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 are the most popular illicit drugs used for recreational purposes worldwide. However, the neurobiological substrate of their mood-altering capacity has not been elucidated so far. Here we report that CB1 cannabinoid receptors are expressed at high levels in certain amygdala nuclei, especially in the lateral and basal nuclei, but are absent in other nuclei (e.g., in the central nucleus and in the medial nucleus). Expression of the CB1 protein was restricted to a distinct subpopulation of GABAergic interneurons corresponding to large cholecystokinin-positive cells. Detailed electron microscopic investigation revealed that CB1 receptors are located presynaptically on cholecystokinin-positive axon terminals, which establish symmetrical GABAergic synapses with their postsynaptic targets. The physiological consequence of this particular anatomical localization was investigated by whole-cell patch-clamp recordings in principal cells of the lateral and basal nuclei. CB1 receptor agonists WIN 55,212-2 and CP 55,940 reduced the amplitude of GABA(A) receptor-mediated evoked and spontaneous IPSCs, whereas the action potential-independent miniature IPSCs were not significantly affected. In contrast, CB1 receptor agonists were ineffective in changing the amplitude of IPSCs in the rat central nucleus and in the basal nucleus of CB1 knock-out mice. These results suggest that cannabinoids target specific elements in neuronal networks of given amygdala nuclei, where they presynaptically modulate GABAergic synaptic transmission. We propose that these anatomical and physiological features, characteristic of CB1 receptors in several forebrain regions, represent the neuronal substrate for endocannabinoids involved in retrograde synaptic signaling and may explain some of the emotionally relevant behavioral effects of cannabinoid exposure.

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.

Spine-free calretinin-immunoreactive (CR-IR) interneurons form a subpopulation of GABAergic cells in the rat hippocampus. A characteristic feature of these cells--located in all areas and layers--is the frequent dendro-dendritic and axo-dendritic contacts they form with each other. In this study we examined in detail the connectivity of these neurons by reconstructing their dendritic and axonal arbor and by identifying their postsynaptic targets. Radially running dendrites of CR-IR cells, located in different layers, intermingled into long braids. An average cell was in contact with dendrites of three to seven other CR-IR cells. Reconstruction of the dendritic trees from six consecutive sections demonstrated that at least 15 cells may participate in a dendro-dendritically connected cluster. Electron microscopical examination revealed that regularly spaced zonula adherentia connect the touching dendrites. The postsynaptic targets of CR-IR neurons have been examined using postembedding immunogold staining for GABA. CR-containing GABA-immunoreactive axons of local origin formed multiple symmetrical synaptic contacts (two to five) exclusively on GABAergic dendrites (CR-negative as well as CR-positive). Two to 10 CR-IR axons may converge onto a single CR-IR neuron, often from cells belonging to the same dendro-dendritically connected cluster. Using double immunocytochemistry, CR-IR cells were shown to heavily innervate calbindin D28k-containing interneurons and VIP-containing basket cells but avoided the parvalbumin-containing basket and axo-axonic cells. The unique connectivity of CR-IR cells may enable them to play a crucial role in the generation of synchronous, rhythmic hippocampal activity by controlling other interneurons terminating on different dendritic and somatic compartments of principal cells.

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Changes in synaptic efficacy are essential for neuronal development, learning and memory formation and for pathological states of neuronal excitability, including temporal-lobe epilepsy. At synapses, where there is a high probability of opening of postsynaptic receptors, all of which are occupied by the released transmitter, the most effective means of augmenting postsynaptic responses is to increase the number of receptors. Here we combine quantal analysis of evoked inhibitory postsynaptic currents with quantitative immunogold localization of synaptic GABA(A) receptors in hippocampal granule cells in order to clarify the basis of inhibitory synaptic plasticity induced by an experimental model of temporal-lobe epilepsy (a process known as kindling). We find that the larger amplitude (66% increase) of elementary synaptic currents (quantal size) after kindling results directly from a 75% increase in the number of GABA(A) receptors at inhibitory synapses on somata and axon initial segments. Receptor density was up by 34-40% and the synaptic junctional area was expanded by 31%. Presynaptic boutons were enlarged, which may account for the 39% decrease in the average number of released transmitter packets (quantal content). Our findings establish the postsynaptic insertion of new GABA(A) receptors and the corresponding increase in postsynaptic responses augmenting the efficacy of mammalian inhibitory synapses.

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 M carbachol in submerged hippocampal slices at 30 Ϯ 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 Ϯ 0.7 Hz, close to the negative peak of the oscillation (0.03 Ϯ 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 Ϯ 2.7 Hz), shortly after the negative peak (1.97 Ϯ 0.95 msec) and were strongly phase-coupled. Dendritic inhibitory interneurons fired at lower frequency (8.4 Ϯ 2.4 Hz) and with less fidelity and a longer delay after the negative peak (4.3 Ϯ 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-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 (CB 1 ) 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 CB 1 revealed axonal and somatic labeling with striking layer specificity: a high density of CB 1 -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 CB 1 was only found in axon terminals, all of which contained GABA and formed symmetric synapses. Double immunostaining also revealed that CB 1 -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 CB 1 -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.