Norbert Hájos

Differences between Somatic and Dendritic Inhibition in the Hippocampus

Hippocampal synaptic inhibition is mediated by distinct groups of inhibitory cells. Some contact pyramidal cells perisomatically, while others terminate exclusively on their dendrites. We examined perisomatic and dendritic inhibition by recording from CA3 inhibitory and pyramidal cells and injecting biocytin to visualize both cells in light and electron microscopy. Single perisomatic inhibitory cells made 2-6 terminals clustered around the soma and proximal pyramidal cell processes. Dendritic cells established 5-17 terminals, usually on different dendrites of a pyramidal cells. Perisomatic terminals were larger than those facing dendritic membrane. Perisomatic inhibitory cells initiated the majority of simultaneous IPSPs seen in nearby pyramidal cells. Single IPSPs initiated by perisomatic sodium-dependent action potentials. Activation of inhibitory fibers terminating on dendrites could suppress calcium-dependent spikes. Thus, distinct inhibitory cells may differentially control dendritic electrogenesis and axonal output of hippocampal pyramidal cells.

Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations

Using a new antibody developed against the C‐terminus of the cannabinoid receptor (CB1), the immunostaining in the hippocampus revealed additional axon terminals relative to the pattern reported previously with an N‐terminus antibody. Due to a greater sensitivity of this antibody, a large proportion of boutons in the dendritic layers displaying symmetrical (GABAergic) synapses were also strongly immunoreactive for CB1 receptors, as were axon terminals of perisomatic inhibitory cells containing cholecystokinin. Asymmetrical (glutamatergic) synapses, however, were always negative for CB1. To investigate the effect of presynaptic CB1 receptor activation on hippocampal inhibition, we recorded inhibitory postsynaptic currents (IPSCs) from principal cells. Bath application of CB1 receptor agonists (WIN55,212‐2 and CP55,940) suppressed IPSCs evoked by local electrical stimulation, which could be prevented or reversed by the CB1 receptor antagonist SR141716A. Action potential‐driven IPSCs, evoked by pharmacological stimulation of a subset of interneurons, were also decreased by CB1 receptor activation. We also examined the effects of CB1 receptor agonists on Ca2+‐independent miniature IPSCs (mIPSC). Both agonists were without significant effect on the frequency or amplitude of mIPSCs. Synchronous gamma oscillations induced by kainic acid in the CA3 region of hippocampal slices were reversibly reduced in amplitude by the CB1 receptor agonist CP 55,940, which is consistent with an action on IPSCs. We used CB1–/– knock‐out mice to confirm the specificity of the antibody and of the agonist (WIN55,212‐2) action. We conclude that activation of presynaptic CB1 receptors decreases Ca2+‐dependent GABA release, and thereby reduces the power of hippocampal network oscillations.

Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic synaptic transmission in the hippocampus

Psychoactive effects of cannabinoids are thought to be mediated, at least in part, by suppression of both glutamate and GABA release via CB1 cannabinoid receptor. Two types of cannabinoid receptor (CB1 and CB2) have been cloned so far. The CB1 receptors are abundantly expressed in the nervous system, whereas CB2 receptors are limited to lymphoid organs (Matsuda et al., 1990; Munro et al., 1993). Immunocytochemical and electrophysiological studies revealed that in the hippocampus CB1 receptors are expressed on axon terminals of GABAergic inhibitory interneurons (Tsou et al., 1999; Katona et al., 1999) and activation of these receptors decreases GABA release (Hájos et al., 2000). Other physiological studies pointed out the involvement of CB1 receptors in the modulation of hippocampal glutamatergic synaptic transmission and long-term potentiation (Stella et al., 1997; Misner and Sullivan, 1999), but anatomical studies could not confirm the existence of CB1 receptors on glutamatergic terminals. Here we examined cannabinoid actions on both glutamatergic and GABAergic synaptic transmission in the hippocampus of wild type (CB1+/+) and CB1 receptor knockout mice (CB1-/-). The synthetic cannabinoid agonist WIN55,212-2 reduced the amplitudes of excitatory postsynaptic currents in both wild type and CB1-/- mice, while inhibitory postsynaptic currents were decreased only in wild type mice, but not in CB1-/- animals. Our findings are consistent with a CB1 cannabinoid receptor-dependent modulation of GABAergic postsynaptic currents, but a novel cannabinoid-sensitive receptor must be responsible for the inhibition of glutamatergic neurotransmission.

Perisomatic Feedback Inhibition Underlies Cholinergically Induced Fast Network Oscillations in the Rat Hippocampus In Vitro

Gamma frequency network oscillations are assumed to be important in cognitive processes, including hippocampal memory operations, but the precise functions of these oscillations remain unknown. Here, we examine the cellular and network mechanisms underlying carbachol-induced fast network oscillations in the hippocampus in vitro, which closely resemble hippocampal gamma oscillations in the behaving rat. Using a combination of planar multielectrode array recordings, imaging with voltage-sensitive dyes, and recordings from single hippocampal neurons within the CA3 gamma generator, active current sinks and sources were localized to the stratum pyramidale. These proximal currents were driven by phase-locked rhythmic inhibitory inputs to pyramidal cells from identified perisomatic-targeting interneurons. AMPA receptor-mediated recurrent excitation was necessary for the synchronization of interneuronal discharge, which strongly supports a synaptic feedback model for the generation of hippocampal gamma oscillations.

Spike Timing of Distinct Types of GABAergic Interneuron during Hippocampal Gamma Oscillations In Vitro

Gamma frequency (30-100 Hz) network oscillations occur in the intact hippocampus during awake, attentive behavior. Here, we explored the underlying cellular mechanisms in an in vitro model of persistent gamma-frequency oscillations, induced by bath application of 20 μm carbachol in submerged hippocampal slices at 30 ± 1°C. Current-source density analysis of the field oscillation revealed a prominent alternating sink-source pair in the perisomatic and apical dendritic regions of CA3. To elucidate the active events generating these extracellular dipoles, we examined the firing properties of distinct neuron types. Visually guided unit recordings were obtained from individual CA3 neurons followed by intracellular labeling for anatomical identification. Pyramidal cells fired at 2.82 ± 0.7 Hz, close to the negative peak of the oscillation (0.03 ± 0.65 msec), and often in conjunction with a negative spike-like component of the field potential. In contrast, all phase-coupled interneurons fired after this negative peak. Perisomatic inhibitory interneurons fired at high frequency (18.1 ± 2.7 Hz), shortly after the negative peak (1.97 ± 0.95 msec) and were strongly phase-coupled. Dendritic inhibitory interneurons fired at lower frequency (8.4 ± 2.4 Hz) and with less fidelity and a longer delay after the negative peak (4.3 ± 1.1 msec), whereas interneurons with cell body in the stratum radiatum often showed no phase relationship with the field oscillation. The phase and spike time data of individual neurons, together with the current-source density analysis, support a synaptic feedback model of gamma oscillations primarily involving pyramidal cells and inhibitory cells targeting their perisomatic region.

Endocannabinoid signaling in rat somatosensory cortex : Laminar differences and involvement of specific interneuron types

Endocannabinoid-mediated retrograde signaling exerts powerful control over synaptic transmission in many brain areas. However, in the neocortex, the precise laminar, cellular, and subcellular localization of the type 1 cannabinoid receptor (CB1) as well as its function has been elusive. Here we combined multiple immunolabeling with whole-cell recordings to investigate the morpho-functional characteristics of cannabinoid signaling in rat somatosensory cortex. Immunostaining for CB1 revealed axonal and somatic labeling with striking layer specificity: a high density of CB1-positive fibers was seen in layers II-III, in layer VI, and in upper layer V, whereas other layers had sparse (layer IV) or hardly any (layer I) staining. Membrane staining for CB1 was only found in axon terminals, all of which contained GABA and formed symmetric synapses. Double immunostaining also revealed that CB1-positive cells formed two neurochemically distinct subpopulations: two-thirds were cholecystokinin positive and one-third expressed calbindin, each subserving specific inhibitory functions in cortical networks. In addition, cannabinoid sensitivity of GABAergic input showed striking layer specificity, as revealed by both electrophysiological and anatomical experiments. We found a unique population of large pyramidal neurons in layer VB that received much less perisomatic innervation from CB1-expressing GABAergic axon terminals and, accordingly, showed no depolarization-induced suppression of inhibition, unlike pyramidal cells in layer II, and a population of small pyramidal cells in layer V. This suggests that inhibitory control of pyramidal cells involved in intracortical or corticostriatal processing is fine-tuned by activity-dependent endocannabinoid signaling, whereas inhibition of pyramidal cells relaying cortical information to lower subcortical effector centers often lacks this plasticity.

Pharmacological separation of cannabinoid sensitive receptors on hippocampal excitatory and inhibitory fibers.

Our earlier studies demonstrated that in the hippocampus, cannabinoids suppress inhibitory synaptic transmission via CB(1) cannabinoid receptors, whereas a novel cannabinoid-sensitive receptor modulates excitatory synapses (Katona, I. et al., Journal of Neuroscience 19 (1999) 4544; Hájos, N. et al., European Journal of Neuroscience 12 (2000) 3239; Hájos, N. et al., Neuroscience 106 (2001) 1). The novel receptor does not correspond to CB(2), since this receptor type is not expressed in the brain (Munro, S. et al., Nature 365 (1993) 61). Recent binding experiments revealed that the synthetic cannabinoid WIN 55,212-2 binds with lower affinity to brain membranes of CB(1) receptor-knockout mice indicating that pharmacological differences exist between these two types of cannabinoid receptors in the hippocampus (Breivogel et al., Molecular Pharmacology 60 (2001) 155). To analyze this difference in detail, we first determined the EC(50) values of WIN 55,212-2 for excitatory and inhibitory transmission in rat hippocampal slices using whole-cell patch-clamp recordings. The estimated EC(50) value for inhibitory postsynaptic currents (IPSC) evoked by electrical stimulation in CA1 pyramidal cells was 0.24 microM, whereas for excitatory postsynaptic currents (EPSC) it was 2.01 microM, respectively. The cannabinoid antagonist, AM251, blocked the WIN 55,212-2-induced inhibition of evoked IPSCs, but not of EPSCs, providing evidence for its selectivity for CB(1). We then tested the hypothesis of whether the cannabinoid effect on hippocampal excitatory neurotransmission is mediated via receptors with an affinity for vanilloid ligands. Co-application of the vanilloid receptor antagonist capsazepine (10 microM) with cannabinoids (WIN55,212-2 or CP55,940) prevented the reduction of EPSCs, but not of IPSCs. The amplitude of evoked EPSCs was also suppressed by superfusion of the vanilloid receptor agonist capsaicin (10 microM), an effect which could also be antagonized by capsazepine. In contrast, capsaicin did not change the amplitude of evoked IPSCs. These results demonstrate that WIN 55,212-2 is an order of magnitude more potent in reducing GABAergic currents via CB(1) than in inhibiting glutamatergic transmission via the new CB receptor. The sensitivity of the new CB receptor (and EPSCs) to vanilloid ligands, but not to the cannabinoid antagonist AM251, represents another pharmacological tool to distinguish the two receptors, since CB(1) (and its effect on IPSCs) is not modulated by vanilloids, but is antagonized by AM251.

Precision and Variability in Postsynaptic Target Selection of Inhibitory Cells in the Hippocampal CA3 Region

Non‐pyramidal cells were filled intracellularly with biocytin in the CA3 region of the guinea‐pig hippocampus in vitro, within or close to stratum pyramidale. On the basis of camera lucida reconstructions and electron microscopy, six different cell types with distinct laminar distribution of axon terminals could be distinguished. The axon of three axo‐axonic cells, three typical basket cells, and atypical basket cells of two types arborized in the perisomatic and proximal dendritlc region of CA3 pyramidal cells. Two cells with axons innervating the distal dendritlc segments of pyramidal cells were also found; one terminated in stratum radiatum and the other in stratum lacunosum‐moleculare. Electron microscopy demonstrated that symmetrical synapses were formed by the labelled boutons on axon initial segments, somata, and proximal or distal dendrites of mostly pyramidal neurons. Axo‐axonic cells showed absolute target selectivity for axon initial segments, whereas for the other cells the distribution of contacted elements was determined by the laminar distribution of axon terminals. In two cases, where additional cells were labelled with biocytin, multiple (up to nine) light microscopically identified contacts (presumed synaptic contacts) were established by the interneurons on several pyramidal cells and on an axo‐axonic cell. Our results show that a restricted set of inhibitory cells, with somata within or close to CA3 stratum pyramldale, possess variable patterns of axonal arborization. Various types of postsynaptic elements are contacted, but precision in selecting certain targets and ignoring others is maintained within a particular cell type and layer. In contrast to the diversity of axonal arbors the structure of the dendritic trees shows no consistent differences, suggesting that the cells may be activated by a similar set of afferents. It seems probable that the innervation of precise regions of postsynaptic pyramidal cells by different types of interneurons–often in conjunction with particular excitatory afferents (Han et at., Eur. J. Neurosci., 5, 395–410, 1993)–underlies functional differences in inhibitory synaptic actions.

Parvalbumin-containing fast-spiking basket cells generate the field potential oscillations induced by cholinergic receptor activation in the hippocampus

Gamma frequency oscillations in cortical regions can be recorded during cognitive processes, including attention or memory tasks. These oscillations are generated locally as a result of reciprocal interactions between excitatory pyramidal cells and perisomatic inhibitory interneurons. Here, we examined the contribution of the three perisomatic interneuron types—the parvalbumin-containing fast-spiking basket cells (FSBCs) and axo-axonic cells (AACs), as well as the cholecystokinin-containing regular-spiking basket cells (RSBCs) to cholinergically induced oscillations in hippocampal slices, a rhythmic activity that captures several features of the gamma oscillations recorded in vivo. By analyzing the spiking activities of single neurons recorded in parallel with local field potentials, we found that all three cell types fired phase locked to the carbachol-induced oscillations, although with different frequencies and precision. During these oscillations, FSBCs fired the most with the highest accuracy compared with the discharge of AACs and RSBCs. In further experiments, we showed that activation of μ-opioid receptors by DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin acetate), which significantly reduced the inhibitory, but not excitatory, transmission, suppressed or even blocked network oscillations both in vitro and in vivo, leading to the desynchronization of pyramidal cell firing. Using paired recordings, we demonstrated that carbachol application blocked GABA release from RSBCs and reduced it from FSBCs and AACs, whereas DAMGO further suppressed the GABA release only from FSBCs, but not from AACs. These results collectively suggest that the rhythmic perisomatic inhibition, generating oscillatory fluctuation in local field potentials after carbachol treatment of hippocampal slices, is the result of periodic GABA release from FSBCs.

Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.

Studies in brain slices have provided a wealth of data on the basic features of neurons and synapses. In the intact brain, these properties may be strongly influenced by ongoing network activity. Although physiologically realistic patterns of network activity have been successfully induced in brain slices maintained in interface‐type recording chambers, they have been harder to obtain in submerged‐type chambers, which offer significant experimental advantages, including fast exchange of pharmacological agents, visually guided patch‐clamp recordings, and imaging techniques. Here, we investigated conditions for the emergence of network oscillations in submerged slices prepared from the hippocampus of rats and mice. We found that the local oxygen level is critical for generation and propagation of both spontaneously occurring sharp wave–ripple oscillations and cholinergically induced fast oscillations. We suggest three ways to improve the oxygen supply to slices under submerged conditions: (i) optimizing chamber design for laminar flow of superfusion fluid; (ii) increasing the flow rate of superfusion fluid; and (iii) superfusing both surfaces of the slice. These improvements to the recording conditions enable detailed studies of neurons under more realistic conditions of network activity, which are essential for a better understanding of neuronal network operation.