Antibodies to Postsynaptic PKC Substrate Neurogranin Prevent Long-term Potentiation in Hippocampal CA1 Neurons

Both pre- and postsynaptic protein kinase C have been implicated in long-term potentiation. Neurogranin (also known as BICKs and RC3) is a neuronal postsynaptic protein kinase C substrate. In the present study we injected monoclonal IgGs that recognize the protein kinase C phosphorylation site in neurogranin and B-50 (GAP-43), and that have been shown to inhibit protein kinase C-mediated B-50 phosphorylation, through a whole-cell clamp pipette into CA1 pyramidal neurons in rat hippocampal slices. Injection of neurogranin IgGs, but not of control IgGs, prevented the induction of tetanus-induced long-term potentiation without affecting posttetanic potentiation. Our results suggest that neurogranin is involved in mechanisms of activity-dependent synaptic plasticity.     

Antagonists of the Platelet-activating Factor Receptor Block Long-term Potentiation in Hippocampal Slices

The effects of antagonists and agonists of the platelet-activating factor (PAF) receptor on the formation of long-term potentiation (LTP) were examined in slices of rat hippocampus. The antagonist trans- BTD (trans-2,5-bis-(3,4,5-trimethoxyphenyl)-1,3-dioxolane) at concentrations of 8–16 μM blocked LTP in field CA1 while the same concentration of a stereo isomer ( cis -BTD) with low affinity for PAF receptors was without effect. CV3988, an antagonist structurally related to PAF, also attenuated LTP. The blockade of LTP by trans-BTD was partially reversed by simultaneous application of the non-metabolizable receptor agonist carbamyl-PAF. Trans-BTD did not change the following physiological measures: (i) paired-pulse facilitation, (ii) responses occurring during the short bursts given to induce LTP, (iii) N -methyl- d -aspartate receptor-mediated responses, and (iv) potentiation measured during the first minute after high-frequency stimulation. It thus appears that trans- BTD interferes with LTP at some step after induction and initial expression. These results suggest that activation of PAF receptors contributes to the stabilization of LTP, possibly via an effect on intracellular calcium levels.     

Characterization of Nerve Growth Factor (NGF) Release from Hippocampal Neurons: Evidence for a Constitutive and an Unconventional Sodium-dependent Regulated Pathway

We investigated the mechanism of neuronal nerve growth factor (NGF) release with regard to the potential function of NGF as a mediator of neuronal plasticity in the CNS. The analysis was performed in hippocampal slices and in primary cultures of hippocampal neurons, transiently transfected with an NGF cDNA construct to increase the level of NGF expression. In both systems there was activity-dependent NGF release initiated by high potassium (KCl), veratridine, glutamate or carbachol. Replacement of 90% of sodium in the medium with N -methyl-glucamine strongly reduced this release. The KCl- and veratridine-initiated NGF release was suppressed by tetrodotoxin; release by glutamate was less sensitive to tetrodotoxin but was sodium-dependent. The glutamate effect could be inhibited by GYK152644, an antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, but not by MK-801, an antagonist of NMDA receptors. The activity-dependent release of NGF did not depend on extracellular Ca²⁺, but was sensitive to the intracellular Ca²⁺ chelator bis-( o -aminophenoxy)-ethane- N,N,N',N' -tetraacetic acid tetra(acetoxymethyl)-ester, and to depletion of intracellular calcium stores. Conversely, mobilization of Ca²⁺ from intracellular stores with caffeine and thapsigargin mimicked the effect of depolarization. Basal NGF release could be reduced by either temperature block (15°C) or tetrodotoxin to ˜50%. The combination of both treatments reduced NGF release to below the detection limit, suggesting that basal release has constitutive and regulated components, the latter presumably resulting from spontaneous activity of interconnected neurons.     

A Role for Nitric Oxide in Long-term Potentiation

Nitric oxide production in the cerebellum and induction of long-term potentiation (LTP) in the hippocampus have some characteristics in common: both phenomena are induced by activation of N -methyl-D-aspartate receptors and both are highly dependent on calcium-mediated processes. Here we provide evidence that endogenous nitric oxide production is necessary for synaptic plasticity in the CA1 hippocampus of the rat. LTP recorded in slices was blocked in a concentration-dependent manner by the nitric oxide synthase inhibitors L- N ^G-nitroarginine and L- N ^G-nitroarginine methyl ester, but L- N ^G-monomethylarginine was only marginally active. Bathing the slices with haemoglobin, a protein that scavenges nitric oxide, also resulted in a concentration-dependent blockade of LTP. Nitric oxide released locally from hydroxylamine produced a stable potentiation of synaptic transmission that was not additive with LTP induced by high-frequency stimulation. These results are fully consistent with the presumed retrograde messenger role of nitric oxide in LTP.     

Maintenance of Modality-specific Connections in the Spinal Cord after Neonatal Nerve Growth Factor Deprivation

We have investigated the properties of antidromically identified lamina I neurons in the rat dorsal horn ( in vivo ) after neonatal administration of antibody to nerve growth factor (anti-NGF). Treatment from postnatal day (P) 2 to P9 yielded normal lamina I cell physiology; most cells responded to mechanical nociception and the remainder had a wide dynamic range (WDR). Extending anti-NGF treatment to P14 reduced the proportion of cells responding to mechanical nociception, increased the proportion of WDR cells, and caused the emergence of cells not driven by cutaneous inputs. Both nociceptive-specific and WDR cells had larger receptive fields, suggestive of enhanced central action of the remaining nociceptive afferents. These findings cannot be explained by direct action of anti-NGF on spinal cord neurons since both P2-9 and P2-14 treatments should have had similar effects given the time course of development of the blood-brain barrier. The results are discussed in terms of previous findings indicating normal numbers of D-hairs and high-threshold mechanoreceptors (HTMRs) after anti-NGF treatment from P2 to P9, but a decline in the number of HTMRs and an increase in the number of D-hairs after treatment from P2 to P14. It is suggested that the reduction in nociceptive neurons and the appearance of neurons not driven by cutaneous stimulation in lamina I results from the reduction in HTMR input. However, D-hair input to lamina I did not increase despite the larger number of these afferents, suggesting that their central action was regulated to maintain appropriate modality relationships between periphery and centre.     

The Immediate Early Gene Arc Is Not Required for Hippocampal Long-Term Potentiation

Memory consolidation is thought to occur through protein synthesis-dependent synaptic plasticity mechanisms such as long-term potentiation (LTP). Dynamic changes in gene expression and epigenetic modifications underlie the maintenance of LTP. Similar mechanisms may mediate the storage of memory. Key plasticity genes, such as the immediate early gene Arc, are induced by learning and by LTP induction. Mice that lack Arc have severe deficits in memory consolidation, and Arc has been implicated in numerous other forms of synaptic plasticity, including long-term depression and cell-to-cell signaling. Here, we take a comprehensive approach to determine if Arc is necessary for hippocampal LTP in male and female mice. Using a variety of Arc knock-out (KO) lines, we found that germline Arc KO mice show no deficits in CA1 LTP induced by high-frequency stimulation and enhanced LTP induced by theta-burst stimulation. Temporally restricting the removal of Arc to adult animals and spatially restricting it to the CA1 using Arc conditional KO mice did not have an effect on any form of LTP. Similarly, acute application of Arc antisense oligodeoxynucleotides had no effect on hippocampal CA1 LTP. Finally, the maintenance of in vivo LTP in the dentate gyrus of Arc KO mice was normal. We conclude that Arc is not necessary for hippocampal LTP and may mediate memory consolidation through alternative mechanisms.SIGNIFICANCE STATEMENT The immediate early gene Arc is critical for maintenance of long-term memory. How Arc mediates this process remains unclear, but it has been proposed to sustain Hebbian synaptic potentiation, which is a key component of memory encoding. This form of plasticity is modeled experimentally by induction of LTP, which increases Arc mRNA and protein expression. However, mechanistic data implicates Arc in the endocytosis of AMPA-type glutamate receptors and the weakening of synapses. Here, we took a comprehensive approach to determine if Arc is necessary for hippocampal LTP. We find that Arc is not required for LTP maintenance and may regulate memory storage through alternative mechanisms.     

Long-Lasting Enhancement of the Membrane-Associated Protein Kinase C Activity in the Hippocampal Kindled Rat

Abstract: We demonstrated that only membrane-associated protein kinase C (PKC) activity increased in the bilateral hippocampus (HIPP) up to 4 weeks and in the amyg-dala/pyriform cortex (AM/PC) at 4 weeks after the last kindled seizure. The enhancement of the membrane-associated PKC activity exceeds the increase in the protein concentration, which was observed in part. The overwhelming increase in the PKC activity should be of significance in the long-term maintenance of the kindling phenomenon.     

Failure to Induce Long-term Depression by an Anti-Correlation Procedure in Area CA1 of the Rat Hippocampal Slice

We have independently in our two laboratories re-examined the report by Stanton and Sejnowski ( Nature , 339 , 215 – 218, 1989) that single stimuli to a test pathway in area CA1 of the hippocampal slice, when delivered between short bursts of stimuli to a second, convergent pathway, produce an associative long-term depression (LTD) in the test pathway. While robust associative LTP was observed when stimuli to the two inputs were correlated in time, the anti-correlation procedure failed to induce LTD; rather, a trend towards potentiation was observed. This result was obtained using both submerged and interface chambers, and in two different strains of rat. A transient depression lasting for a few minutes could usually be elicited by strong tetanic stimulation; this depression was not restricted to activated pathways.     

Membrane Arachidonic Acid Concentration Correlates with Age and Induction of Long-term Potentiation in the Dentate Gyrus in the Rat

We examined the induction and maintenance of long-term potentiation (LTP) in vivo in the dentate gyrus of 4-month-old and 22-month-old urethane-anaesthetized rats. High-frequency stimulation of the perforant path induced an immediate increase in the slope of the population excitatory postsynaptic potential (EPSP), which was sustained in the 4-month-old animals for the duration of the experiment (45 min post-tetanus). In the 22-month-old group, the mean slope of the population EPSP decreased almost to baseline by the end of the experiment. Examination of the individual records indicated that LTP was sustained for the duration of the experiment in half of the 22-month-old animals, while in the others only post-tetanic potentiation was observed. Membrane arachidonic acid concentration was reduced in aged compared with young animals and was lowest in the subgroup of aged animals which failed to sustain LTP. Potassium-stimulated, calcium-dependent release of glutamate was also decreased in aged compared with young animals, but LTP was associated with an increase in glutamate release in the 4-month-old group and 22-month-old subgroup in which LTP was successfully sustained; no change was observed in the 22-month-old group in which LTP was not sustained. The results indicate a correlation between membrane arachidonic acid concentration, glutamate release and ability to sustain LTP in aged animals.     

The Relative Contribution of NMDA Receptor Channels in the Expression of Long-term Potentiation in the Hippocampal CA1 Region

Long-term potentiation (LTP) was studied in the hippocampal CA1 region of guinea-pigs using a solution containing 0.1 mM magnesium and 10 μM of the non- N -methyl- d -aspartate (non-NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), leaving an NMDA-mediated field excitatory postsynaptic potential (EPSP). Brief high-frequency afferent tetanization induced a substantial synapse-specific potentiation of the NMDA EPSP with a time course closely resembling that previously described for LTP of the non-NMDA-mediated EPSP. This NMDA EPSP potentiation was occluded by prior induction of LTP in normal solution. Using a solution containing 0.1 mM magnesium and 1 μM CNQX, the EPSP was composed of both a non-NMDA- and an NMDA-mediated component which could be measured separately and in parallel. Manipulations that cause increased transmitter release, such as phorbol ester application and changes in stimulation frequency, enhanced the two measures nearly equally. Afferent tetanization induced an increase of both EPSP components, with a similar time course, the NMDA component showing a relative increase of about one-third of that of the non-NMDA one. These results suggest that, to the extent that LTP is based on an increased release of transmitter, the mechanism exhibits features distinct from those underlying other forms of enhanced release.     

Basic Fibroblast Growth Factor Opens Calcium-Permeable Channels in Quail Mesencephalic Neural Crest Neurons

In order to investigate the action of basic fibroblast growth factor (bFGF) in the nervous system, we have studied the ionic signals elicited by this peptide in cultured quail mesencephalic neural crest neurons using patch-clamp and cytofluorimetric techniques. In this preparation stimulation with bFGF induced, with a delay of some tens of seconds, an inward cationic current. Single-channel experiments provided evidence for the activation of a calcium-permeable channel. In single-cell cytofluorimetric measurements, a sustained rise in [Ca²⁺Ii was observed, which was dependent on the presence of external calcium. These events may play a role in the developmental effects of bFGF.     

The NMDA Receptor Antagonist CPP Suppresses Long-term Potentiation in the Rat Hippocampal — accumbens Pathway In Vivo

Excitation of afferent fibres originating in the ventral subiculum of the hippocampus through stimulation of the fimbria elicits field potentials in the nucleus accumbens. When recorded in the dorsomedial aspect of the nucleus accumbens, the evoked field responses consisted of an early, negative-going component (Nl) with a peak latency of 8–10 ms, followed by a second negative-going peak (N2) with a latency of 22–24 ms. The N1 response reflects monosynaptic activation of nucleus accumbens neurons; the N2 component appears to be polysynaptic in origin. In control rats, high-frequency stimulation of the fimbria (three trains at 250 Hz, 250 ms, delivered at 50 min intervals) resulted in a long-lasting potentiation of both the N1 and N2 components. The magnitude of potentiation exhibited by the polysynaptic N2 response was typically greater than that of the monosynaptically evoked N1 response. Following delivery of the first train, the amplitude of the N1 and N2 components was increased by -20 and 50% respectively. Administration of the competitive N-methyl-d -aspartate (NMDA) receptor antagonist 3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP, 10 mg/kg i.p.) had no significant effects on the evoked nucleus accumbens responses. High-frequency stimulation failed to produce a significant increase in the amplitude of either the N1 or the N2 response when delivered 45–60 min after CPP administration. To test whether the suppressant effects of CPP were time-dependent, two further high-frequency trains were applied 90 and 180 min after administration of the drug. Significant increases in the amplitude of the N1 and N2 components were observed only after the third train, delivered 180 min after CPP injection. These results demonstrate that high-frequency stimulation of hippocampal afferents to the nucleus accumbens induces LTP in both a monosynaptic and a polysynaptic pathway. In both cases, the induction of LTP is suppressed in a time-dependent manner by the competitive NMDA receptor antagonist CPP. Thus, NMDA receptor activation appears to be prerequisite for the induction of LTP in the hippocampus - accumbens pathway.     

移植齿状回颗粒细胞重建海马神经网络的方法研究

目的:探讨新生大鼠齿状回颗粒细胞在培养的海马组织切片上迁移特征。方法:从生后3d的绿色荧光蛋白(GFP)基因导入的SD大鼠海马组织中分离颗粒细胞层组织,分别移植到生后7d的宿主(野生型)SD大鼠海马组织切片的不同区域,施行共同培养6d,观察表达GFP的齿状回颗粒细胞的迁移方向和纤维投射。结果:当移植物移植到齿状回门区附近时,表达GFP的齿状回颗粒细胞迁移到宿主海马组织的齿状回门区和颗粒细胞层,并发出树突和轴突分别投射到齿状回的内分子层和CA3区。结论:移植的齿状回颗粒细胞在海马组织切片上迁移并发出神经纤维投射到CA3区,提示移植的齿状回颗粒细胞可能参与海马的局部神经网络。     

Positive Feedback between Acetylcholine and the Neurotrophins Nerve Growth Factor and Brain-derived Neurotrophic Factor in the Rat Hippocampus

In the rat hippocampus, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are synthesized by neurons in an activity-dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up-regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high-affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.     

Long-term Potentiation and Field EPSPs in the Lateral and Medial Perforant Paths in the Dentate Gyrus In Vitro: a Comparison

The entorhinal cortex projects monosynaptically to the granule cells in the dentate gyrus via the lateral and medial perforant paths. These two subdivisions of the perforant path differ with respect to synaptic properties, and recent studies suggest that they also differ with respect to long-term potentiation (LTP). In the present study, using the in vitro slice preparation of the guinea-pig hippocampus, field excitatory postsynaptic potentials (EPSPs) and LTP in the lateral and medial perforant paths were compared. The two pathways were distinguished on the basis of their different termination in the dendritic layer, their different pharmacology and short-term synaptic facilitation. The field EPSP [obtained in the presence of γ-aminobutyric acid (GABA) A and B receptor antagonists] consisted of a non- N -methyl- d -aspartate (NMDA) component with different time characteristics in the two pathways, the decay being monoexponential in the lateral perforant path and biexponential in the medial one. In addition, the field EPSP in both pathways contained a small NMDA-mediated component that could also be observed after complete blockade of the non-NMDA one. LTP induction in both lateral and medial perforant paths was facilitated by blockade of GABA_A inhibition, showed associative properties, and was blocked by NMDA receptor antagonists. Following the induction event, LTP in both pathways developed to a peak value within 30–40 s, and the stability of LTP was correlated with the amount of postsynaptic, but not presynaptic, activity during the induction event. During blockade of GABA_A inhibition the opioid receptor antagonist naloxone and the β-adrenergic antagonist timolol had no effect on the magnitude or stability of LTP. It is concluded that LTP in the lateral and medial perforant paths does not differ with respect to induction mechanisms and early temporal characteristics.     

Time-related Changes in Basal Phosphoinositide Turnover after Induction of Long-term Potentiation in the Dentate Gyrus are Blocked by Commissural Stimulation

We have examined basal phosphoinositide turnover in synaptosomes obtained from the dentate gyrus of anaesthetized rats in which long-term potentiation was induced unilaterally in perforant path-granule cell synapses. Relative to the unpotentiated side, [3H]myo-inositol labelling of inositol phosphates was significantly enhanced 45 min and 3 h after induction of long-term potentiation, but reduced after 2.5 min. Similarly, [14C]arachidonic acid labelling of 1,2-diacylglycerol was increased 45 min and 3 h after induction of long-term potentiation, but reduced after 2.5 min. In a second series of experiments, induction of long-term potentiation was blocked by stimulation of the commissural projection to granule cells. In synaptosomes prepared from this tissue, there was no difference in phosphoinositide turnover between tetanized and control sides at any of the three post-tetanic intervals. We conclude that in the dentate gyrus, long-term potentiation is associated with an increase in phosphoinositide turnover which is established between 2.5 min and 45 min post-tetanus and which persists for at least 3 h.     

Hippocampal unit-behavior correlations during classical conditioning

The correspondence that develops over the course of classical conditioning between the temporal distribution of increased unit activity in the rabbit hippocampus and the amplitude-time distribution of the behavioral nictitating membrane response is analyzed. Results reveal a high degree of correspondence between neural and behavioral measures. The real time correlation between the within-trial probability of increased hippocampal unit discharge and amplitude-time course of the nictitating membrane response grows substantially with learning. Further analyses reveal that this apparent increase in correlation results from a growth in amount of hippocampal unit activity per se (i.e., a differentiation of the hippocampal unit response from background firing rates), rather than an increase in the correspondence between cellular and behavioral measures (i.e. a repatterning of hippocampal discharges to more accurately code spatio-temporal aspects of the behavioral response). These and other results indicate that the neuronal ‘temporal model’ of the behavioral response either develops within the hippocampus from the first few conditioning trials or develops first in entorhinal cortex to subsequently influence hippocampal discharge patterns. On the other hand, the increase in amount of hippocampal unit activity developing with conditioning appears to occur within the hippocampus.     

Nerve Growth Factor and Basic Fibroblast Growth Factor Protect Cholinergic Neurons Against Quinolinic Acid Excitotoxicity in Rat Neostriatum

In the present work we have characterized a possible mechanism leading to the early survival of neostriatal cholinergic neurons after quinolinic acid injection. Different doses of quinolinic acid were injected in rat neostriatum and two different parameters were analysed 7 days after the lesion: choline acetyltransferase (ChAT) activity and nerve growth factor (NGF) levels. We have observed that ChAT activity decreased (until 68 nmol quinolinic acid) and NGF levels increased (until 34 nmol quinolinic acid) in a dose-dependent manner. In order to characterize the time-course of the lesion on NGF levels and ChAT activity, and the possible protective effect of NGF and basic fibroblast growth factor (bFGF) on cholinergic neurons, we have used the quinolinic acid dose (68 nmol) at which the first decrease of ChAT activity was observed. ChAT activity and NGF levels showed different patterns of response to quinolinic acid injection, since the maximal effect was reached at 1 day for ChAT activity and at 2 days for NGF levels. NGF or bFGF simultaneously injected with quinolinic acid (68 nmol) completely prevented the decrease in ChAT activity in a dose-dependent manner but NGF was more effective than bFGF. Furthermore, differences observed in ChAT activity after NGF but not bFGF treatment were correlated with changes in the number of ChAT immunoreactive cells. Finally, we have also observed that, although bFGF alone was not able to modify NGF levels, bFGF simultaneously injected with quinolinic acid produced an increase of NGF levels higher than that observed after quinolinic acid injection alone. Our results show that NGF and bFGF protect striatal cholinergic neurons against quinolinic acid injury, and bFGF is able to potentiate the increase of NGF after the lesion, suggesting a cooperative action between different trophic factors in neuronal protection after excitotoxic injury. Thus, administration of trophic factors may be relevant in the prevention and treatment of neurodegenerative disorders, such as Huntington's disease.     

NMDA Receptor-dependent Transient Homo- and Heterosynaptic Depression in Picrotoxin-treated Hippocampal Slices

Extracellular recording was used to study the effects of high-frequency (tetanic) stimulation on excitatory synaptic transmission in the CA1 region of rat hippocampal slices in the presence of the _γ-aminobutyric acid (GABA) type A antagonist, picrotoxin (50 _γM). Under these conditions tetanic stimulation (100 Hz, 1 s) at the test intensity resulted in homosynaptic long-term potentiation (LTP). In contrast, tetanic stimulation of higher intensity (100 Hz, 1 s, double test intensity) resulted in homo- and heterosynaptic depression which recovered within 45 min. A transient (1–3 min) negative shift in DC potential and a transient (5–10 min) depression of the homosynaptic fibre volley occurred immediately following the higher intensity tetanus. The DC shift, induction of homo- and heterosynaptic depression and depression of the fibre volley were reversibly prevented by the N -methyl- d -aspartate (NMDA) receptor antagonist, d -2-amino-5-phosphonopentanoate (AP5; 20 _γM) but were not prevented by a variety of L-type calcium channel antagonists. Transient (30 - 45 min) synaptic depression of pharmacologically isolated NMDA receptor-mediated field excitatory postsynaptic potentials also occurred following tetanic stimulation (100 Hz, 1 s) at double test intensity. These results demonstrate an NMDA receptor-dependent form of reversible synaptic depression in the CA1 region of the hippocampus.