Norepinephrine stimulation of adenylate cyclase potentiates protein kinase C action: electrophysiological studies in the dentate gyrus

Protein kinase C and norepinephrine-stimulated adenylate cyclase have been linked to formation of long-term potentiation in the dentate gyrus. However, little is known about second messenger system interactions regulating synaptic transmission. In electrophysiological studies of the dentate gyrus, we observe synergistic interactions between norepinephrine and phorbol esters, activators of protein kinase C. Norepinephrine markedly potentiates the block of adenosine's inhibitory action by phorbol esters. Norepinephrine's action is mimicked by the beta-adrenoceptor agonist isoproterenol and blocked by the beta-adrenoceptor antagonists timolol and propranolol. Forskolin also mimicks norepinephrine's action. Accordingly, norepinephrine's potentiation of protein kinase C appears to be mediated by the cyclic AMP second messenger system. This mechanism may contribute to norepinephrine's facilitation of long-term potentiation in the dentate gyrus.     

Running induces widespread structural alterations in the hippocampus and entorhinal cortex

Physical activity enhances hippocampal function but its effects on neuronal structure remain relatively unexplored outside of the dentate gyrus. Using Golgi impregnation and the lipophilic tracer DiI, we show that long-term voluntary running increases the density of dendritic spines in the entorhinal cortex and hippocampus of adult rats. Exercise was associated with increased dendritic spine density not only in granule neurons of the dentate gyrus, but also in CA1 pyramidal neurons, and in layer III pyramidal neurons of the entorhinal cortex. In the CA1 region, changes in dendritic spine density are accompanied by changes in dendritic arborization and alterations in the morphology of individual spines. These findings suggest that physical activity exerts pervasive effects on neuronal morphology in the hippocampus and one of its afferent populations. These structural changes may contribute to running-induced changes in cognitive function.     

Expression of long-term potentiation in aged rats involves perforated synapses but dendritic spine branching results from high-frequency stimulation alone

Evidence for morphological substrates of long-term changes in synaptic efficacy is controversial, partly because it is difficult to employ an unambiguous control. We have used a high-frequency stimulation protocol in vivo to induce long-term potentiation (LTP) in the hippocampal dentate gyrus of aged (22-month-old) rats and have found a clear distinction between animals that sustain LTP and those that fail to sustain it. The "failure group" was used as a specific/"like-with-like" control for morphological changes associated with the expression of LTP per se. Quantitative optical and electron microscopy was used to analyze large populations of dendritic spines and excitatory perforant path synapses; LTP was found to be associated with an increase in numbers of segmented (perforated) postsynaptic densities in spine synapses. In contrast, an increase in the number of branched spines appears to result from high-frequency stimulation alone. These data shed light on the current controversy about the expression mechanism of LTP.     

Impairment of in vivo theta-burst long-term potentiation and network excitability in the dentate gyrus of synaptopodin-deficient mice lacking the spine apparatus and the cisternal organelle

The function of the spine apparatus in dendritic spines and the cisternal organelles in axon initial segments is little understood. The actin-associated protein, synaptopodin, is essential for the formation of these organelles which are absent in synaptopodin -/- mice. Here, we used synaptopodin -/- mice to explore the role of the spine apparatus and the cisternal organelle in synaptic plasticity and local circuit excitability in response to activation of the perforant path input to the dentate gyrus in vivo. We found impaired long-term potentiation following theta-burst stimulation, whereas tetanus-evoked LTP was unaffected. Furthermore, paired-pulse inhibition of the population spike was reduced and granule cell excitability was enhanced in mutants, hence revealing an impairment of local network inhibition. In summary, our data represent the first electrophysiological evidence that the lack of the spine apparatus and the cisternal organelle leads to a defect in long-term synaptic plasticity and alterations in local circuit control of granule cell excitability under adult in vivo conditions.     

Long-term potentiation in the rat dentate gyrus is associated with enhanced Arc/Arg3.1 protein expression in spines, dendrites and glia

Electron microscopic immunocytochemical methods were used to determine the localization, subcellular distribution and expression of activity-regulated cytoskeletal protein (Arc/Arg3.1) in dentate gyrus after unilateral induction of long-term potentiation (LTP) in the perforant pathway of anaesthetized rats. At 2 h post-induction, immunoreaction product was visible in the dentate gyrus in both the granule cell and molecular layers. Arc expression was higher in the potentiated than the unstimulated contralateral hemisphere. Single-section electron microscopy analysis in unstimulated tissue and in tissue prepared 2 and 4 h after LTP induction showed Arc immunoreactivity (Arc-IR) in dendrites, dendritic spines and glia. Arc-IR was associated with synaptic and non-synaptic plasma membrane apposed to axon terminals and with cytoplasmic organelles, including the cytoskeleton. Arc-IR was also present in neuronal perikarya and there was occasional labelling of nuclei and axons. At 2 h post-LTP induction, there were significant increases in Arc-IR within the granule cell and molecular layers of the dentate gyrus and particularly within the middle molecular layer relative to the inner and outer molecular layers. This increase in Arc expression 2 h after LTP induction was blocked by the N-methyl-D-aspartate receptor antagonist (RS)-3-2-carboxypiperazin-4-yl-propyl-1-phosphonic acid. In animals killed 4 h after LTP induction, Arc expression had declined and differences between the potentiated and unpotentiated hemispheres were no longer significant. Our data provide ultrastructural evidence for a transient LTP-associated increase in the expression of Arc protein in the middle molecular layer of the dentate gyrus, with preferential targeting to dendrites, dendritic spines and glia.     

Ultrastructural evidence for presynaptic mu opioid receptor modulation of synaptic plasticity in NMDA-receptor-containing dendrites in the dentate gyrus

Physiological studies have demonstrated that long-term potentiation (LTP) induction in N-methyl-D-aspartate (NMDA) receptor containing dentate granule cells following lateral perforant path stimulation is opioid dependent, involving mu-opioid receptors (MORs) on gamma-aminobutyric acid (GABA)-ergic neurons. To determine the cellular relationships of MORs to postsynaptic NMDA receptor-containing dendrites, immunoreactivity (-I) against MOR and the NMDA receptor subunit 1 (NMDAR1) was examined in the outer molecular layer of the dentate gyrus using electron microscopy. MOR-I was predominantly in axons and axon terminals. NMDAR1-I was almost exclusively in spiny dendrites, but was also in a few terminals. Using immunogold particles to localize precisely NMDAR1, one-third of the NMDAR1-I was detected on the dendritic plasmalemma; in dendritic spines plasmalemmal immunogold particles were near synaptic densities. Many MOR-labeled axons and terminals contacted NMDAR1-labeled dendrites. MOR-labeled terminals formed symmetric (inhibitory-type) synapses on NMDAR1-labeled dendritic shafts or nonsynaptically contacted NMDAR1-labeled shafts and spines. MOR-labeled axons often abutted NMDAR1-containing dendritic spines which received asymmetric (excitatory-type) synapses from unlabeled terminals. Occasionally, MOR-labeled terminals and dendrites were apposed to NMDAR1-containing terminals. These results provide anatomical evidence that endogenous enkephalins or exogenous opioid agonists could inhibit GABAergic terminals that modulate granule cell dendrites, thus boosting depolarizing events in granule cells and facilitating the activation of NMDA receptors located on their dendrites.     

High-frequency discharge of dentate granule cells, but not long-term potentiation, induces c-fos protein

Competence genes, such as c-fos, may play key roles in information storage in the nervous system by linking relatively brief extracellular signals to long-term changes in the neuron. In support of this idea we, and others, have shown that the c-fos protein occurs in adult mammalian neurons and that higher levels of the protein are induced in certain brain regions after kindled or metrazol-induced seizures in mice and rats, sensory stimulation and mechanical damage in spinal cord neurons, and after depolarization in PC12 cells. Here we report that a massive induction of c-fos protein is observed in dentate granule cells in four conditions that result in repetitive firing: localized seizure discharges; high frequency antidromic activation; orthodromic activation in the presence of iontophoresed bicuculline; and frequency potentiation. However, stimulation of the perforant path with high frequency trains that produced long-term potentiation at the perforant path-granule cell synapse did not reliably induce c-fos in the dentate gyrus. These findings suggest that c-fos induction can follow repetitive neuronal discharge but is not involved in long-term potentiation.     

Norepinephrine regulates long-term potentiation of both the population spike and dendritic EPSP in hippocampal dentate gyrus

Hippocampal slices from norepinephrine (NE)-depleted rats exhibited marked reductions in long-term potentiation (LTP) of both the population spike and dendritic EPSP in the dentate gyrus. In contrast, depletion of serotonin (5-hydroxytryptamine, 5-HT) had no effect on either population spike or EPSP-LTP. In addition, superfusion of slices with NE produced potentiation of both the granule cell population spike and dendritic EPSP which persisted long after NE washout. These data support a role for NE in regulating long-term plasticity of both granule cell action potential firing and dendritic EPSPs.     

Protein kinase C and ERK involvement in dendritic spine plasticity in cultured rodent hippocampal neurons

The roles of protein kinase C and the MAP-kinase extracellular receptor kinase in structural changes associated with long-term potentiation of network activity were examined in cultured hippocampal neurons. A brief exposure to a conditioning medium that favours activation of the N-methyl-d-aspartate receptor caused a rapid and specific increase in staining of neurons for the phosphorylated form of extracellular receptor kinase as well as of cyclic AMP response element binding protein. Exposure of the cultures to the conditioning medium was followed by a protein synthesis-dependent formation of novel dendritic spines. An extracellular receptor kinase antagonist PD98059 blocked the phosphorylated form of extracellular receptor kinase response and the formation of novel spines. A selective protein kinase C agonist, phorbol 12-myristate 13-acetate, caused activation of extracellular receptor kinase and formation of novel spines. The protein kinase C antagonist GF109203x blocked the phosphorylated form of extracellular receptor kinase response and the subsequent spine formation by phorbol 12-myristate 13-acetate. Both the conditioning medium and phorbol 12-myristate 13-acetate caused a delayed increase in mean amplitude of miniature excitatory postsynaptic currents recorded in the hippocampal neurons. These results indicate that activation of extracellular receptor kinase mediates the effect of a conditioning protocol on the formation of dendritic spines. The formation of novel spines was associated with long-term increase in network activity and functional synaptic connectivity among the cultured neurons.     

Long lasting changes in the spontaneous activity of hippocampal neurons following stimulation of the entorhinal cortex

The spontaneous activity of hippocampal and denate units was investigated in acute and chronic experiments on adult rats. Spontaneous unitary discharges from both regions were measured before and after brief tetanic stimulation of the entorhinal cortex. An increase in the frequency of spontaneous unit activity was obtained in units recorded from the dentate gyrus and CA3 field of the hippocampus for up to 20 min following stimulation. In chronic recordings of physiologically identified dentate units from freely moving rats similar results were obtained. The findings are discussed with reference to long lasting potentiation of synaptic activity in the perforant path-dentate granule cell connection.     

Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus

Eph receptors and their ephrin ligands assume various roles during central nervous system development. Several of these proteins are also expressed in the mature brain, and notably in the hippocampus, where EphA4 and ephrins have been shown to influence dendritic spine morphology and long-term potentiation (LTP). To examine the cellular and subcellular localization of EphA4 in adult rat ventral hippocampus, we used light and electron microscopic immunocytochemistry with a specific polyclonal antibody against EphA4. After immunoperoxidase labeling, EphA4 immunoreactivity was found to be enriched in the neuropil layers of CA1, CA3, and dentate gyrus. In all examined layers of these regions, myelinated axons, small astrocytic leaflets, unmyelinated axons, dendritic spines, and axon terminals were immunolabeled in increasing order of frequency. Neuronal cell bodies and dendritic branches were immunonegative. EphA4-labeled dendritic spines and axon terminals corresponded to 9-19% and 25-40% of the total number of spines and axon terminals, respectively. Most labeled spines were innervated by unlabeled terminals, but synaptic contacts between two labeled elements were seen. The vast majority of synaptic junctions made by labeled elements was asymmetrical and displayed features of excitatory synapses. Immunogold labeling of EphA4 was located mostly on the plasma membrane of axons, dendritic spines, and axon terminals, supporting its availability for surface interactions with ephrins. The dual preferential labeling of EphA4 on pre- or postsynaptic specializations of excitatory synapses in adult rat hippocampus is consistent with roles for this receptor in synaptic plasticity and LTP.     

Activation of latent precursors in the hippocampus is dependent on long-term potentiation

The recent discovery of a large latent population of precursor cells in the dentate gyrus of adult mice led us to investigate whether activation of this population is regulated by synaptic activity, thereby explaining the observation that environmental signals can affect neurogenesis. Using a variety of stimulation protocols, we found that only a long-term potentiation (LTP)-inducing protocol activated the latent precursor pool, leading to increased neurogenesis in the dentate gyrus. LTP induced by high-frequency stimulation (HFS) of the perforant pathway in vivo produced a two-fold increase in the number of neurospheres cultured from the stimulated hippocampus, compared with the unstimulated hippocampus. No increase in neurosphere number or neurogenesis was observed when the HFS failed to induce LTP. These results show that LTP can activate latent neural precursor cells in the adult mouse dentate gyrus, thereby providing a direct mechanism for regulating activity-driven neurogenesis. In the future, it may be possible to utilize such learning- or stimulation-induced neurogenesis to overcome disorders characterized by neuronal loss.     

Regional- and age-dependent reduction in trkB receptor expression in the hippocampus is associated with altered spine morphologies.

BACKGROUND: Changes in densities and in the morphology of dendritic spines in the hippocampus are linked to hippocampal long-term potentiation (LTP), spatial learning, and depression. Decreased brain-derived neurotrophic factor (BDNF) levels seem to contribute to depression. Through its receptor trkB, BDNF is also involved in hippocampal LTP and hippocampus-dependent learning. Conditionally gene-targeted mice in which the ablation of trkB is restricted to the forebrain and occurs only during postnatal development display impaired learning and LTP. METHODS: To examine whether there is a link among impaired hippocampal synaptic plasticity, altered spines, and trkB receptors, we performed a quantitative analysis of spine densities and spine length in the hippocampal area CA1 and the dentate gyrus in conditional mutant mice (trkB(lox/lox)CaMKII-CRE mice). TrkB protein and mRNA levels were assayed using Western blot and in situ hybridization analysis. RESULTS: Fifteen-week-old mutant mice exhibit specific reductions in spine densities and a significant increase in spine length of apical and basal dendrites in area CA1. These alterations correlate with a time- and region-specific reduction in full-length trkB mRNA in the hippocampus. CONCLUSIONS: TrkB functions in structural remodeling of hippocampal dendritic spines, which in turn may affect synaptic transmission and plasticity.     

Effects of protein undernutrition on the dentate gyrus in rats of three age groups

The effect of an 8% casein and a control 25% casein diet on the granule cells in the dorsal blade of the dentate gyrus of the rat hippocampal formation was studied at 30, 90 and 220 days of age. Female rats were fed either an 8% or 25% casein diet 5 weeks prior to conception and the litters were maintained on these respective diets until killed. In rapid-Golgi-impregnated cells, we measured major and minor axes of the soma of the dentate granule neurons, the number of spines on 50-microns segments of proximal, middle and terminal regions of the largest dendrite per granule cell and the number of dendrites intersecting 8 concentric rings 38 microns apart. At all 3 ages studied undernourished rats showed, when compared to controls, significant reductions of the major and minor axes of the somata and significant reductions in the number of spines on dendrites in the middle and terminal dendritic segments. Dendritic branching was significantly reduced in undernourished rats compared to controls in all but the 4th concentric rings, with the greatest effect being seen on the outer 3 concentric rings at 90 and 220 days of age. The location of the deficit in dendritic synaptic spines and the greatest deficit in dendritic branching correspond to the sites of termination of the lateral and medial perforant pathway projection to the dentate gyrus on the terminal and middle dendritic segments of the granule cells. The deficits noted in the granule cells of the dentate gyrus in this study were more severe than those found in our previous studies on the effect of the low protein diet in these same rats on visual cortical pyramidal cells and on the 3 cell types in the nucleus raphe dorsalis and nucleus locus coeruleus.     

Long-term potentiation in intact infant rat hippocampus

In rats 14-28 days of age, high-frequency stimulation of the perforant path granule cell synapse in the dentate gyrus produced long-term potentiation of the population spike that was comparable in magnitude (150-250% of baseline) and duration (120 min) to that produced in adult animals with the same stimulation paradigm. In contrast, potentiation of the excitatory postsynaptic potential occurred inconsistently.     

Granule cell loss and dendritic regrowth in the hippocampal dentate gyrus of the rat after chronic alcohol consumption

The effects of chronic alcohol consumption (CAC) on the relative number of dentate gyrus granule cells and their dendritic trees, were studied in animals fed alcohol for 6, 12 and 18 months and in their respective controls. The granule cell density was estimated with the unbiased disector method. Following 6 months of alcohol consumption, the thickness of the dentate gyrus granular layer and the relative number of dentate granule cells were significantly decreased when compared with controls. The granule cell dendritic arborizations showed an increase of their dendritic extent in alcohol-treated rats. No significant differences were found in the density of dendritic spines between alcohol-fed and control animals. These results indicate the existence of hippocampal granule cell dendritic regrowth in alcohol-fed rats, probably occurring as a compensatory response to the granule cell deficit which follows the alcohol-induced granule cell degeneration. These degenerative and regenerative changes might have functional implications for the organization of the synaptic hippocampal circuitry after long periods of alcohol consumption.     

Impairment of long-term potentiation and paired-pulse facilitation in rat hippocampal dentate gyrus following developmental lead exposure in vivo

Neonatal rats were exposed to lead from parturition to weaning via the milk of dams drinking 0.2% lead acetate solution. The alterations of long-term potentiation (LTP) and paired-pulse facilitation (PPF) of hippocampal dentate gyrus in adult rats (90–115 days) following developmental lead exposure were studied in vivo. Input/output (I/O) function, paired-pulse facilitation (PPF), excitatory postsynaptic potential (EPSP) and population spike (PS) amplitude were measured in the dentate gyrus (DG) in response to stimulation applied to the lateral perforant path. The results showed that LTP was induced in control rats with an average PS potentiation of 321.1±50.0% (n=18), which was significantly greater than the increase in PS potentiation (173.5±30.0%, n=17, p<0.001) in lead-exposed rats after tetanizing stimulation. The mean EPSP potentiation increased to 172.4±27.0% (n=18) in control and 138.8±21.4% (n=17) in lead-exposed rats after tetanizing stimulation. The lead-induced impairment of LTP of PS potentiation was more serious than that of EPSP potentiation. Following pairs stimulation of perforant fiber at 250 μA and an interpulse interval (IPI) of 10–1000 ms, the average peak facilitation of PS was 211.3±25.0% (n=13) in control and 187.7±23.0% (n=11) in lead-exposed rats. The average facilitation period duration of PS was 243.0±35.8 ms (n=13) in control and 138.0±24.4 ms (n=11) in lead-exposed rats. These results suggested that developmental lead exposure in neonatal rats caused impairments in LTP and PPF of hippocampal dentate gyrus.     

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.