Development Of Long-term Potentiation Research Articles

An Optogenetic Approach to Studies of the Mechanisms of Heterosynaptic Plasticity in Neocortical Neurons

Heterosynaptic plasticity is a poorly studied type of synaptic plasticity in which synapses not active during the plasticity induction process are modified. The main methodological problem in studying the mechanisms of heterosynaptic plasticity using conventional techniques are the need for large numbers of experiments because of the probabilistic nature of the development of plastic changes in the synaptic inputs to neurons after intracellular tetanization – the most widely used approach to inducing heterosynaptic plasticity. To overcome this problem, we report here use of a method of delivering optogenetic stimulation to a multitude of presynaptic neurons converging on a single postsynaptic neuron. Our studies using rat brain slices showed that intracellular tetanization of a pyramidal neuron in layer 5 of the neocortex leads to the development of depression or potentiation of a proportion of the synaptic inputs from above-lying pyramidal cells in layers 2/3 expressing light-activated protein channelrhodopsin-2. Use of this stimulation method was found to allow a single experiment to yield sufficient data for statistical evaluation of the correlation between the paired pulse ratio and the amplitude of changes in EPSP after intracellular tetanization. We also showed that so-called “silent” synapses were not activated on development of long-term potentiation induced by intracellular tetanization.

Activated leukocyte cell adhesion molecule is involved in excitatory synaptic transmission and plasticity in the rat spinal dorsal horn.

Activated leukocyte cell adhesion molecule (ALCAM), a member of type I transmembrane immunoglobulin superfamily of cell adhesion molecule, is expressed in the surface membrane of various cell types including neurons. In the spinal cord dorsal horn (DH), the first gate for the sensory and pain transmission to the brain, the expression and function of ALCAM have not been known yet. Therefore, we here investigate the synaptic function of ALCAM in the substantia gelatinosa (lamina II) of the spinal DH, as well as its expression in the DH. Bath-application of ALCAM/Fc or CD6/Fc, the recombinant human IgG1-Fc chimeric proteins, specifically potentiated C-fiber-mediated excitatory synaptic transmission and predominantly increased spontaneous release of glutamate. In addition, the development of long-term potentiation, a form of synaptic plasticity, at excitatory synapses was significantly inhibited in the presence of the recombinant proteins. The functional roles of ALCAM in the spinal DH were further supported by immunohistochemical analysis; it showed that ALCAM intensely expressed through laminae I/II with the exception of lateral portion of the dorsal part of inner lamina II and distinctly co-localized with molecular markers of C-fibers, such as peptidergic calcitonin gene-related protein and transient receptor potential vanilloid type 1 and non-peptidergic isolectin B4. This study, for the first time, suggests the modulatory roles of ALCAM in the excitatory synaptic transmission and plasticity in the rat spinal DH.

Effects of copper on viability and functional properties of hippocampal neurons in vitro

Copper (Cu2+) is an essential metal presented in the mammalian brain and released from synaptic vesicles following neuronal depolarization. However, the disturbance of Cu2+ homeostasis results in neurotoxicity. In our study we performed for the first time a combined functional investigation of cultured hippocampal neurons under Cu2+ exposure, its effect on spontaneous spike activity of hippocampal neuronal network cultured on multielectrode array (MEA), and development of long-term potentiation (LTP) in acute hippocampal slices in the presence of Cu2+. Application of 0.2mM CuCl2 for 24h reduced viability of cultured neurons to 40±6%, whereas 0.01mM CuCl2 did not influence significantly on the neuronal survival. However, exposure to the action of 0.01mM Cu2+ resulted in pronounced reduction of network spike activity and abolished LTP induced by high-frequency stimulation of Schaffer's collaterals in CA1 pyramidal neurons of hippocampal slices. Antioxidant Trolox, the hydrosoluble vitamin E analogue, prevented neurotoxic effect and alterations of network activity under Cu2+ exposure, but didn't change the impairment of LTP in Cu2+-exposured hippocampal slices. We hypothesized that spontaneous network neuronal activity probably is one of the potential targets of Cu2+-induced neurotoxicity, in which free radicals can be involved. At the same time, it may be suggested that Cu2+-induced alterations of long-lasting trace processes (like LTP) are not mediated by oxidative damage.

Use of Fluorescence Anisotropy to Explore the Subunit Composition of Ca2+/Calmodulin Protein Kinase II Holoenzymes

Ca/calmodulin dependent protein kinase II (CaMKII) is a serine/threonine kinase which is highly enriched in the CNS and thought to be important in the development of long term potentiation. Several variants of CaMKII exist but alpha and beta subunits predominate in brain tissue. Though it is commonly believed that alpha and beta subunits combine to form heteromeric holoenzymes comprised of 12 subunits, this has not been directly demonstrated in vivo. Therefore, we used FRET imaging to directly examined the proximity of alpha and beta isoforms when they assemble to form heterologous holoenzymes. Specifically we used both time-resolved and steady-state fluorescence anisotropy measurements. Energy migration FRET (emFRET; FRET between like fluorophores) is observed in holoenzymes in which the C termini have been tagged with Venus. Fluorescence anisotropy is one of the few techniques which can detect emFRET and provide information of the number of fluorophores undergoing energy transfer. Venus tagged alpha isoforms were coexpressed with Amber (a point mutation in Venus that destroys the fluorophore) tagged beta isoforms and energy migration was seen to decrease indicating that holoenzymes are composed of both alpha and beta subunits in close proximity. Fluorescence anisotropy signatures of Venus tagged holoenzymes were invariant across a broad range of expression levels suggesting that inter-holoenzyme emFRET was not occurring. We used anisotropy “standards” consisting of concatamers of Venus and Amber to facilitate the interpretation of anisotropy curves in terms of the number of fluorescent proteins participating in emFRET. Together, these experiments indicate that alpha and beta subunits can coexist in the same holoenzyme. The availability of emFRET “standards” to the general scientific community should aid in the interpretation of anisotropy decay data.

Vagus nerve stimulation potentiates hippocampal LTP in freely-moving rats

Previous studies have demonstrated that electrical stimulation of the vagus nerve (VNS) delivered at a moderate intensity following a learning experience enhances memory in laboratory rats and human subjects, while VNS at lower or higher intensities has little or no effect. This finding suggests that VNS may affect memory processes by modulating neural plasticity in brain structures associated with memory storage such as the hippocampus. To test this hypothesis, the present study investigated the modulatory effect of VNS on the development of long-term potentiation (LTP) in the dentate gyrus of freely-moving rats. Rats receiving 0.4 mA VNS showed enhanced potentiation of the population spike amplitude for at least 24 h after tetanus relative to the sham-stimulation group. In contrast, no such effect was observed with 0.2 mA VNS. Stimulation at 0.8 mA had a short-term effect and tended to enhance early LTP, but to a lesser extent than did 0.4 mA. The 0.4 mA stimulation was the same intensity that was previously shown to enhance retention performance in an inhibitory avoidance task. These findings suggest that the neural mechanisms underlying the mnemonic effect of VNS may involve modulating synaptic plasticity in the hippocampus. These data also suggest that neural activity in the vagus nerve, occurring as a result of changes in peripheral state, is an important mechanism by which emotional experiences and arousal can enhance the storage of memories of those experiences.

Pyroglutamyl-Asparagine Amide Normalizes Long-Term Potentiation in Rat Hippocampal Slices

Preapplication of peptide piracetam analogue pyroglutamyl-asparagine amide to rat hippocampal slices facilitates long-term potentiation of focal responses in the CA1 field after weak tetanization of the synaptic input (30 pulses, 100 Hz). This treatment normalized the development of long-term potentiation after standard tetanization (100 pulses, 100 Hz) impaired by ethanol.

Behavioral Depression-Related Modifications of the Properties of Glutamatergic Synapses in the Basolateral Amygdalar Nucleus in Rats

Behavioral depression evoked by social isolation of experimental rats was accompanied by changes in the properties of glutamatergic synapses in the basolateral amygdalar nucleus, BLAN; the amplitude of field EPSP (fEPSP) decreased, long-term potentiation was intensified, while long-term depression of synaptic transmission was weakened. These modifications could be reproduced in vitro by application of 100 nM deoxycorticosterone or, partially, of 1 μM prednisolone to brain slices of intact rats, but were absent in the case of application of 100 nM dexamethasone. Observed in vivo social isolation-evoked changes in the properties of glutamatergic synapses could be prevented by subcutaneous injections of 2 mg/kg spironolactone (twice a day over 72 h of isolation). In vitro, 2 μM spironolactone eliminated modulating effects of deoxycorticosterone and inverted effects of prednisolone on the plastic properties of glutamatergic synapses. Inhibitors of transcription and ribosomal protein synthesis, doxorubicin (100 μM) and levomycetin (2 mM), respectively, prevented effects of deoxycorticosterone on synaptic transmission. The influence of the latter hormone on synaptic transmission in the BLAN was suppressed by an inhibitor of protein kinase C, polymixin B (50 μM), but significantly increased by an inhibitor of tyrosine phosphatases, sodium orthovanadate (1 mM). Suppression of synaptic transmission, which was evoked by deoxycorticosterone, was not accompanied by changes in the paired-pulse facilitation and amplitude of the NMDA component of fEPSP, while the amplitude of the AMPA component decreased. We suppose that these changes are based on intensification of synthesis of the proteins involved in endocytosis of the AMPA receptors, which results in intensification of endocytosis and a drop in the number of AMPA receptors in the postsynaptic thickenings. These changes of the fundamental properties of synaptic transmission facilitate the development of long-term potentiation of synaptic transmission, but weaken long-term depression of the transmission. The pattern of deoxycorticosterone-induced modifications of the plastic properties of glutamatergic synapses corresponds well to the main statements of the theory of metaplasticity.

Role of nitric oxide in short-term potentiation and long-term facilitation: involvement of NO in breathing stability.

It has been well documented that increases in breathing in response to a variety of stimuli may often outlast the actual period of stimulation in various species (see references1, 2). For instance, after a brief hypoxic challenge, ventilation slowly returns to baseline values over a period of minutes, a phenomenon often termed as “short-term potentiation (STP).” On the other hand, after episodes of recurrent hypoxic challenges, depending on species, breathing may remain elevated as long as an hotir. This long lasting enhancement of respiration that follows recurrent episodes of hypoxia is referred to as “long-term facilitation (LTF).” It has been proposed that STP and LTF are critical for maintaining stability of breathing, especially in situations involving acute changes in arterial blood gases2, and may involve brainstem neurons1,2. Recent studies on hippocampal neurons suggest that nitric oxide (NO) is critical for the development of long term potentiation (LTP) associated with learning and memory3,4. Given that NOS-1, the enzyme that generates NO, is co-localized with 5-HT in raphe neurons5, 6, which has been shown to be important for the generation of LTF2, as well as our recent observation that NOS-1 mutant mice exhibit breathing instability during hypoxia7, prompted us to examine whether NO generated by NOS-1 plays a role in the development STP and/or LTF.

The effects of forelimb deafferentation on the post-natal development of synaptic plasticity in the hippocampus.

The effects of partial deafferentation of the forelimb on the development of long-term potentiation in the hippocampus of rats aged 13-18 days were studied. Long-term potentiation in hippocampus field CA1 was of greater amplitude and duration in control rats at 16-18 days of post-natal ontogenesis than in adult animals. Partial deafferentation by section of the median nerve in the forelimb on the 13th day of life led to the disappearance of this excess at 16-18 days. The peak in synaptic plasticity occurred later in operated animals--on day 17--and was much less marked than in controls. The decreases in the amplitude and duration of long-term potentiation in hippocampal field CA1 in operated animals provides evidence for a decrease in the sensitivity and/or number of NMDA receptors. This suggests that partial deafferentation of one limb may lead not to a decrease but to an increase in spike and synaptic activity in the hippocampus, which in normal conditions may affect the maturation of the plastic properties of synaptic transmission associated with the expression and positions of NMDA receptors. The level of long-term potentiation in sham-operated rats was significantly greater than in controls of the same age. This significant increase in NMDA-dependent long-term potentiation may be explained by a decrease in the level of activation due to anesthesia. It is suggested that the decrease in the spike activity of cells receiving signals from the median nerve may be compensated for by activation of other specific and non-specific inputs.

Corpus callosum and cerebral laterality in a modular brain model

This paper elaborates the function of corpus callosum in the brain model that contains encoding and modulating axons: the former encode data as presynaptic axonal ‘on-off’ patterns, and the latter help the former convert data into long-term memory through the development of long-term potentiation and depression. It is hypothesized that callosal axons transfer data codes as interhemispheric memory. Bisection of corpus callosum cuts off interhemispheric data transfer and results in strange-hand syndrome, decreased attention and difficulties in acquiring new interdependent bimanual skills. While uniting two hemispheres for a unitary consciousness, corpus callosum contributes to two similar sets of integrated abstract memory, one in each hemisphere. Therefore, it takes bilateral cerebral lesions to manifest a failure of converting short-term memory into long-term memory. The asymmetric callosal data transfer may correlate with cerebral laterality where a cerebral function, such as language, is conducted mainly in one hemisphere for the benefit of less interhemispheric data-traffic. Complete lateralization of a cerebral function is the rare occasion when the specialized neuron groups (modules) for that function all reside in one hemisphere. It is possible that many cerebral functions including language are incompletely lateralized, and corpus callosum links the non-lateralized modules with the lateralized ones. The more the cerebral lateralization, the fewer the non-lateralized modules to be linked, and the smaller the corpus callosum.

Thyrotropin-releasing hormone inhibits long-term potentiation in synaptic systems of rat hippocampus

The effects of thyrotropin-releasing hormone (TRH) on long-term potentiation of field responses in mossy fibers—CA3 and Shaffer collaterals—CA1 synaptic systems were studied on rat hippocampal slices. Incubation with micromolar concentrations of TRH inhibited the development of long-term potentiation in both synaptic systems. It is suggested that this phenomenon underlies the antiamnesic effect of TRH.

Effects of potentiated antibodies to brain specific protein S100 on the dynamics of long-term potentiation in hippocampal slices

Native monoclonal antibodies to neurospecific S100 protein completely prevented the development of long-term potentiation (LTP) in rat hippocampal slices. Potentiated antiserum prepared by multiple dilutions according to homeopathic procedure (1:1012) did not affect LTP, but abolished the effect of native antiserum when applied 20 min prior to it. Neither nonimmune rabbit serum at the same dilution nor homeopathic solvent modified the development of LTP in hippocampal slices.

45Ca exchange in rat cortex slices in conditions of long-term potentiation.

Existing data on the role of Ca2+ ions in the development of long-term potentiation were used as a basis for studying changes in different Ca2+ compartments in cells in living rat olfactory cortex slices during potentiation. The kinetics of 45Ca2+ exchange were studied at 5, 15, and 30 min of potentiation. During the induction phase (1-5 min) of long-term potentiation, the fraction of tightly-bound intracellular Ca2+ decreased. There were no changes in the content of Ca2+ ions in other fractions at this stage. During maintenance of potentiation, which lasted 15-25 min, Ca2+ levels in the extracellular and intracellular compartments did not differ from controls. At 30 min, during extinction of long-term potentiation, there was a significant redistribution of Ca2+ in cells: the levels of free and loosely-bound Ca increased, as did extracellular Ca2+.

FK960, a novel potential anti-dementia drug, augments long-term potentiation in mossy fiber-CA3 pathway of guinea-pig hippocampal slices

Our previous studies have demonstrated that FK960 (FR59960; N-(4-acetyl-1-piperazinyl)- p-fluorobenzamide monohydrate), a novel antidementia piperazine derivative, exerts beneficial effects on memory deficits in various animal models of amnesia in rats [M. Yamazaki, N. Matsuoka, N. Maeda, Y. Ohkubo, I. Yamaguchi, FK960 N-(4-acetyl-1-piperazinyl)- p-fluorobenzamide monohydrate ameliorates the memory deficits in rats through a novel mechanism of action, J. Pharmacol. Exp. Ther., 279 (1996) 1157–1173.] and in rhesus monkeys [N. Matsuoka, T.G. Aigner, FK960 [ N-(4-acetyl-1-piperazinyl)- p-fluorobenzamide monohydrate], a novel potential antidementia drug, improves visual recognition memory in rhesus monkeys: comparison with physostigmine, J. Pharmacol. Exp. Ther., 280 (1997) 1201–1209]. To clarify the synaptic mechanisms of its antiamnesic action, FK960 was investigated for its effects on the development of long-term potentiation (LTP) in guinea-pig hippocampal slices. The magnitude of LTP of population spike recorded in CA3 pyramidal neurons was significantly augmented by perfusing FK960 (10 −9–10 −6 M) for 25 min before and during tetanic stimulation of the mossy fibers, whereas the basal amplitude of population spikes before tetanus was hardly affected by the drug. The dose–response curve was bell-shaped with a maximal augmentation at 10 −7 M. Scopolamine (10 −6 M) per se had little effect on the magnitude of LTP in the mossy fiber-CA3 pathway, but significantly attenuated its enhancement by FK960 (10 −7 M). In hippocampal slices from animals treated with cysteamine (200 mg/kg, s.c.), which was shown to deplete the hippocampal somatostatin, FK960 (10 −7 M) hardly affected the LTP. These results suggest that FK960 enhances the magnitude of LTP in the mossy fiber-CA3 pathway through an activation of the cholinergic–somatostatinergic link in the hippocampal formation. Furthermore, it can be postulated that the drug regulates the cognitive function by modulating directly synaptic plasticity in the hippocampal neuronal network.

Augmentation of LTP induced by Primed–Bursts tetanic stimulation in hippocampal CA1 area of morphine dependent rats

The effects of chronic morphine administration on the development of Long-term potentiation (LTP) were investigated at the Schaffer collateral-CA1 pyramidal cell synapses of the rat hippocampal slices using primed-bursts tetanic stimulation. Significant enhancement of orthodromic population spike (OPS) was found for all stimulus intensities after tetanic stimulation. OPS enhancement was greatest when tested with low to mid-range stimulus intensities (25 and 50 μA). There was also significant decrease in OPS delay. These responses were similar in slices from both control and morphine dependent rats. At all delivered stimulus intensities, the amount of LTP of OPS in slices from dependent rats was larger than that of control slices. However, these differences in LTP of OPS were significant at low stimulus intensities. These findings suggest that chronic morphine administration had induced changes in CA1 neurocircuitry which modulated synaptic plasticity during high frequency stimulation and appeared as augmented LTP.

Role of the phosphoinositide signal system and methylation of phosphatidylethanolamine in the development of long-term post-tetanic potentiation in rats.

High-frequency stimulation eliciting long-term post-tetanic potentiation of neuronal excitation in slices of the rat olfactory cortex was accompanied by changes in the metabolism of phospholipid components of cell membranes. At the first stage of the development of long-term potentiation (10 min after tetanization), there was a reduction in phosphoinositide metabolism. The maintenance phase of the potentiated state (30 min after tetanization) was associated with a three-fold increase in the incorporation of 14C-labeled groups from adenosylmethionine into phosphatidylethanolamine methylation products and with normalization of phosphoinositide metabolism. Sixty minutes after tetanization, when potentiation had decayed, there was activation of phosphoinositide metabolism and the intensity of phosphatidylethanolamine methylation returned to the control level. It is suggested that the phosphoinositide system plays an important role in the induction of long-term potentiation, as well as at the stage of recovery of normal neuronal excitability, while the long-term maintenance phase of elevated neuronal excitability was associated with long-lasting changes in the level of phosphatidylethanolamine methylation. The effect of glutamate receptor agonists on the carbachol-stimulated phosphoinositide response in potentiated slices was found to differ from that in nonpotentiated slices. The development of the long-term potentiated state is thus accompanied by a modulatory action of glutamate on the phosphoinositide response.

Atropine inhibits associative potentiation in the hippocampus.

The effects of the muscarinic receptor blocker atropine on associative long-term potentiation were studied in the CA1 region of the rat hippocampus. Focal excitatory post-synaptic potentials (EPSP) were recorded in living slices. Local application of atropine (0.1 mM) significantly inhibited associative long-term potentiation (40-60 min after tetanization) in the "weak" input (str. radiatum 128 +/- 10% compared with 168 +/- 9% in controls). Long-term potentiation at the "strong" input (str. oriens) was increased relative to the control (from 137 +/- 13% to 158 +/- 4%). These results indicate that endogenous acetylcholine aids the development of long-term potentiation in synapses with low levels of activation.

Na+,K(+)-ATPase activity in neurons and glial cells of the olfactory cortex of the rat brain during the development of long-term potentiation.

Na+,K(+)-ATPase and Mg(2+)-ATPase activities were studied in neurons and glial cells of the olfactory cortex of the rat by quantitative cytophotometry in conditions of long-term potentiation (LTP), and significant changes in direction and extent were found. Na+,K(+)-ATPase activity decreased in neurons in the first 15 min after LTP, with subsequent elevation by 30 min. Mg(2+)-ATPase activity remained unchanged in these conditions. Glial cells showed significant increases in Na+,K(+)-ATPase activity in the initial period after LTP, with return to control by 30 min. Again, there were no significant changes in Mg(2+)-ATPase activity. The formation and persistence of LTP in neurons and glial cells was accompanied by significant changes in Na+,K(+)-ATPase activity, which were reciprocal in nature.

Inhibition of long-term potentiation development in rat hippocampal slice by alpha 2-macroglobulin, an acute-phase protein in the brain.

Alpha-2-macroglobulin (alpha 2M) in the rat and human brain is an acute-phase protein synthesized primarily by astrocytes, and it has been implicated in Alzheimer's disease and other neuropathological processes. The activated forms of alpha 2M, but not the native form, can suppress the neurite outgrowth of the central neurons, presumably through binding to neurotrophic factors and through direct inhibition of neurotrophic factor receptor signal transduction. Since neurotrophic factors are known to be involved in synaptic plasticity, we tested the effect of both the native and methylamine-activated (MA-alpha 2M) forms of alpha 2M on long-term potentiation (LTP) in area CA1 of adult rat hippocampal slice. Neither native alpha 2M nor MA-alpha 2M had an effect on baseline synaptic transmission. LTP induced by 200-Hz trains in the presence of 1.4 microM or 0.14 microM native alpha 2M was indistinguishable from control LTP. Although the presence of MA-alpha 2M at the same concentrations did not interfere with LTP induction, the development and maintenance of potentiation was blocked in a concentration-dependent time course. Results of this study indicate that the accumulation and activation of alpha 2M with inflammatory neuropathologies such as Alzheimer's disease can inhibit synaptic plasticity, which might partly account for the memory deficits seen in these patients.

The biochemistry of learning and memory.

An overview of some of the biochemical and molecular events involved in the process of learning and memory are presented in a short review. Two invertebrate models of learning are considered: the gill-withdrawal reflex of Aplysia and avoidance learning in Drosophila melanogaster. Particular attention is paid to the biochemical mechanisms underlying both the development of long-term potentiation (LTP) and passive avoidance learning (PAL) in the young chick. The role of several biological molecules in learning and memory are considered, for example, protein kinase C (PKC), Ca(++)-Calmodulin kinase II (CaMKII), GAP-43, and glutamate receptors.