Increased O-GlcNAcylation rapidly decreases GABAAR currents in hippocampus but depresses neuronal output.

O-GlcNAcylation, a post-translational modification involving O-linkage of ß-N-acetylglucosamine to Ser/Thr residues on target proteins, is increasingly recognized as a critical regulator of synaptic function. Enzymes that catalyze O-GlcNAcylation are found at both presynaptic and postsynaptic sites, and O-GlcNAcylated proteins localize to synaptosomes. An acute increase in O-GlcNAcylation can affect neuronal communication by inducing long-term depression (LTD) of excitatory transmission at hippocampal CA3-CA1 synapses, as well as suppressing hyperexcitable circuits in vitro and in vivo. Despite these findings, to date, no studies have directly examined how O-GlcNAcylation modulates the efficacy of inhibitory neurotransmission. Here we show an acute increase in O-GlcNAc dampens GABAergic currents onto principal cells in rodent hippocampus likely through a postsynaptic mechanism, and has a variable effect on the excitation/inhibition balance. The overall effect of increased O-GlcNAc is reduced synaptically-driven spike probability via synaptic depression and decreased intrinsic excitability. Our results position O-GlcNAcylation as a novel regulator of the overall excitation/inhibition balance and neuronal output.

Simvastatin-mediated enhancement of long-term potentiation is driven by farnesyl-pyrophosphate depletion and inhibition of farnesylation.

Simvastatin (SV), a competitive inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase and a widely prescribed treatment for hypercholesterolemia, exerts numerous positive pleiotropic effects that are thought to occur independent of its cholesterol-lowering properties. In previously published work, we have shown that chronic SV treatment rescues cognitive function in a transgenic mouse model of Alzheimer's disease, and enhances learning and memory in non-transgenic mice without affecting total brain cholesterol and amyloid-beta levels. More recently, we demonstrated the ability of SV to enhance long-term potentiation (LTP) in the CA1 region of the hippocampus in slices from wild-type C57BL/6 mice via a mechanism dependent upon phosphatidylinositol 3-kinase (PI3-K)/Akt activation during LTP induction. The present study was conducted to better understand the molecular mechanisms underlying SV-induced enhancement of LTP. Specifically, it was found that inhibiting production of isoprenoid intermediates in the biosynthetic pathway for cholesterol triggers the downstream events leading to enhanced LTP. Interestingly, two major isoprenoid intermediates exhibit differential effects. Replenishment of farnesyl pyrophosphate, but not geranylgeranyl pyrophosphate, abolished the LTP-enhancing ability of SV. In parallel to this finding, inhibiting farnesylation, but not geranylgeranylation, replicated the enhancement of LTP caused by SV. Finally, inhibiting farnesylation promotes the activation of Akt during the induction phase. Together, these results suggest that SV enhances LTP in CA1 by modulating isoprenylation-dependent molecular pathways downstream of farnesyl transferase. These findings will aid in the identification of novel therapeutic targets that modulate synaptic and cognitive function.

Sympathetic sprouting in visual cortex stimulated by cholinergic denervation rescues expression of two forms of long-term depression at layer 2/3 synapses.

Cholinergic innervation of hippocampus and cortex is required for some forms of learning and memory. Several reports have shown that activation of muscarinic m1 receptors induces a long-term depression (mLTD) at glutamate synapses in hippocampus and in several areas of cortex, including perirhinal and visual cortices. This plasticity likely contributes to cognitive function dependent upon the cholinergic system. In rodent models, degeneration of hippocampal cholinergic innervation following lesion of the medial septum stimulates sprouting of adrenergic sympathetic axons, originating from the superior cervical ganglia (SCG), into denervated hippocampal subfields. We previously reported that this adrenergic sympathetic sprouting occurs simultaneously with a reappearance of cholinergic fibers in hippocampus and rescue of mLTD at CA3-CA1 synapses. Because cholinergic neurons throughout basal forebrain degenerate in aging and Alzheimer's disease, it is critical to determine if this compensatory sprouting occurs in other regions impacted by cholinergic cell loss. To this end, we investigated whether lesion of the nucleus basalis magnocellularis (NbM) to cholinergically denervate cortex stimulates adrenergic sympathetic sprouting and the accompanying increase in cholinergic innervation. Further, we assessed whether the presence of sprouting positively correlates with the ability of glutamate synapses in acute visual cortex slices to express mLTD and low frequency stimulation induced LTD (LFS LTD), another cholinergic dependent form of plasticity in visual cortex. We found that both mLTD and LFS LTD are absent in animals when NbM lesion is combined with bilateral removal of the SCG to prevent possible compensatory sprouting. In contrast, when the SCG remain intact to permit sprouting in animals with NbM lesion, cholinergic fiber density is increased concurrently with adrenergic sympathetic sprouting, and mLTD and LFS LTD are preserved. Our findings suggest that autonomic compensation for central cholinergic degeneration is not specific to hippocampus, but is a general repair mechanism occurring in other brain regions important for normal cognitive function.

Simvastatin enhances hippocampal long-term potentiation in C57BL/6 mice.

Statins inhibit 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway, and they are widely used to control plasma cholesterol levels and prevent cardiovascular disease. However, emerging evidence indicates that the beneficial effects of statins extend to the CNS. Statins have been shown to improve the outcome of stroke and traumatic brain injury, and statin use has been associated with a reduced prevalence of Alzheimer's disease (AD) and dementia. However, prospective studies with statins in AD have produced mixed results. Recently, we reported that simvastatin, a widely used statin in humans, enhances learning and memory in non-transgenic mice as well as in transgenic mice with AD-like pathology on a mixed genetic background. However, the cellular and molecular mechanisms underlying the beneficial effects of simvastatin on learning and memory remain elusive. The present study was undertaken to investigate the effect of acute simvastatin treatment on hippocampal long-term potentiation (LTP), a cellular model of learning and memory, in brain slices from C57BL/6 mice. Our results demonstrate that a prolonged in vitro simvastatin treatment for 2-4 h, but not a short-term 20-min exposure, significantly increases the magnitude of LTP at CA3-CA1 synapses without altering basal synaptic transmission or the paired-pulse facilitation ratio in hippocampal slices. Furthermore, we show that phosphorylation of Akt (protein kinase B) is increased significantly in the CA1 region following 2-hour treatment with simvastatin, and that inhibition of Akt phosphorylation suppresses the simvastatin-induced enhancement of LTP. These findings suggest activation of Akt as a molecular pathway for augmented hippocampal LTP by simvastatin treatment, and implicate enhancement of hippocampal LTP as a potential cellular mechanism underlying the beneficial effects of simvastatin on cognitive function.

Functionally distinct groups of interneurons identified during rhythmic carbachol oscillations in hippocampus in vitro.

During distinct behavioral states, the hippocampus exhibits characteristic rhythmic electrical activity. Evidence in vivo suggests that both principal pyramidal cells and GABAergic interneurons participate in generating oscillations. We found that during rhythmic oscillations in area CA3, functionally distinct classes of interneurons could be identified, although all recorded interneurons had similar dendritic and axonal arbors. One group of interneurons was powerfully excited by CA3 pyramidal cells, whereas two other interneuron groups were relatively unaffected by pyramidal cell firing. One of these groups of interneurons was potently inhibited by other local interneurons during the pyramidal cell bursts. Our findings emphasize that morphologically similar cells are wired together very differently within the local circuit. The classes of hippocampal interneurons we have tentatively defined may be used during distinct behavioral states to switch the local network from one oscillatory state to another.

Hippocampal interneurons are excited via serotonin-gated ion channels.

Hippocampal interneurons are excited via serotonin-gated ion channels. J. Neurophysiol. 78: 2493-2502, 1997. Serotonergic neurons of the median raphe nucleus heavily innervate hippocampal GABAergic interneurons located in stratum radiatum of area CA1, suggesting that this strong subcortical projection may modulate interneuron excitability. Using whole cell patch-clamp recording from interneurons in brain slices, we tested the effects of serotonin (5-HT) on the physiological properties of these interneurons. Serotonin produces a rapid inward current that persists when synaptic transmission is blocked by tetrodotoxin and cobalt, and is unaffected by ionotropic glutamate and gamma-aminobutyric acid (GABA) receptor antagonists. The 5-HT-induced current was independent of G-protein activation. Pharmacological evidence indicates that 5-HT directly excites these interneurons through activation of 5-HT3 receptors. At membrane potentials negative to -55 mV, the current-voltage (I-V) relationship of the 5-HT current displays a region of negative slope conductance. Therefore the response of interneurons to 5-HT strongly depends on membrane potential and increases greatly as cells are depolarized. Removal of extracellular calcium, but not magnesium, increases the amplitude of 5-HT-induced currents and removes the region of negative slope conductance, thereby linearizing the I-V relationship. The axons of 5-HT-responsive interneurons ramify widely within CA1; some of these interneurons also project to and arborize extensively in the dentate gyrus. The organization of these inhibitory connections strongly suggests that these cells regulate excitability of both CA1 pyramidal cells and dentate granule cells. As our results indicate that 5-HT may mediate fast excitatory synaptic transmission onto these interneurons, serotonergic inputs can simultaneously modulate the output of both hippocampus and dentate gyrus.

Hippocampal interneurons express a novel form of synaptic plasticity.

Individual GABAergic interneurons in hippocampus can powerfully inhibit more than a thousand excitatory pyramidal neurons. Therefore, control of interneuron excitability provides control over hippocampal networks. We have identified a novel mechanism in hippocampus that weakens excitatory synapses onto GABAergic interneurons. Following stimulation that elicits long-term potentiation at neighboring synapses onto excitatory cells, excitatory synapses onto inhibitory interneurons undergo a long-term synaptic depression (interneuron LTD; iLTD). Unlike most other forms of hippocampal synaptic plasticity, iLTD is not synapse specific: stimulation of an afferent pathway triggers depression not only of activated synapses but also of inactive excitatory synapses onto the same interneuron. These results suggest that high frequency afferent activity increases hippocampal excitability through a dual mechanism, simultaneously potentiating synapses onto excitatory neurons and depressing synapses onto inhibitory neurons.

Nicotinic receptor activation facilitates GABAergic neurotransmission in the avian lateral spiriform nucleus.

Whole-cell patch clamp recordings were performed in embryonic chick brain slices to characterize responses to nicotinic receptor activation in the mesencephalic lateral spiriform nucleus. Using intracellular recording, we previously reported the presence of functional high-affinity nicotinic sites in this nucleus that are insensitive to blockade with kappa- and alpha-bungarotoxin. We now report that nicotinic agonists not only produce an inward current in these cells, but also elicit a massive increase in the frequency of spontaneous postsynaptic currents without changing the amplitude distribution or risetime and decay kinetics of these events. The nicotinic receptor antagonist, dihydro-beta-erythroidine, blocks both the postsynaptic inward current and the enhancement of spontaneous postsynaptic currents. The spontaneous currents reverse at or near the chloride ion equilibrium potential and are completely blocked by 10 microM bicuculline, indicating that these events are likely to be GABAergic inhibitory postsynaptic currents. The nicotinic agonist-induced enhancement in inhibitory postsynaptic current frequency is blocked by 1.0 microM tetrodotoxin, demonstrating that the effect is mediated through the activation of voltage-dependent sodium channels. Nicotinic receptors are widely distributed in the central nervous system and in some cases are thought to modulate the release of various neurotransmitters. Our results show that activation of nicotinic receptors facilitates inhibitory neurotransmission in the avian lateral spiriform nucleus by increasing the frequency of spontaneous GABAergic postsynaptic currents. These data support a role for nicotinic receptors in the regulation of GABA release from nerve terminals in this nucleus.

Electrophysiological evidence for presynaptic nicotinic receptors in the avian ventral lateral geniculate nucleus.

1. Whole-cell patch-clamp recording in embryonic chick brain slices was used to examine the effect of nicotinic receptor activation on synaptic activity in neurons of the ventral lateral geniculate nucleus (LGNv). In LGNv neurons with input resistances < 250 M omega, bath perfusion of the nicotinic agonist, carbachol (10-30 microM), in the presence of 0.5-1.0 microM tetrodotoxin (TTX) and 1.0 microM atropine, produced a dramatic increase in the frequency of spontaneous postsynaptic currents (n = 8/8), while eliciting little or no direct postsynaptic response. The nicotinic antagonist, dihydro-beta-erythroidine (DH beta E; 30-100 microM) had no effect on basal synaptic currents, but blocked the carbachol-induced enhancement of spontaneous currents, demonstrating that activation of nicotinic receptors is responsible for this effect. 2. The gamma-aminobutyric acid A (GABAA) receptor antagonist, bicuculline (10-20 microM), blocked the basal spontaneous synaptic currents (n = 4) as well as the carbachol-induced enhancement of these events (n = 3), indicating that these currents are likely to be GABAergic inhibitory postsynaptic currents (IPSCs). 3. Because the nicotinic agonist-induced increase in IPSC frequency occurred during blockade of synaptic transmission with TTX, the enhancement of synaptic activity is not dependent upon action potential propagation. This is in marked contrast to our results in chick lateral spiriform neurons in which nicotinic agonist-induced increases in spontaneous GABAergic IPSCs were completely abolished in the presence of TTX. The data indicate that the nicotinic receptors mediating the increase in IPSC frequency in the LGNv are likely to be located directly on presynaptic nerve terminals.

Different responses to opioids measured in terminals and somas of Edinger-Westphal neurons.

Neurotransmitter receptors on the axon terminals of a neuron can be located a considerable distance away from comparable receptors on the cell body or dendrites of the same neuron. We examined the effects of activating either nerve terminal receptors or those located on or near the somas of chick Edinger-Westphal neurons. Cell body responses were measured via intracellular recording in a brain slice preparation. To measure nerve terminal responses, intracellular recordings were obtained from the large, calyciform nerve endings in intact ciliary ganglia, which emanate from neurons of the lateral Edinger-Westphal nucleus. Cell bodies of Edinger-Westphal neurons responded to leucine-enkephalin with a dose-dependent hyperpolarization that was associated with a decrease in input resistance. In spontaneously active Edinger-Westphal somas, leucine-enkephalin caused marked inhibition of suprathreshold and subthreshold activity, indicating that, as with a number of other central neurons, the major effect of opioids was to reduce excitability. The response to opioids was sensitive to naloxone (1 microM) and was a direct effect, since it was not blocked by either 0.5 microM tetrodotoxin or 100 microM cadmium. More selective mu ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin) and delta ([D-Ser2]-leucine-enkephalin-Thr and [D-Pen2,5]-enkephalin) opioid agonists produced effects similar to those of leucine-enkephalin. Opioids produced strikingly different effects in the nerve terminals of Edinger-Westphal neurons, where the major effect was a depolarization associated with a decrease in input resistance. The effects of opioids in the terminals were reduced in a low sodium buffer, indicating that they were dependent on the presence of extracellular sodium.(ABSTRACT TRUNCATED AT 250 WORDS)

The cannabinoid receptor: biochemical and cellular properties in neuroblastoma cells.

The cannabinoid receptor that has been pharmacologically characterized for hypothermia, spontaneous activity, analgesia and catalepsy in rodents is the same pharmacological receptor that inhibits adenylate cyclase in vitro. The inhibition of adenylate cyclase by the cannabinoid receptor results from an interaction with Gi, based on the biochemical kinetic properties of the response, the sensitivity to pertussis toxin ADP-ribosylation, and the thermodynamic characteristics of the response. From precedents based on studies of the well-characterized G protein coupled receptors, rhodopsin and the beta-adrenergic receptor, we can predict the tertiary structure of the cannabinoid receptor. Three sites of potential glycosylation are present on the receptor. However, treatment of N18TG2 neuroblastoma cells with tunicamycin to prevent glycosylation of newly synthesized receptors failed to alter cannabinoid-induced inhibition of cyclic AMP accumulation. The cannabinoid response was rapidly desensitized (within 1/2 h). Treatment of cells with tunicamycin failed to alter agonist-induced desensitization processes. These findings can be more veraciously interpreted as we gain a better understanding of the cellular dynamics of the cannabinoid receptor.