Inflammation Subverts Hippocampal Synaptic Plasticity in Experimental Multiple Sclerosis

Abnormal use-dependent synaptic plasticity is universally accepted as the main physiological correlate of memory deficits in neurodegenerative disorders. It is unclear whether synaptic plasticity deficits take place during neuroinflammatory diseases, such as multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). In EAE mice, we found significant alterations of synaptic plasticity rules in the hippocampus. When compared to control mice, in fact, hippocampal long-term potentiation (LTP) induction was favored over long-term depression (LTD) in EAE, as shown by a significant rightward shift in the frequency–synaptic response function. Notably, LTP induction was also enhanced in hippocampal slices from control mice following interleukin-1β (IL-1β) perfusion, and both EAE and IL-1β inhibited GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) without affecting glutamatergic transmission and AMPA/NMDA ratio. EAE was also associated with selective loss of GABAergic interneurons and with reduced gamma-frequency oscillations in the CA1 region of the hippocampus. Finally, we provided evidence that microglial activation in the EAE hippocampus was associated with IL-1β expression, and hippocampal slices from control mice incubated with activated microglia displayed alterations of GABAergic transmission similar to those seen in EAE brains, through a mechanism dependent on enhanced IL-1β signaling. These data may yield novel insights into the basis of cognitive deficits in EAE and possibly of MS.     

Involvement of Nitric Oxide in Adenosine Release in the Developing and Adult Mouse Hippocampus

The novel type of neurotransmitter/neuromodulator nitric oxide (NO) is linked to activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptors and has been shown to modify transmitter release in the brain. The inhibitory neuromodulator adenosine has been thought to act as an endogenous neuroprotectant against cerebral ischemia and neuronal damage. The effects of NO-generating compounds on the release of preloaded [3H]adenosine from hippocampal slices from developing (7-day-old) and adult (3-month-old) mice were investigated, using a superfusion system, under normal conditions and in vitro ischemia. The release of adenosine was markedly potentiated at both ages by the NO-producing compounds S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and hydroxylamine. The evoked releases were reduced by the NO synthase inhibitors nitroarginine and 7-nitroindazole at both ages. They were also reduced by the inhibitor of soluble guanylyl cyclase 1H-(1,2,4-oxadiazolo(4,3a)quinoxalin-1-one (ODQ) in adults, indicating that the NO/cGMP pathway is involved in this release. Release of adenosine was also evoked when the cGMP levels were increased by superfusing slices with the phosphodiesterase inhibitor zaprinast. The markedly enhanced adenosine release under ischemic conditions was further potentiated by the ionotropic glutamate receptor agonists and NO-generating compounds, whereas zaprinast and ODQ had no effect, rendering unlikely the involvement of cGMP in the ischemic release. Moreover, NO was able to provoke substantial release of adenosine in the presence of NMDA under both normal and ischemic conditions, which could significantly add to the neuroprotective potential of this neuromodulator in both adult and developing hippocampus.     

Nitric oxide deficit in chronic intermittent hypoxia impairs large conductance calcium-activated potassium channel activity in rat hippocampal neurons

Sleep apnea associated with chronic intermittent hypoxia (IH) impairs hippocampal functions but the pathogenic mechanisms involving dysfunction of nitric oxide (NO) and ionic channels remain unclear. We examined the hypothesis that hippocampal NO deficit impairs the activity of large conductance calcium-activated potassium (BK) channels in rats with chronic IH, mimicking conditions in patients with sleep apnea. A patch-clamp study was performed on hippocampal CA1 neurons acutely dissociated from IH and control rats. The levels of endogenous NO and intracellular calcium in the CA1 region of the hippocampal slices were measured respectively by electrochemical microsensors and spectrofluorometry. We found that the open probability of BK channels remarkably decreased in the CA1 pyramidal neurons in a time-dependent manner with the IH treatment, without changes in the unitary conductance and reversal potential. NO donors, SNP or DETA/NO, significantly restored the activity of BK channels in the IH neurons, which was prevented by blockade of S-nitrosylation with NEM or MTSES but not by inhibition of the cGMP pathway with ODQ or 8-bromo-cGMP. Endogenous NO levels were substantially lowered in the IH hippocampus during resting and hypoxia. Also, the level of protein expression of neuronal NO synthase was markedly lessened in the IH neurons with decreased intracellular calcium response to hypoxia. Collectively, the results suggest that the IH-induced NO deficit mediated by a down-regulation of the expression of neuronal NO synthase plays a causative role in the impaired activity of BK channels, which could account for the hippocampal injury in patients with sleep apnea.     

Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.     

Actin-associated neurabin-protein phosphatase-1 complex regulates hippocampal plasticity

Protein phosphatase-1 (PP1) has been implicated in the control of long-term potentiation (LTP) and depression (LTD) in rat hippocampal CA1 neurons. PP1 catalytic subunits associate with multiple postsynaptic regulatory subunits, but the PP1 complexes that control hippocampal LTP and LTD in the rat hippocampus remain unidentified. The neuron-specific actin-binding protein, neurabin-I, is enriched in dendritic spines, and tethers PP1 to actin-rich postsynaptic density to regulate morphology and maturation of spines. The present studies utilized Sindbis virus-mediated expression of wild-type and mutant neurabin-I polypeptides in organotypic cultures of rat hippocampal slices to investigate their role in synaptic plasticity. While wild-type neurabin-I elicited no change in basal synaptic transmission, it enhanced LTD and inhibited LTP in CA1 pyramidal neurons. By comparison, mutant neurabins, specifically those unable to bind PP1 or F-actin, decreased basal synaptic transmission, attenuated LTD and increased LTP in slice cultures. Biochemical and cell biological analyses suggested that, by mislocalizing synaptic PP1, the mutant neurabins impaired the functions of endogenous neurabin-PP1 complexes and modulated LTP and LTD. Together, these studies provided the first biochemical and physiological evidence that a postsynaptic actin-bound neurabin-I-PP1 complex regulates synaptic transmission and bidirectional changes in hippocampal plasticity.     

Nitric Oxide Regulates Cyclic GMP-Dependent Protein Kinase Phosphorylation in Rat Brain

Abstract: Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.     

Presynaptic CaMKII is necessary for synaptic plasticity in cultured hippocampal neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is very critical for synaptic plasticity and memory formation. Although significant progress has been made in understanding the role of postsynaptic CaMKII in synaptic plasticity, very little is known about its presynaptic function during plasticity changes. Here we report that KN-93, a membrane-permeable CaMKII inhibitor, blocked glutamate-induced increases in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the number of presynaptic functional boutons in cultured hippocampal pyramidal neurons. In addition, presynaptic injection of the membrane-impermeable CaMKII inhibitor peptide 281-309 blocked synaptic plasticity induced by tetanus, glutamate, or NO/cGMP pathway activation as expressed by long-lasting increases in EPSC amplitude and functional presynaptic boutons. Presynaptic injection of CaMKII itself coupled with weak tetanus produced an immediate and long-lasting enhancement of EPSC amplitude. Thus, the present results conclusively prove that presynaptic CaMKII is essential for synaptic plasticity in cultured hippocampal neurons.     

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum: an immunocytochemical and pharmacological study

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1muM), expected to generate local NO concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent activation of ionotropic glutamate NMDA/non-NMDA receptors, in cerebellar slices from adult rat. We have found that low concentration of exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner. Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP production in Purkinje cells in rat cerebellar cortex.     

Hysteresis of synaptic effectiveness--one of the possible mechanisms for associative learning?

The conditions on the occurrence of hysteresis of the amount of phosphorylated receptors were determined depending on the rhythmic frequency of presynaptic neuronal activity using the full and simplified models of synaptic plasticity for pyramidal hippocampal neurones. It is assumed that this phenomenon lies at the basis of the conservation of long-term potentiation.     

Sources and targets of nitric oxide signalling in insect nervous systems

Nitric oxide (NO) is a membrane permeant signalling molecule which activates soluble guanylyl cyclase and leads to the formation of cyclic GMP (cGMP) in target cells. In the nervous system, NO/cGMP signalling is thought to play essential roles in synaptic plasticity during development and also in the mature animal. This review summarizes neurochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of insects. The anatomical localization of donor and target cells suggests functions in olfaction, vision, and mechanosensation. Behavioural assays have uncovered contributions of NO signalling in oxygen sensing, habituation to chemosensory stimuli, and associative memory formation. During development, NO regulates cell proliferation, axonal outgrowth, and synaptic maturation. The cellular distribution of NO-responsive cells suggests that NO can serve as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger irrespective of conventional synaptic connectivity.     

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.     

Taurine Release in the Developing and Adult Mouse Hippocampus: Involvement of Cyclic Guanosine Monophosphate

The inhibitory neuromodulator taurine is involved in osmoregulation and cell volume adjustments in the central nervous system. In addition, taurine protects neural cells from excitotoxicity and prevents harmful metabolic events evoked by cell-damaging conditions. The release of taurine in nervous cell preparations is greatly enhanced by glutamate receptor agonists and various cell-damaging conditions. NO-generating compounds also increase taurine release in the mouse hippocampus. The further involvement of the NO/cGMP pathway and protein kinases in preloaded [³H]taurine release from hippocampal slices from adult (3-month-old) and developing (7-day-old) mice in normoxia and in ischemia was now studied using a superfusion system. The release was enhanced by 8-Br-cGMP and the phosphodiesterase inhibitor 2-(2-propyloxyphenyl)-8-azapurin-6-one (zaprinast), particularly in the immature hippocampus, indicating that increased cGMP levels induce taurine release. The release was also increased by the inhibitor of soluble guanylyl cyclase, 1H-(1,2,4)oxadiazolo-(4,3a)quinoxalin-1-one (ODQ) and the protein kinase C activator 4-phorbol 12-myristate 13-acetate (PMA), but only in the adult hippocampus. The ischemia-induced release was also enhanced by increased cGMP levels in both adult and developing mice, whereas protein kinase inhibitors had no effects in any conditions. The results demonstrate that cGMP is able to modulate hippocampal taurine release in both adult and developing mice, the rise in cGMP levels evoking taurine release in normoxia and in ischemia. This could be part of the neuroprotective properties of taurine, being thus important particularly in cell-damaging conditions and in preventing excitotoxicity.     

A critical role for the glial-derived neuromodulator D-serine in the age-related deficits of cellular mechanisms of learning and memory

Age-associated deficits in learning and memory are closely correlated with impairments of synaptic plasticity. Analysis of N-methyl-D-aspartate receptor (NMDAr)-dependent long-term potentiation (LTP) in CA1 hippocampal slices indicates that the glial-derived neuromodulator D-serine is required for the induction of synaptic plasticity. During aging, the content of D-serine and the expression of its synthesizing enzyme serine racemase are significantly decreased in the hippocampus. Impaired LTP and NMDAr-mediated synaptic potentials in old rats are rescued by exogenous D-serine. These results highlight the critical role of glial cells and presumably astrocytes, through the availability of D-serine, in the deficits of synaptic mechanisms of learning and memory that occur in the course of aging.     

Simvastatin Treatment Enhances NMDAR-Mediated Synaptic Transmission by Upregulating the Surface Distribution of the GluN2B Subunit

The ramifications of statins on plasma cholesterol and coronary heart disease have been well documented. However, there is increasing evidence that inhibition of the mevalonate pathway may provide independent neuroprotective and procognitive pleiotropic effects, most likely via inhibition of isoprenoids, mainly farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). FPP and GGPP are the major donors of prenyl groups for protein prenylation. Modulation of isoprenoid availability impacts a slew of cellular processes including synaptic plasticity in the hippocampus. Our previous work has demonstrated that simvastatin (SV) administration improves hippocampus-dependent spatial memory, rescuing memory deficits in a mouse model of Alzheimer’s disease. Treatment of hippocampal slices with SV enhances long-term potentiation (LTP), and this effect is dependent on the activation of Akt (protein kinase B). Further studies showed that SV-induced enhancement of hippocampal LTP is driven by depletion of FPP and inhibition of farnesylation. In the present study, we report the functional consequences of exposure to SV at cellular/synaptic and molecular levels. While application of SV has no effect on intrinsic membrane properties of CA1 pyramidal neurons, including hyperpolarization-activated cyclic-nucleotide channel-mediated sag potentials, the afterhyperpolarization (AHP), and excitability, SV application potentiates the N-methyl D-aspartate receptor (NMDAR)-mediated contribution to synaptic transmission. In mouse hippocampal slices and human neuronal cells, SV treatment increases the surface distribution of the GluN2B subunit of the NMDAR without affecting cellular cholesterol content. We conclude that SV-induced enhancement of synaptic plasticity in the hippocampus is likely mediated by augmentation of synaptic NMDAR components that are largely responsible for driving synaptic plasticity in the CA1 region.     

Maternal infection and fever during late gestation are associated with altered synaptic transmission in the hippocampus of juvenile offspring rats

Prenatal exposure to infection is known to affect brain development and has been linked to increased risk for schizophrenia. The goal of this study was to investigate whether maternal infection and associated fever near term disrupts synaptic transmission in the hippocampus of the offspring. We used LPS to mimic bacterial infection and trigger the maternal inflammatory response in near-term rats. LPS was administered to rats on embryonic days 15 and 16 and hippocampal synaptic transmission was evaluated in the offspring on postnatal days 20-25. Only offspring from rats that showed a fever in response to LPS were tested. Schaffer collateral-evoked field excitatory postsynaptic potentials (fEPSPs) and fiber volleys in CA1 of hippocampal slices appeared smaller in offspring from the LPS group compared with controls, but, when the fEPSPs were normalized to the amplitude of fiber volleys, they were larger in the LPS group. In addition, intrinsic excitability of CA1 pyramidal neurons was heightened, as antidromic field responses in the LPS group were greater than those from control. Short-, but not long-term plasticity was impaired since paired-pulse facilitation of the fEPSP was attenuated in the LPS group, whereas no differences in long-term potentiation were noted. These results suggest that LPS-induced inflammation during pregnancy produces in the offspring a reduction in presynaptic input to CA1 with compensatory enhancements in postsynaptic glutamatergic response and pyramidal cell excitability. Neurodevelopmental disruption triggered by prenatal infection can have profound effects on hippocampal synaptic transmission, likely contributing to the memory and cognitive deficits observed in schizophrenia.     

Nitric oxide: an unconventional messenger in the nervous system of an orthopteroid insect

Nitric oxide (NO) is a membrane-permeant messenger molecule generated from the amino acid L-arginine. NO can activate soluble guanylyl cyclase leading to the formation of cyclic GMP (cGMP) in target cells. In the nervous system, NO/cGMP signalling is thought to play essential roles in synaptic plasticity during development and also in the mature animal. This paper examines biochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of the locust, a commonly used neurobiological preparation. Biochemical investigations suggest that an identical enzyme is responsible for both NO synthase (NOS) and NADPH-diaphorase activity after tissue fixation. Immunocytochemical staining of an olfactory center in the locust brain shows that NOS-immunoreactivity colocalizes with NADPH-diaphorase at the cellular level. The cytochemical staining of NO donor and target cells in adult animals suggests functions in olfaction, vision, and sensorimotor integration. During development, NO is implicated in axonal outgrowth and synaptogenesis. The cellular distribution of NO-responsive cells in neural circuits reflects potential functions of NO as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger independent of conventional synaptic connectivity.     

Soluble guanylyl cyclase during postnatal porcine pulmonary maturation

The nitric oxide (NO)/cGMP pathway plays a key role in the regulation of pulmonary vascular tone during the transition from the fetal to the neonatal circulation, and it is impaired in pathophysiological conditions such as pulmonary hypertension. In the present study, we have analyzed the changes in the function and expression of soluble guanylyl cyclase (sGC) in pulmonary arteries during early postnatal maturation in isolated third-branch pulmonary arteries from newborn (3-18 h of age) and 2-wk-old piglets. The expression of sGC beta(1)-subunit in pulmonary arteries increased with postnatal age both at the level of mRNA and protein. The catalytic region of porcine sGC beta(1) was sequenced, showing a 92% homology with the human sequence. This age-dependent increase in sGC expression correlated with increased vasorelaxant responses to the physiological sGC activator NO and to the exogenous sGC activator YC-1, but not to the membrane-permeable cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate. In conclusion, an increased expression of sGC in pulmonary conduit arteries from 2-wk-old compared with newborn piglets explains, at least partly, the age-dependent increase in the vasorelaxant response of NO and other activators of sGC.     

C-Type natriuretic peptide modulates pre- and postsynaptic properties in hippocampal area CA1 in vitro

The cGMP producing natriuretic peptide receptor B (NPR-B) and its ligand C-type natriuretic peptide (CNP) are widely distributed in the brain and are highly expressed in the hippocampal regions CA1-CA3. To date only limited functional data is available concerning the physiological effects of the peptide hormone in the hippocampus. Therefore, we were interested in how bath application of the peptide hormone might influence synaptic plasticity following high frequency stimulation (HFS). We found that CNP application decreased the population spike (PS) amplitude after HFS, thereby affecting long-term potentiation (LTP) in acute hippocampal slices. To investigate the molecular consequences of CNP application leading to a decrease in PS amplitude, we further analyzed the impact of the hormone on the number of presynaptic synapsin I clusters and number of postsynaptic AMPA receptor subunit GluR1 clusters as well as their co-localization in a primary hippocampal cell culture system. The observed pre-and postsynaptic effects after CNP stimulation of the cGMP pathway in hippocampal cell cultures may underlie the effect of the peptide hormone on LTP.     

ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC₅₀ values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N^ω-propyl-L-arginine, suggesting the involvement of P2X₇Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.