Catecholaminergic innervation of pyramidal and GABAergic nonpyramidal neurons in the rat hippocampus

Summary This study describes the catecholaminergic innervation of rat hippocampal neurons at the electron microscopic level by using an antibody against tyrosine hydroxylase (TH) and immunocytochemical techniques. In a first series of experiments, the course and distribution as well as the synaptic contacts of TH-immunoreactive fibers were analyzed with the peroxidase-antiperoxidase (PAP) method. Next, peroxidase immunostaining of TH fibers was combined with glutamate decarboxylase (GAD) immunostaining, using avidinated ferritin as a second electrondense marker. Our results demonstrate that TH-immunostained terminals establish asymmetric synaptic contacts with spines of pyramidal neurons, and symmetric synaptic contacts with cell bodies and dendritic shafts of ferritin-labeled GAD-immunoreactive nonpyramidal cells.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.     

Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex

The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine-coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3-4 h and neurons can be maintained in culture for up to 4 weeks.     

Effect of cannabinoids on glycine-activated currents in pyramidal neurons of the rat hippocampus

Using electrophysiological techniques (a patch-clamp technique in the whole-cell configuration and intracellular perfusion of neurons), we studied the effect of cannabinoids on the characteristics of glycine-activated currents in freshly isolated pyramidal neurons of the rat hippocampus. We found that endocannabinoids (anandamide and 2-arachidonoyl glycerol), as well as a synthetic cannabinoid, WIN 55,212-2, when applied in physiological concentrations, decreased the amplitude of glycine-activated currents. The agents under study accelerated the kinetics of activation and desensitization of glycine-induced Cl⁻ currents. The characteristics of the currents recovered after washout from cannabinoids. Changes in the kinetics of desensitization of glycine-activated currents depended noticeably on the holding potential; at positive potentials the sensitivity to cannabinoids was higher. These effects of cannabinoids were also observed in the presence of antagonists of CB1/CB3 receptors and an inhibitor of G proteins, GDPβS. These data indicate that under our experimental conditions cannabinoids exerted direct effects on glycine receptors. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 15–21, January–February, 2007.     

Rapid spinogenesis of pyramidal neurons induced by activation of glucocorticoid receptors in adult male rat hippocampus

Modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. Dendritic spines are essential for memory storage processes. Here, we investigated the effect of dexamethasone (DEX), a specific agonist of glucocorticoid receptor (GR), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of Lucifer Yellow-injected spines in slices. The application of 100 nM DEX (stressful high concentration) induced rapid modulation of the density and morphology of dendritic spines in CA1 pyramidal neurons within 1h. The total spine density increased from 0.88 spines/microm (control) to 1.36 spines/microm (DEX-treated). DEX significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. Because the presence of 10 microM cycloheximide, an inhibitor of protein synthesis, did not suppress the DEX effect, these responses are probably non-genomic. Western immunoblot analysis demonstrated the localization of classical type GR in Triton-insoluble synaptosomal fractions (enriched in postsynaptic membranes) from hippocampal slices, suggesting a possible action site of DEX at spines.     

Activation of glutamate ionotropic connections of sensorimotor cortex neurons during conditioning

Changes in conditioned impulse reactions of neurons in sensorimotor cortex were studied during microiontophoretic application of glutamatergic and GABA ergic agonistic and antagonistic drugs. It was shown that ionotropic glutamate receptors (AMPA and NMDA) are activated by a conditioned stimulus. Not only large pyramidal neurons of deep cortical layers but surrounding short-axon inhibitory interneurons are involved in the reaction. It was shown that the activity of pyramidal neurons is under a constant inhibitory control from surrounding interneurons. This inhibition is involved in organization of excitatory cortical responses during conditioning.     

Postnatal changes in the overall postsynaptic currents evoked in CA1 pyramidal neurons of the rat hippocampus

Evoked fast postsynaptic currents (fPSCs) during the postnatal development of rats (postnatal day 6-70, P6-P70) were systematically examined in hippocampal CA1 pyramidal neurons using whole-cell recordings with biocytin-filled electrodes. Focal stimulation of the stratum radiatum in the CA1 region elicited fPSCs in 80% of the neurons P6-7, 90% of P9-10, and 100% of > or =P11. In neurons P6-7, the fPSCs were exclusively inward and had multiple (on average 5.6) peaks. The fPSCs increased in amplitude with the growth of dendritic arborization, but decreased in the number of peaks. A distinct outward fPSC following the inward fPSC emerged in neurons > or =P11 and was abolished by bicuculline (50 microM). Bicuculline increased the amplitude and duration of the initial inward fPSC (fEPSC) in all age groups and characteristically recruited the polysynaptic second component of fEPSCs in neurons P11-P21. No spontaneous periodic inward current was detected in any age group after blocking GABAA receptors. The coapplication of DL-2-amino-5-phosphonopentanoic acid (AP5, 100 microM) with bicuculline did not eliminate the polysynaptic second component, but the second component was only elicited in slices in which the CA3 region was kept intact. Moreover, the bicuculline- and AP5-resistant second component was due to the burst activity of CA3 pyramidal neurons, which were excited through excitatory recurrents of the Schaffer collaterals. Plausible physiological functions of the generation of the second component in vivo were discussed.     

Donepezil in low micromolar concentrations modulates voltage-gated potassium currents in pyramidal neurons of rat hippocampus

Donepezil is a cholinesterase inhibitor widely used for the treatment of Alzheimer’s disease. Voltage-gated K⁺-channels are discussed as possible targets for the drug, but the results obtained by different authors are contradictory. In the present study performed on pyramidal cells isolated from rat’s hippocampus, we investigated the effect of donepezil on delayed rectifier K⁺-current (I_K(DR)) and transient outward K⁺-current (I_K(A)) using patch-clamp technique. The inhibitory effect of donepezil on I_K(DR) was found in all the cells tested, but its strength varied in different cells. Two groups of neurons were differing in their sensitivity to donepezil: more sensitive (IC₅₀ = 8.9 μM) and less sensitive (IC₅₀ = 114.9 μM). The effect of the drug on I_K(DR) was rapid, reversible and voltage-dependent, increasing with depolarization. Donepezil modulated I_K(A) in two different ways: in some cells it suppressed the current with the IC₅₀ value of 23.4 μM, while in other cells it augmented the current with a bell-shaped dose–response curve. Maximal (about twofold) enhancement of I_K(A) amplitude was caused by 10 μM donepezil. Augmentation of I_K(A) increased with membrane depolarization. Our results show for the first time that voltage-dependent potassium channels in mammals’ neurons are effectively modulated by low micromolar concentrations of donepezil.     

Suppression of A-type potassium current in pyramidal neurons of the rat hippocampus, induced by pinacidil and its fluorine derivatives

With the use of a patch-clamp technique in the whole-cell configuration, we studied the effects of pinacidil and its fluorine derivatives on A-type potassium current (I _A) through the membrane of pyramidal neurons of the rat hippocampus. Hydrogen peroxide (10 mM) exerted no influence on the rate of inactivation ofI _A; therefore, this current is probably mediated by Shal Kv4.2 potassium channels. Pinacidil demonstrated the properties of a weakI _A blocker: in the 500 μM concentration it blocked about 45% of the current, while 50 μM of pinacidil fluorine derivatives were capable of blocking up to 30% ofI _A. The effects of pinacidil and its derivatives showed no dependence on the stimulating potential. A similar pattern of the effects of pinacidil fluorine derivatives, which are an order of magnitude stronger than those of pinacidil itself, allows us to suppose that the imine nitrogen of the tested compounds is significantly more involved in the molecular interaction with the site of an A-type potassium channel than the pyridine nitrogen.     

M-channels modulate the intrinsic excitability and synaptic responses of layer 2/3 pyramidal neurons in auditory cortex

Neurons in the auditory cortex are believed to utilize temporal patterns of neural activity to accurately process auditory information but the intrinsic neuronal mechanism underlying the control of auditory neural activity is not known. The slowly activating, persistent K⁺ channel, also called M-channel that belongs to the Kv7 family, is already known to be important in regulating subthreshold neural excitability and synaptic summation in neocortical and hippocampal pyramidal neurons. However, its functional role in the primary auditory cortex (A1) has never been characterized. In this study, we investigated the roles of M-channels on neuronal excitability, short-term plasticity, and synaptic summation of A1 layer 2/3 regular spiking pyramidal neurons with whole-cell current-clamp recordings in vitro. We found that blocking M-channels with a selective M-channel blocker, XE991, significantly increased neural excitability of A1 layer 2/3 pyramidal neurons. Furthermore, M-channels controled synaptic responses of intralaminar-evoked excitatory postsynaptic potentials (EPSPs); XE991 significantly increased EPSP amplitude, decreased the rate of short-term depression, and increased the synaptic summation. These results suggest that M-channels are involved in controlling spike output patterns and synaptic responses of A1 layer 2/3 pyramidal neurons, which would have important implications in auditory information processing.     

Activation of Rho GTPases by synaptic transmission in the hippocampus

Ras-related GTPases of the Rho family, such as RhoA and RhoB, are well-characterised mediators of morphological change in peripheral tissues via their effects on the actin cytoskeleton. We tested the hypothesis that Rho family GTPases are involved in synaptic transmission in the CA1 region of the hippocampus. We show that GTPases are activated by synaptic transmission. RhoA and RhoB were activated by low frequency stimulation, while the induction of long-term potentiation (LTP) by high frequency stimulation was associated with specific activation of RhoB via NMDA receptor stimulation. This illustrates that these GTPases are potential mediators of synaptic transmission in the hippocampus, and raises the possibility that RhoB may play a role in plasticity at hippocampal synapses during LTP.     

Estrogen induces rapid decrease in dendritic thorns of CA3 pyramidal neurons in adult male rat hippocampus

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Thorns of thorny excrescences of CA3 hippocampal neurons are post-synaptic regions whose presynaptic partners are mossy fiber terminals. Here we demonstrated the rapid effect of estradiol on the density of thorns of thorny excrescences, by imaging Lucifer Yellow-injected CA3 neurons in adult male rat hippocampal slices. The application of 1nM estradiol induced rapid decrease in the density of thorns on pyramidal neurons within 2h. The estradiol-mediated decrease in the density of thorns was blocked by CNQX (AMPA receptor antagonist) and PD98059 (MAP kinase inhibitor), but not by MK-801 (NMDA receptor antagonist). ERalpha agonist PPT induced the same suppressive effect as that induced by estradiol on the density of thorns, but ERbeta agonist DPN did not affect the density of thorns. Note that a 1nM estradiol treatment did not affect the density of spines in the stratum radiatum and stratum oriens. A search for synaptic ERalpha was performed using purified RC-19 antibody. The localization of ERalpha (67kDa) in the CA3 mossy fiber terminals and thorns was demonstrated using immunogold electron microscopy. These results imply that estradiol drives the signaling pathway including ERalpha and MAP kinase.     

Effect of a non-hydrolyzable analog of diadenosine polyphosphates on NMDA-mediated currents in isolated pyramidal neurons of the rat hippocampus

Using a patch-clamp technique under voltage clamp conditions, we studied the effect of a non-hydrolyzable analog of diadenosine polyphosphates (AppCH₂ppAs) on chemoactivated transmembrane currents through NMDA channels (NMDA currents) in isolated pyramidal neurons of the rat hippocampal CA3 zone. In 55.7% of the cases, AppCH₂ppAs caused an increase in the peak amplitude of the currents induced by application of aspartate. In 39.5% of the cases, the agent exerted no effect, while in 4.8% these currents were suppressed. When studying the pharmacological effect of an increase in the amplitude of NMDA currents, we found that potentiation of these currents is mediated, first of all, by activation of P₂ purinoceptors and is prevented by a blocker of tyrosine kinases, genistein. Receptor-channel NMDA complexes, due to their ability to be blocked by divalent cations, also contribute to the above effect of AppCH₂ppA. Based on the data obtained, we conclude that AppCH₂ppA influences NMDA receptors via activation of the P₂ receptors and subsequent activation of tyrosine kinases; this leads to the modification of receptor-channel NMDA complexes and to the removal of their tonic blocking by zinc ions. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 205–210, May–June, 2006.     

Analysis of excitatory and inhibitory components of postsynaptic currents recorded in the pyramidal neurons and interneurons of the rat hippocampus

Postsynaptic currents recorded in the whole-cell configuration with patch-clamp method are actually the sum ofexcitatory (EPSC) and inhibitory (IPSC) components. An approach has been developed allowing the quantitative evaluation of the amplitude and the time course of EPSC and IPSC without treatment of the brain slice with pharmacological inhibitors. The approach is based on the substantial difference in the equilibrium potential values of incoming cationic and anionic currents as the existence of linear regions of corrent-voltage dependence of these currents. The comparison of the results obtained with the classical pharmacological method and with the suggested one demonstrated their coincidence. It allows analysing the postsynaptic currents in sigle neurons without altering the synaptic transmission in the whole brain slice. The contribution of inhibitory currents in the composite synaptic response of intemeurons turned out to be smaller in comparison with pyramidal neurons of CA1 field of the rat hippocampus.