of development but remains present in adulthood. Moreover, monocular enucleation leads to an increase in pGAP-43 expression in the deafferented colliculus. Taken together these results suggest a role for pGAP-43 in retinotectal morphological plasticity observed both during normal development and after monocular enucleation.

Critical Involvement of Postsynaptic Protein Kinase Activation in Long-Term Potentiation at Hippocampal Mossy Fiber Synapses on CA3 Interneurons

Hippocampal mossy fiber (MF) synapses on area CA3 lacunosum-moleculare (L-M) interneurons are capable of undergoing a Hebbian form of NMDA receptor (NMDAR)-independent long-term potentiation (LTP) induced by the same type of high-frequency stimulation (HFS) that induces LTP at MF synapses on pyramidal cells. LTP of MF input to L-M interneurons occurs only at synapses containing mostly calcium-impermeable (CI)-AMPA receptors (AMPARs). Here, we demonstrate that HFS-induced LTP at these MF-interneuron synapses requires postsynaptic activation of protein kinase A (PKA) and protein kinase C (PKC). Brief extracellular stimulation of PKA with forskolin (FSK) alone or in combination with 1-Methyl-3-isobutylxanthine (IBMX) induced a long-lasting synaptic enhancement at MF synapses predominantly containing CI-AMPARs. However, the FSK/IBMX-induced potentiation in cells loaded with the specific PKA inhibitor peptide PKI(6-22) failed to be maintained. Consistent with these data, delivery of HFS to MFs synapsing onto L-M interneurons loaded with PKI(6-22) induced posttetanic potentiation (PTP) but not LTP. Hippocampal sections stained for the catalytic subunit of PKA revealed abundant immunoreactivity in interneurons located in strata radiatum and L-M of area CA3. We also found that extracellular activation of PKC with phorbol 12,13-diacetate induced a pharmacological potentiation of the isolated CI-AMPAR component of the MF EPSP. However, HFS delivered to MF synapses on cells loaded with the PKC inhibitor chelerythrine exhibited PTP followed by a significant depression. Together, our data indicate that MF LTP in L-M interneurons at synapses containing primarily CI-AMPARs requires some of the same signaling cascades as does LTP of glutamatergic input to CA3 or CA1 pyramidal cells.