Effects of insulin on long-term potentiation in hippocampal slices from diabetic rats

AIMS/HYPOTHESIS: Cognitive deficits occur commonly in diabetic patients. It is unclear whether these impairments result from hypoglycaemia during intensive insulin therapy, or from the diabetes itself. The aim of this study was to examine if impaired energy utilization resulting from insulin deficiency contributes to impaired long-term potentiation (reflecting impaired synaptic plasticity). As long-term potentiation is considered a candidate cellular mechanism underlying learning and memory, understanding how diabetes alters long-term potentiation may provide insight into mechanisms producing cognitive deficits in diabetes. METHODS: Electrophysiologic recordings were used to study long-term potentiation in the CA1 region of hippocampal slices from healthy rats and rats with streptozotocin-induced diabetes. RESULTS: Long-term potentiation was difficult to induce in slices from diabetic rats in standard recording buffer (contains 10 mmol/l glucose). In slices from diabetic rats, increasing extracellular glucose failed to recover long-term potentiation induction, but 10 mmol/l pyruvate added to standard buffer enabled long-term potentiation induction. Moreover, incubation of slices from diabetic rats with insulin enabled long-term potentiation induction in standard buffer. Acute administration of streptozotocin alone did not impair long-term potentiation in slices from healthy animals, and changing extracellular glucose concentrations over the range of 5 mmol/l to 30 mmol/l did not alter long-term potentiation in slices from control rats. CONCLUSIONS/INTERPRETATION: These observations suggest that impaired energy utilization from insulin deficiency, rather than the accompanying hyperglycaemia, impair long-term potentiation in diabetes. Impaired hippocampal synaptic plasticity could contribute to learning and cognitive impairment in diabetic patients.     

Inhibition of the production and maintenance of long-term potentiation in rat hippocampal slices by a monoclonal antibody

Monoclonal antibodies generated to 5-day-old postnatal rat dentate gyri were tested for their effects on long-term potentiation (LTP) in rat hippocampal slices. One antibody, B6E11, was found to block the production of LTP and to suppress established LTP in both area CA1 of the hippocampus and the dentate gyrus. In CA1, B6E11 was effective only when applied to the apical dendrites synapsing with the potentiating input. The production of LTP could not be blocked if B6E11 was applied to the cell bodies or to the basal dendrites of CA1. In field CA1, B6E11 had no effect on the production of short-term potentiation. Another monoclonal antibody from the same panel as B6E11, of the same immunoglobulin class, which also binds to hippocampal neurons similarly to B6E11, had no effect on LTP production. These results supply evidence that B6E11 modulates LTP by an interaction with a specific cell-surface protein associated with the dendrites of neurons in both dentate gyrus and CA1 regions.     

Biphasic requirement for geranylgeraniol in hippocampal long-term potentiation

Mice deficient in cholesterol 24-hydroxylase exhibit reduced rates of cholesterol synthesis and other non-sterol isoprenoids that arise from the mevalonate pathway. These metabolic abnormalities, in turn, impair learning in the whole animal and hippocampal long-term potentiation (LTP) in vitro. Here, we report pharmacogenetic experiments in hippocampal slices from wild-type and mutant mice that characterize the dependence of LTP on the non-sterol isoprenoid, geranylgeraniol. Addition of geranylgeraniol to slices from 24-hydroxylase knockout mice restores LTP to wild-type levels; however, farnesol, a chemically related compound, does not substitute for geranylgeraniol nor does another animal model of impaired LTP (apolipoprotein E deficiency) respond to this isoprenoid. The requirement for geranylgeraniol is independent of acute protein isoprenylation as judged in experiments employing cell-permeable inhibitors of protein farnesyl transferase and geranylgeranyl transferase enzymes and in mutant mice hypomorphic for geranylgeranyltransferase II. Time course studies show that geranylgeraniol acts within 5 min and at 2 different times during the establishment of LTP: just before electrical stimulation and approximately 15 min thereafter. Localized delivery of geranylgeraniol to the dendritic trees of CA1 hippocampal neurons via the recording electrode is sufficient to restore LTP in slices from 24-hydroxylase knockout mice. We conclude that geranylgeraniol acts specifically and quickly to affect LTP in the Schaffer collaterals of the hippocampus.     

Impaired monosynaptic potentiation in in vitro hippocampal slices from aged, memory-deficient rats.

Neurophysiological experiments were conducted in vitro on 400 mu thick transverse hippocampal slices from aged and young rats. These slices exhibit neurophysiological responses similar to those of intact hippocampus. The aged rats have previously been found to exhibit impaired retention. Synaptic responses of the Schaffer collateral system were not found to be different between aged and young slices when elicited by very low frequency (0.3 Hz) electrical stimulation. However, the aged slices exhibited marked deficits in frequency and posttetanic potentiation in response to repetitive stimulation (15 Hz). This deficit was interpreted as resulting from an increased tendency to synaptic depression, rather than from impaired potentiation processes. The possibility of a relationship of these physiological deficits in hippocampal synaptic plasticity to the deficits in behavioral plasticity found in these aged animals is considered.     

Rapid activation of hippocampal casein kinase II during long-term potentiation.

Several studies suggest that protein kinase C and type II Ca2+/calmodulin-dependent protein kinase are activated during induction of long-term potentiation (LTP). We now report that casein kinase II (CK-II), which is present in high concentration in the hippocampus, is also activated in the CA1 region during LTP. CK-II activity increased within 2 min after a train of high-frequency electrical stimulations and reached a maximum (2-fold increase) 5 min later before returning to baseline value. The stimulated protein kinase activity, which was blocked by a selective antagonist of N-methyl-D-aspartate receptors, exhibited specific properties of CK-II, including phosphorylation of the specific substrates of CK-II, marked inhibition by a low heparin concentration, and the use of GTP as a phosphate donor. CK-II activity was also selectively and rapidly augmented in another form of LTP produced by bath application of tetraethylammonium; this LTP (called LTPk) is Ca2+ dependent but N-methyl-D-aspartate independent. Phosphorylation of casein that was not inhibited by heparin (i.e., casein kinase I) remained unchanged. We suggest that an increase in CK-II activity is important in LTP induction.     

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

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

Age-related deterioration of long-term potentiation in the CA3 and CA1 regions of hippocampal slices from the senescence-accelerated mouse

Long-term potentiation (LTP) is one candidate for the mechanism underlying memory storage. In the present study, we carried out electrophysiological studies on hippocampal slices prepared from the senescence-accelerated mouse (SAM-P/8), a strain which shows accelerated senescence and failure of certain types of learning in behavioral tests. The findings were compared with those noted in the SAM-R/1 substrain without severe symptoms of senescence. No significant differences were found between SAM-R/1 and SAM-P/8 of the same ages in responses in the absence of tetanic stimulation, and in LTP after tetanic stimulation. However, there were marked decreases in the degree of potentiation with aging in both strains.     

Frequency-dependent associative long-term potentiation at the hippocampal mossy fiber-CA3 synapse.

The mossy fiber-CA3 synapse displays an N-methyl-D-aspartate-receptor-independent mu-opioid-receptor-dependent form of long-term potentiation (LTP) that is thought not to display cooperativity or associativity with coactive afferents. However, because mossy fiber LTP requires repetitive synaptic activity for its induction, we reevaluated cooperativity and associativity at this synapse by using trains of mossy fiber stimulation. Moderate-, but not low-, intensity trains induced mossy fiber LTP, indicating cooperativity. Low-intensity mossy fiber trains that were normally ineffective in inducing LTP could induce mossy fiber LTP when delivered in conjunction with trains delivered to commissural-CA3 afferents. Associative mossy fiber LTP also could be induced with single mossy fiber pulses when delivered with commissural trains in the presence of a mu-opioid-receptor agonist. Our findings suggest a frequency-dependent variation of Hebbian associative LTP induction that is regulated by the release of endogenous opioid peptides.     

Synaptic disinhibition during maintenance of long-term potentiation in the CA1 hippocampal subfield.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is widely believed to occur through a strengthening of efficacy of excitatory synapses between afferent fibers and pyramidal cells. An alternative mechanism of LTP, reduction of efficacy of synaptic inhibition, was examined in the present report. The present study demonstrates that the maintenance of LTP in the CA1 hippocampal subfield of guinea pigs is accompanied by impairment of type A gamma-aminobutyric acid (GABA) receptor function, particularly at apical dendritic sites of CA1 pyramidal cells. Enhanced excitability of GABAergic interneurons during LTP represents a strengthening of inhibitory efficacy. The net effect of opposite modifications of synaptic inhibition during LTP of CA1 pyramidal cells is an overall impairment of the strength of GABAergic inhibition, and disinhibition could contribute importantly to CA1 pyramidal cell LTP.     

Cognitive deficits and impaired hippocampal long-term potentiation in KATP-induced DEND syndrome

ATP-sensitive potassium (K_ATP) gain-of-function (GOF) mutations cause neonatal diabetes, with some individuals exhibiting developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome. Mice expressing K_ATP-GOF mutations pan-neuronally (nK_ATP-GOF) demonstrated sensorimotor and cognitive deficits, whereas hippocampus-specific hK_ATP-GOF mice exhibited mostly learning and memory deficiencies. Both nK_ATP-GOF and hK_ATP-GOF mice showed altered neuronal excitability and reduced hippocampal long-term potentiation (LTP). Sulfonylurea therapy, which inhibits K_ATP, mildly improved sensorimotor but not cognitive deficits in K_ATP-GOF mice. Mice expressing K_ATP-GOF mutations in pancreatic β-cells developed severe diabetes but did not show learning and memory deficits, suggesting neuronal K_ATP-GOF as promoting these features. These findings suggest a possible origin of cognitive dysfunction in DEND and the need for novel drugs to treat neurological features induced by neuronal K_ATP-GOF.

ATP-sensitive potassium (K_ATP) channels are a unique link between cellular metabolism and membrane excitability. K_ATP gain-of-function (GOF) mutations have been identified as the most common cause of neonatal diabetes (1, 2), which, in many cases, manifests neurological features in a novel syndrome known as developmental delay, epilepsy, and neonatal diabetes (DEND) (3–6). Neurological symptoms of DEND include motor and developmental delays, severe epileptic phenotypes, and lifelong intellectual disabilities (7, 8). Diabetic features arise from suppression of insulin secretion by expression of K_ATP-GOF channels in pancreatic insulin-producing β-cells, and mice pan-neuronally expressing a DEND-associated K_ATP-GOF mutation showed sensorimotor deficits attributed to loss of excitability in cerebellar Purkinje neurons (9, 10). However, the involvement of K_ATP-GOF mutations in other neurological features as well as the treatability of these features remain unknown.K_ATP channels are hetero-octameric complexes comprising four pore-forming Kir6.x and four sulfonylurea receptor subunits, with Kir6.2 and SUR1 compositions predominating in neurons of the hippocampus and cerebellum (11, 12) as well as in pancreatic insulin-producing β-cells (13). SUR1 subunits provide pharmacological sensitivity to K_ATP channel openers (diazoxide) and blockers (e.g., sulfonylureas such as glibenclamide and tolbutamide). Kir6.2 and SUR1 subunits each contain RKR endoplasmic reticulum retention motifs, with the expression of both subunits required to form functional channels (14). Mice globally lacking K_ATP demonstrate spatial learning deficits (15, 16), intrahippocampal application of the K_ATP channel opener diazoxide impairs spatial learning and memory (17), and intraseptal application of glibenclamide improved spatial memory defects induced by galanin or morphine in rats (18, 19). K_ATP currents regulate spike rates and spontaneous bursting activity in hippocampal CA1/CA3 neurons (20) and gate epileptic seizures (21), suggesting that neurological features may arise from alterations to excitability in hippocampal neurons (10). In human neonatal diabetes, sulfonylureas are effective in normalizing blood glucose (22) and often successful in restoring muscular tone, but they are not nearly as effective in treating neurological, especially cognitive, features of DEND (4, 23–25). These findings raise questions about the pathophysiology of DEND, particularly the relative contributions of neuronal and pancreatic expression of K_ATP-GOF channels in the development of neurological features. Here, we explored the origin, underlying mechanisms, and treatability of the cognitive deficits of DEND in mouse models expressing K_ATP-GOF channels in central neurons (pan-neuronal or hippocampus specific) or in pancreatic β-cells.     

An ADP-ribosyltransferase as a potential target for nitric oxide action in hippocampal long-term potentiation.

Recent studies of long-term potentiation (LTP) in the CA1 region of the hippocampus have demonstrated that nitric oxide (NO) may be involved in some forms of LTP and have suggested that postsynaptically generated NO is a candidate to act as a retrograde messenger. However, the molecular target(s) of NO in LTP remain to be elucidated. The present study examined whether either of two potential NO targets, a soluble guanylyl cyclase or an ADP-ribosyltransferase (ADPRT; EC 2.4.2.31) plays a role in LTP. The application of membrane-permeant analogs of cGMP did not produce any long-lasting alterations in synaptic strength. In addition, application of a cGMP-dependent protein kinase inhibitor did not prevent LTP. We found that the CA1 tissue from hippocampus possesses an ADPRT activity that is dramatically stimulated by NO and attenuated by two different inhibitors of mono-ADPRT activity, phylloquinone and nicotinamide. The extracellular application of these same inhibitors prevented LTP. Postsynaptic injection of nicotinamide failed to attenuate LTP, suggesting that the critical site of ADPRT activity resides at a nonpostsynaptic locus. These results suggest that ADP-ribosylation plays a role in LTP and are consistent with the idea that an ADPRT may be a target of NO action.     

Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine

Repetitive correlated activation of pre- and postsynaptic neurons induced long-term potentiation (LTP) of synaptic transmission among hippocampal neurons grown on a layer of astrocytes (mixed cultures) but not among neurons cultured in glial conditioned medium. Supplement of D-serine, an agonist for the glycine-binding site of N-methyl-D-aspartate (NMDA) receptors, enhanced NMDA receptor activation and enabled LTP induction in glial conditioned medium cultures. The induction of LTP in both mixed cultures and hippocampal slices was suppressed by NMDA receptor antagonists, glycine-binding-site blockers of NMDA receptors, or an enzyme that degrades endogenous D-serine. By providing extracellular D-serine that facilitates activation of NMDA receptors, astrocytes thus play a key role in long-term synaptic plasticity.     

A hebbian form of long-term potentiation dependent on mGluR1a in hippocampal inhibitory interneurons.

Hippocampal inhibitory interneurons play important roles in controlling the excitability and synchronization of pyramidal cells, but whether they express long-term synaptic plasticity that contributes to hippocampal network function remains uncertain. We found that pairing postsynaptic depolarization with theta-burst stimulation induced long-term potentiation (LTP) of putative single-fiber excitatory postsynaptic currents in interneurons. Either postsynaptic depolarization or theta-burst stimulation alone failed to induce LTP. LTP was expressed as a decrease in failure rates and an increase in excitatory postsynaptic current amplitude, independent of N-methyl-d-aspartate receptors, and dependent on metabotropic glutamate receptors subtype 1a. LTP was induced specifically in interneurons in stratum oriens and not in interneurons of stratum radiatum/lacunosum-moleculare. Thus, excitatory synapses onto specific subtypes of inhibitory interneurons express a new form of hebbian LTP that will contribute to hippocampal network plasticity.     

Deficits in memory and hippocampal long-term potentiation in mice with reduced calbindin D28K expression.

The influx of calcium into the postsynaptic neuron is likely to be an important event in memory formation. Among the mechanisms that nerve cells may use to alter the time course or size of a spike of intracellular calcium are cytosolic calcium binding or "buffering" proteins. To consider the role in memory formation of one of these proteins, calbindin D28K, which is abundant in many neurons, including the CA1 pyramidal cells of the hippocampus, transgenic mice deficient in calbindin D28K have been created. These mice show selective impairments in spatial learning paradigms and fail to maintain long-term potentiation. These results suggest a role for calbindin D28K protein in temporally extending a neuronal calcium signal, allowing the activation of calcium-dependent intracellular signaling pathways underlying memory function.     

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.     

The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses

Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-F(c) chimeric proteins show that constructs containing Ig1-3 or Ig1 domains, but not Ig2-3 domains mediate homophilic adhesion. These data suggest that neuroplastin plays an essential role in implementing long-term changes in synaptic activity, possibly by means of a homophilic adhesion mechanism.     

Dendritic K+ channels contribute to spike-timing dependent long-term potentiation in hippocampal pyramidal neurons

We investigated the role of A-type K(+) channels for the induction of long-term potentiation (LTP) of Schaffer collateral inputs to hippocampal CA1 pyramidal neurons. When low-amplitude excitatory postsynaptic potentials (EPSPs) were paired with two postsynaptic action potentials in a theta-burst pattern, N-methyl-d-aspartate (NMDA)-receptor-dependent LTP was induced. The amplitudes of the back-propagating action potentials were boosted in the dendrites only when they were coincident with the EPSPs. Mitogen-activated protein kinase (MAPK) inhibitors PD 098059 or U0126 shifted the activation of dendritic K(+) channels to more hyperpolarized potentials, reduced the boosting of dendritic action potentials by EPSPs, and suppressed the induction of LTP. These results support the hypothesis that dendritic K(+) channels and the boosting of back-propagating action potentials contribute to the induction of LTP in CA1 neurons.     

Transient expansion of synaptically connected dendritic spines upon induction of hippocampal long-term potentiation

Dendritic spines are small protrusions from dendritic shafts that contain the postsynaptic sites of glutamatergic synapses in the brain. Spines undergo dramatic activity-dependent structural changes that are particularly prominent during neuronal development. Although changes in spine shape or number have been proposed to contribute to forms of synaptic plasticity that underlie learning and memory, the extent to which spines remain plastic in the adult brain is unclear. We find that induction of long-term potentiation (LTP) of synaptic transmission in acute hippocampal slices of adult mice evokes a reliable, transient expansion in spines that are synaptically activated, as determined with calcium imaging. Similar to LTP, transient spine expansion requires N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ influx and actin polymerization. Moreover, like the early phase of LTP induced by the stimulation protocol, spine expansion does not require Ca2+ influx through L-type voltage-gated Ca2+ channels nor does it require protein synthesis. Thus, transient spine expansion is a characteristic feature of the initial phases of plasticity at mature synapses and so may contribute to synapse remodeling important for LTP.     

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.