Potent depression of stimulus evoked field potential responses in the medial entorhinal cortex by serotonin.

1. The entorhinal cortex (EC), main input structure to the hippocampus, gets innervated by serotonergic terminals from the raphe nuclei and expresses 5-HT-receptors at high density. Using extra- and intracellular recording techniques we here investigated the effects of serotonin on population and cellular responses within the EC. 2. Stimulation in the lateral entorhinal cortex resulted in complex field potential responses in the superficial EC. The potentials are composed of an early antidromic and a late orthodromic component reflecting the efferent and afferent circuitry. 3. Serotonin (5-HT) reduced synaptic potentials of the stimulus evoked extracellular field potential at all concentrations tested (0. 1 - 100 microM; 59%-depression by 10 microM serotonin), while the antidromic response was not significantly changed by up to 50 microM 5-HT. Depression of field potential responses by serotonin was associated with a significant increase in paired-pulse facilitation from 1.15 to 1.88. 4. The effects of serotonin on field potential responses were mimicked by 5-HT1A-receptor agonists (8-OH-DPAT, 5-CT) and partially prevented by the 5-HT1A-receptor antagonist (S-UH-301). Moreover, the 5-HT1A-receptor antagonist WAY100635 reduced the effect of 5-CT. 5. Fenfluramine, a serotonin releaser, mimics the effects of serotonin on stimulus-evoked field potential responses, indicating that synaptically released serotonin can produce the changes in reactivity to afferent stimulation. 6. Depression of isolated AMPA-receptor mediated EPSCs by serotonin as well as fenfluramine was associated with an increase in paired pulse facilitation, indicating a presynaptic locus of action. 7. We conclude that physiological concentrations of serotonin potently suppresses excitatory synaptic transmission in the superficial entorhinal cortex by a presynaptic mechanism.     

Carbachol-induced changes in excitability and [Ca2+]i signalling in projection cells of medial entorhinal cortex layers II and III

The entorhinal cortex (EC) is a major gateway for sensory information into the hippocampus and receives a cholinergic input from the forebrain. Therefore, we studied muscarinic effects on excitability and intracellular Ca2+ signalling in layer II stellate and layer III pyramidal projection neurons of the EC. In both classes of neurons, local pressure-pulse application of carbachol (1 mM) caused small, atropine-sensitive membrane depolarizations that were not accompanied by any detectable changes in [Ca2+]i. At a higher concentration (10 mM), carbachol induced a larger membrane depolarization associated with synaptic oscillations and epileptiform activity in both classes of neurons. In contrast to the intrinsic theta rhythm in stellate cells with one dominant peak frequency at approximately 7 Hz, the synaptically mediated oscillation induced by carbachol showed three characteristic peaks in the theta and gamma frequency range at approximately 11, 23 and 40 Hz. Although carbachol-induced epileptiform activity was associated with increases in intracellular free Ca2+ in both layer II and III cells, the observed [Ca2+]i accumulation was significantly larger in layer III than in layer II cells. Responses to intracellular current injections showed differences in Ca2+ accumulation in layer II and III cells at the same membrane potentials, suggesting a dominant expression of low- and high-voltage-activated Ca2+ channels in these layer II and III cells, respectively. In conclusion, we present evidence for significant differences in the [Ca2+]i regulation between layer II stellate and layer III pyramidal cells of the medial EC.     

Deficiency of Renal Cortical EGF Increases ENaC Activity and Contributes to Salt-Sensitive Hypertension

Various stimuli, including hormones and growth factors, modulate epithelial sodium channels (ENaCs), which fine-tune Na⁺ absorption in the kidney. Members of the EGF family are important for maintaining transepithelial Na⁺ transport, but whether EGF influences ENaC, perhaps mediating salt-sensitive hypertension, is not well understood. Here, the ENaC inhibitor benzamil attenuated the development of hypertension in Dahl salt-sensitive rats. Feeding these salt-sensitive rats a high-salt diet led to lower levels of EGF in the kidney cortex and enhanced the expression and activity of ENaC compared with feeding a low-salt diet. To directly evaluate the role of EGF in the development of hypertension and its effect on ENaC activity, we infused EGF intravenously while continuously monitoring BP of the salt-sensitive rats. Infusion of EGF decreased ENaC activity, prevented the development of hypertension, and attenuated glomerular and renal tubular damage. Taken together, these findings indicate that cortical EGF levels decrease with a high-salt diet in salt-sensitive rats, promoting ENaC-mediated Na⁺ reabsorption in the collecting duct and the development of hypertension.More than 76 million American adults have high BP¹ and the likelihood of developing hypertension significantly increases with age. Nearly 40% of African Americans aged >20 years exhibit hypertension and nearly 70% of these individuals have a form of hypertension that is highly sensitive to salt intake. A reduced ability to maintain sodium homeostasis and normal levels of arterial pressure is a hallmark of all forms of hypertension.² In the kidney, discretionary Na⁺ reabsorption in response to endocrine input to the aldosterone-sensitive distal nephron (ASDN) is a determinant of the pressure-natriuresis relationship, which is of fundamental importance in the long-term control of arterial pressure.^2,3 Although sodium transport in ASDN accounts for a small proportion of renal sodium transport (<10%), ENaC activity is the rate-limiting step for this discretionary Na⁺ reabsorption.⁴The Dahl salt-sensitive (SS) rat strain used in this study is a genetic animal model of hypertension and kidney disease that reveals disease traits similar to those observed in humans. This inbred strain exhibits a low-renin, sodium-sensitive form of hypertension that is associated with severe and progressive proteinuria, glomerulosclerosis, and renal interstitial fibrosis.^5–7The EGF and related hormones are multipotent agents^8–11 involved in regulation of various renal functions and, particularly, ion channel activity. For instance, EGF stimulation rapidly induces TRPC5¹¹ and TRPM6¹⁰ channel translocation to the plasma membrane. Members of the EGF family play an important role in the expansion of renal cysts¹² and promote glomerular injury and renal failure in rapidly progressive crescentic glometulonephritis.¹³ Moreover, Groenestege et al. described a mutation in the pro-EGF encoding gene, which is responsible for development of isolated autosomal recessive hypomagnesemia, associated with renal Mg²⁺ wasting.¹⁴A role for EGF and its related growth factors in the regulation of ENaC-mediated sodium absorption has been proposed, although contradictory results have been observed with respect to ENaC activity and sodium transport.^15–17 Although EGF was shown to stimulate ENaC-mediated renal salt absorption in some studies, others reported that EGF decreases sodium transport and ENaC activity.¹⁷ Our recent data show that EGF and its related growth factors (TGF-α, HB-EGF, and amphiregulin) have a biphasic effect on sodium absorption as represented by the experiments on cultured murine mpkCCD_c14 principal cells.¹⁵ Basolateral application of the EGF family growth factors to polarized mpkCCD_c14 principal cells grown on permeable supports acutely increases Na⁺ reabsorption, whereas chronic treatment of the monolayers with EGF and its related growth factors leads to significant inhibition of ENaC-mediated transport.¹⁵ Similar observations were made in Xenopus laevis A6 principal cells.¹⁶ There are a number of potential mechanisms mediating downstream signaling. Thus, it was proposed that EGF effects could be mediated by either the extracellular signal–regulated kinase 1/2 and mitogen-activated protein kinase pathway, Akt,^16,18 or reactive oxygen species production.¹⁹ENaC dysfunction leads to disturbances in total body Na⁺ homeostasis associated with abnormal regulation of BP as observed in patients with Liddle’s syndrome and pseudohypoaldosteronism type 1.²⁰ However, the exact role of ENaC in mechanisms mediating salt-sensitive hypertension remains unclear. It was proposed that the expression of ENaC is abnormally regulated by dietary sodium in SS rats, and this abnormal expression is one of the factors causing salt-sensitive hypertension.^21–24 Here we confirm that ENaC expression is upregulated on a high-salt (HS) diet and provide direct evidence that ENaC activity is abnormally upregulated by dietary sodium in hypertensive SS rats, and this enhanced activity is one of the major factors causing salt-sensitive hypertension. In addition, our studies identify EGF as a key molecular substrate for a mechanism that diminishes development of salt-sensitive hypertension.     

Neuronal Ca2+ sensor VILIP-1 leads to the upregulation of functional α4β2 nicotinic acetylcholine receptors in hippocampal neurons

The neuronal Ca²⁺-sensor protein VILIP-1, known to affect clathrin-dependent receptor trafficking, has been shown to interact with the cytoplasmic loop of the α4-subunit of the α4β2 nicotinic acetylcholine receptor (nAChR), which is the most abundant nAChR subtype with high-affinity for nicotine in the brain. The α4β2 nAChR is crucial for nicotine addiction and the beneficial effects of nicotine on cognition. Its dysfunction has been implicated in frontal lobe epilepsy, Alzheimer's disease and schizophrenia. Here we report that overexpression of VILIP-1 enhances ACh responsiveness, whereas siRNA against VILIP-1 reduces α4β2 nAChR currents of hippocampal neurons. The underlying molecular mechanism likely involves enhanced constitutive exocytosis of α4β2 nAChRs mediated by VILIP-1. The two interaction partners co-localize in a Ca²⁺-dependent manner with syntaxin-6, a Golgi-SNARE protein involved in trans-Golgi membrane trafficking. Thus, we speculate that regulation of VILIP-1-expression might modulate surface expression of ligand-gated ion channels, such as the α4β2 nAChRs, possibly comprising a novel form of physiological up-regulation of ligand-gated ion channels.     

Comparison of effects induced by toxic applications of kainate and glutamate and by glucose deprivation on area CA1 of rat hippocampal slices

Baseline and stimulus-induced changes in [Ca²⁺]_o and [K⁺]_o as well as field potentials (fp's) were studied during application of the excitatory amino acids kainate or glutamate, or during glucose deprivation in area CA1 and CA3 of rat hippocampal slices. Bath application of kainate in concentrations of 1, 2, 5, 8 and 10 mM induced a sudden rapid fall of [Ca²⁺]_o in area CA1, associated with a negative shift of the slow fp. Kainate induced disappearance of stratum radiatum (SR) as well as alveus stimulation-evoked postsynaptic fp's, with partial recovery after application of up to 2 mM kainate, but no recovery after 5 mM kainate. Only afferent volleys and repetitive SR stimulation-induced decreases of [Ca²⁺]_o recovered after 5 mM kainate. Similar observations were made with glutamate. Only when glutamate was applied with 20 mM, irreversible disappearance of postsynaptic fp's was noted. Glucose deprivation for 60–90 min led to an initial slow decline of [Ca²⁺]_o in area CA1 and CA3, associated with increases in [K⁺]_o, but no significant changes in the fp baseline. Before reaching the lowest level in [Ca²⁺]_o, stimulation of afferent and efferent fibres in area CA1 and CA3 evoked epileptiform discharges. After reaching the lowest level in [Ca²⁺]_o, all postsynaptic potential components were irreversibly abolished, sparing afferent volleys and SR stimulation-induced decreases in [Ca²⁺]_o. The application of the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 μM) and

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-2-amino-5-phosphonovalerate (2APV, 30 μM) during glucose deprivation did not prevent irreversible loss of alveus and SR stimulation-induced postsynaptic signals. These findings suggest that glutamate release during glucose deprivation is not the main factor of acute cell damage.     

Increased inhibitory input to CA1 pyramidal cells alters hippocampal gamma frequency oscillations in the MK-801 model of acute psychosis

The phencyclidine compound MK-801 can induce psychosis with symptoms which closely resemble those observed in an acute schizophrenic episode. Here we used an in vitro model of psychosis after systemic administration of MK-801. We found that kainate-induced gamma frequency field oscillations in animals previously exposed to MK-801 have significantly higher power than in control animals. The intrinsic membrane properties of pyramidal cells, such as membrane input resistance and time constant, were not found to be different. In contrast, the MK-801 cells exhibited significantly more depolarized resting membrane potentials than control cells. We propose cellular alterations in Na+-K+-pump activity and increases in phasic inhibition in MK-801 cells to be the respective underlying mechanisms responsible for the more depolarized resting membrane potentials and the increased power of gamma frequency oscillations observed in MK-801 pretreated animals.     

On the formation of gamma-coherent cell assemblies by oriens lacunosum-moleculare interneurons in the hippocampus

Gamma frequency (30-80 Hz) network oscillations have been observed in the hippocampus during several behavioral paradigms in which they are often modulated by a theta frequency (4-12 Hz) oscillation. Interneurons of the hippocampus have been shown to be crucially involved in rhythms generation, and several subtypes with distinct anatomy and physiology have been described. In particular, the oriens lacunosum-moleculare (O-LM) interneurons were shown to synapse on distal apical dendrites of pyramidal cells and to spike preferentially at theta frequency, even in the presence of gamma-field oscillations. O-LM cells have also recently been shown to present higher axonal ramification in the longitudinal axis of the hippocampus. By using a hippocampal network model composed of pyramidal cells and two types of interneurons (O-LM and basket cells), we show here that the O-LM interneurons lead to gamma coherence between anatomically distinct cell modules. We thus propose that this could be a mechanism for coupling longitudinally distant cells excited by entorhinal cortex inputs into gamma-coherent assemblies.     

Far field effects of seizure like events induced by application of 4-AP in combined entorhinal cortex hippocampal slices

Epileptiform activity induced by 4-AP in hippocampal area CA1 is characterised by short recurrent discharges. These are occasionally superimposed by slow field potential (fp) shifts. Simultaneous recordings of fps and [K(+)](o) in area CA1 and temporal cortex showed a slow fp shift in both regions, but associated rises in [K(+)](o) occurred only in the cortex. Slow fps in area CA1 persisted after disruption of the perforant path, but were abolished after removal of the adjacent cortex from the hippocampus. These findings suggest that slow fps in CA1 can represent far field effects of seizure like events generated in neighbouring cortex.     

Kindling alters entorhinal cortex-hippocampal interaction by increased efficacy of presynaptic GABA(B) autoreceptors in layer III of the entorhinal cortex

We studied the effect of kindling, a model of temporal lobe epilepsy, on the frequency-dependent information transfer from the entorhinal cortex to the hippocampus in vitro. In control rats repetitive synaptic activation of layer III projection cells resulted in a frequency dependent depression of the synaptic transfer of action potentials to the hippocampus. One-to-two-days after kindling this effect was strongly reduced. Although no substantial change in synaptic inhibition upon single electrical stimulation was detected in kindled rats, there was a significant depression in the prolonged inhibition following high frequency stimulation. In kindled animals, paired-pulse depression (PPD) of stimulus-evoked IPSCs in layer III neurons was significantly stronger than in control rats. The increase of PPD is most likely caused by an increased presynaptic GABA(B) receptor-mediated autoinhibition. In kindled animals activation of presynaptic GABA(B) receptors by baclofen (10 microM) suppressed monosynaptic IPSCs significantly more than in control rats. In contrast, activation of postsynaptic GABA(B) receptors by baclofen was accompanied by comparable changes of the membrane conductance in both animal groups. Thus, in kindled animals activation of the layer III-CA1 pathway is facilitated by an increased GABA(B) receptor-mediated autoinhibition leading to an enhanced activation of the monosynaptic EC-CA1 pathway.