Yannis Dalezios

Complementary Roles of Cholecystokinin- and Parvalbumin-Expressing GABAergic Neurons in Hippocampal Network Oscillations

In the hippocampal CA1 area, a relatively homogenous population of pyramidal cells is accompanied by a diversity of GABAergic interneurons. Previously, we found that parvalbumin-expressing basket, axo-axonic, bistratified, and oriens-lacunosum moleculare cells, innervating different domains of pyramidal cells, have distinct firing patterns during network oscillations in vivo. A second family of interneurons, expressing cholecystokinin but not parvalbumin, is known to target the same domains of pyramidal cells as do the parvalbumin cells. To test the temporal activity of these independent and parallel GABAergic inputs, we recorded the precise spike timing of identified cholecystokinin interneurons during hippocampal network oscillations in anesthetized rats and determined their molecular expression profiles and synaptic targets. The cells were cannabinoid receptor type 1 immunopositive. Contrary to the stereotyped firing of parvalbumin interneurons, cholecystokinin-expressing basket and dendrite-innervating cells discharge, on average, with 1.7 ± 2.0 Hz during high-frequency ripple oscillations in an episode-dependent manner. During theta oscillations, cholecystokinin-expressing interneurons fire with 8.8 ± 3.3 Hz at a characteristic time on the ascending phase of theta waves (155 ± 81°), when place cells start firing in freely moving animals. The firing patterns of some interneurons recorded in drug-free behaving rats were similar to cholecystokinin cells in anesthetized animals. Our results demonstrate that cholecystokinin- and parvalbumin-expressing interneurons make different contributions to network oscillations and play distinct roles in different brain states. We suggest that the specific spike timing of cholecystokinin interneurons and their sensitivity to endocannabinoids might contribute to differentiate subgroups of pyramidal cells forming neuronal assemblies, whereas parvalbumin interneurons contribute to synchronizing the entire network.

Neuronal Diversity in GABAergic Long-Range Projections from the Hippocampus

The formation and recall of sensory, motor, and cognitive representations require coordinated fast communication among multiple cortical areas. Interareal projections are mainly mediated by glutamatergic pyramidal cell projections; only few long-range GABAergic connections have been reported. Using in vivo recording and labeling of single cells and retrograde axonal tracing, we demonstrate novel long-range GABAergic projection neurons in the rat hippocampus: (1) somatostatin- and predominantly mGluR1α-positive neurons in stratum oriens project to the subiculum, other cortical areas, and the medial septum; (2) neurons in stratum oriens, including somatostatin-negative ones; and (3) trilaminar cells project to the subiculum and/or other cortical areas but not the septum. These three populations strongly increase their firing during sharp wave-associated ripple oscillations, communicating this network state to the septotemporal system. Finally, a large population of somatostatin-negative GABAergic cells in stratum radiatum project to the molecular layers of the subiculum, presubiculum, retrosplenial cortex, and indusium griseum and fire rhythmically at high rates during theta oscillations but do not increase their firing during ripples. The GABAergic projection axons have a larger diameter and thicker myelin sheet than those of CA1 pyramidal cells. Therefore, rhythmic IPSCs are likely to precede the arrival of excitation in cortical areas (e.g., subiculum) that receive both glutamatergic and GABAergic projections from the CA1 area. Other areas, including the retrosplenial cortex, receive only rhythmic GABAergic CA1 input. We conclude that direct GABAergic projections from the hippocampus to other cortical areas and the septum contribute to coordinating oscillatory timing across structures.

Metabotropic glutamate receptor 8-expressing nerve terminals target subsets of GABAergic neurons in the hippocampus.

Presynaptic metabotropic glutamate receptors (mGluRs) show a highly selective expression and subcellular location in nerve terminals modulating neurotransmitter release. We have demonstrated that alternatively spliced variants of mGluR8, mGluR8a and mGluR8b, have an overlapping distribution in the hippocampus, and besides perforant path terminals, they are expressed in the presynaptic active zone of boutons making synapses selectively with several types of GABAergic interneurons, primarily in the stratum oriens. Boutons labeled for mGluR8 formed either type I or type II synapses, and the latter were GABAergic. Some mGluR8-positive boutons also expressed mGluR7 or vasoactive intestinal polypeptide. Interneurons strongly immunopositive for the muscarinic M2 or the mGlu1 receptors were the primary targets of mGluR8-containing terminals in the stratum oriens, but only neurochemically distinct subsets were innervated by mGluR8-enriched terminals. The majority of M2-positive neurons were mGluR8 innervated, but a minority, which expresses somatostatin, was not. Rare neurons coexpressing calretinin and M2 were consistently targeted by mGluR8-positive boutons. In vivo recording and labeling of an mGluR8-decorated and strongly M2-positive interneuron revealed a trilaminar cell with complex spike bursts during theta oscillations and strong discharge during sharp wave/ripple events. The trilaminar cell had a large projection from the CA1 area to the subiculum and a preferential innervation of interneurons in the CA1 area in addition to pyramidal cell somata and dendrites. The postsynaptic interneuron type-specific expression of the high-efficacy presynaptic mGluR8 in both putative glutamatergic and in identified GABAergic terminals predicts a role in adjusting the activity of interneurons depending on the level of network activity.

Different Fear States Engage Distinct Networks within the Intercalated Cell Clusters of the Amygdala.

Although extinction-based therapies are among the most effective treatments for anxiety disorders, the neural bases of fear extinction remain still essentially unclear. Recent evidence suggests that the intercalated cell masses of the amygdala (ITCs) are critical structures for fear extinction. However, the neuronal organization of ITCs and how distinct clusters contribute to different fear states are still entirely unknown. Here, by combining whole-cell patch-clamp recordings and biocytin labeling with full anatomical reconstruction of the filled neurons and ultrastructural analysis of their synaptic contacts, we have elucidated the cellular organization and efferent connections of one of the main ITC clusters in mice. Our data showed an unexpected heterogeneity in the axonal pattern of medial paracapsular ITC (Imp) neurons and the presence of three distinct neuronal subtypes. Functionally, we observed that the Imp was preferentially activated during fear expression, whereas extinction training and extinction retrieval activated the main ITC nucleus (IN), as measured by quantifying Zif268 expression. This can be explained by the IPSPs evoked in the IN after Imp stimulation, most likely through the GABAergic monosynaptic innervation of IN neurons by one subtype of Imp cells, namely the medial capsular-projecting (MCp)–Imp neurons. MCp–Imp neurons also target large ITC cells that surround ITC clusters and express the metabotropic glutamate receptor 1α. These findings reveal a distinctive participation of ITC clusters to different fear states and the underlying anatomical circuitries, hence shedding new light on ITC networks and providing a novel framework to elucidate their role in fear expression and extinction.

High level of mGluR7 in the presynaptic active zones of select populations of GABAergic terminals innervating interneurons in the rat hippocampus

The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a‐positive) were analysed. The presynaptic active zones of most GAD‐(77%) or GABA‐positive (94%) synaptic boutons on interneurons innervated by mGluR7a‐enriched glutamatergic terminals (mGluR7a‐decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a‐immunonegative. The mGluR7a‐decorated cells were mostly somatostatin‐ and mGluR1α‐immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP‐positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin‐positive synaptic terminals were rare on mGluR7a‐decorated cells, but on these neurons 73% of them were mGluR7a‐immunopositive. Some typeu2003II synapses innervating interneurons were immunopositive for mGluR7b, as were some typeu2003I synapses. Because not all target cells of VIP‐positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target‐cell‐specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7‐decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.

Depression of GABAergic input to identified hippocampal neurons by group III metabotropic glutamate receptors in the rat

The release of GABA in synapses is modulated by presynaptic metabotropic glutamate receptors (mGluRs). We tested whether GABA release to identified hippocampal neurons is influenced by group III mGluR activation using the agonist L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) on inhibitory postsynaptic currents (IPSCs) evoked in CA1 interneurons and pyramidal cells. In interneurons, characterized with biocytin and immunolabelling for somatostatin, evoked IPSCs were depressed by 50u2003µm L‐AP4 (activating mGluR4 and 8) to 68u2003±u20036% of control, but they were rarely depressed in pyramidal cells (96u2003±u20034% of control). At 300–500u2003µm concentration (activating mGluR4, 7 and 8), L‐AP4 depressed IPSCs in both interneurons (to 70u2003±u20036%) and pyramidal cells (to 67u2003±u20034%). The change in trial‐to‐trial variability and in paired‐pulse depression indicated a presynaptic action. In interneurons, the degree of IPSC depression was variable (to 9–87%), and a third of IPSCs were not affected by L‐AP4. The L‐AP4‐evoked IPSC depression was blocked by LY341495. The depression of IPSCs was similar in O‐LM cells and other interneurons. The lack of cell‐type selectivity and the similar efficacy of different concentrations of L‐AP4 suggest that several group III mGluRs are involved in the depression of IPSCs. Electron microscopic immunocytochemistry confirmed that mGluR4, mGluR7a and mGluR8a occur in the presynaptic active zone of GABAergic terminals on interneurons, but not on those innervating pyramidal cells. The high variability of L‐AP4‐evoked IPSC suppression is in line with the selective expression of presynaptic mGluRs by several distinct types of GABAergic neuron innervating each interneuron type.

Distinct dendritic arborization and in vivo firing patterns of parvalbumin-expressing basket cells in the hippocampal area CA3.

Hippocampal CA3 area generates temporally structured network activity such as sharp waves and gamma and theta oscillations. Parvalbumin-expressing basket cells, making GABAergic synapses onto cell bodies and proximal dendrites of pyramidal cells, control pyramidal cell activity and participate in network oscillations in slice preparations, but their roles in vivo remain to be tested. We have recorded the spike timing of parvalbumin-expressing basket cells in areas CA2/3 of anesthetized rats in relation to CA3 putative pyramidal cell firing and activity locally and in area CA1. During theta oscillations, CA2/3 basket cells fired on the same phase as putative pyramidal cells, but, surprisingly, significantly later than downstream CA1 basket cells. This indicates a distinct modulation of CA3 and CA1 pyramidal cells by basket cells, which receive different inputs. We observed unexpectedly large dendritic arborization of CA2/3 basket cells in stratum lacunosum moleculare (33% of length, 29% surface, and 24% synaptic input from a total of ∼35,000), different from the dendritic arborizations of CA1 basket cells. Area CA2/3 basket cells fired phase locked to both CA2/3 and CA1 gamma oscillations, and increased firing during CA1 sharp waves, thus supporting the role of CA3 networks in the generation of gamma oscillations and sharp waves. However, during ripples associated with sharp waves, firing of CA2/3 basket cells was phase locked only to local but not CA1 ripples, suggesting the independent generation of fast oscillations by basket cells in CA1 and CA2/3. The distinct spike timing of basket cells during oscillations in CA1 and CA2/3 suggests differences in synaptic inputs paralleled by differences in dendritic arborizations.

Rhythmically Active Enkephalin-Expressing GABAergic Cells in the CA1 Area of the Hippocampus Project to the Subiculum and Preferentially Innervate Interneurons

Enkephalins (ENKs) are endogenous opioids that regulate synaptic excitability of GABAergic networks in the cerebral cortex. Using retrograde tracer injections in the subiculum, we identified a hippocampal population of ENK-expressing projection neurons. In situ hybridization for GAD shows that ENK-expressing cells are a small GABAergic subpopulation. Furthermore, by extracellular recording and juxtacellular labeling in vivo, we identified an ENK-expressing cell in stratum radiatum of the CA1 area by its complete axodendritic arborization and characteristic spike timing during network oscillations. The somatodendritic membrane was immunopositive for mGluR1α, and there was both a rich local axon in CA1 and subicular-projecting branches. The boutons showed cell-type- and layer-specific innervation, i.e., interneurons were the main targets in the alveus, both interneurons and pyramidal cell dendrites were innervated in the other layers, and interneurons were exclusive targets in the subiculum. Parvalbumin-, but not somatostatin-, calbindin-, or cholecystokinin-expressing interneurons were preferred synaptic targets. During network activity, the juxtacellularly labeled ENK-expressing cell was phase modulated throughout theta oscillations, but silenced during sharp-wave/ripple episodes. After these episodes the interneuron exhibited rebound activity of high-frequency spike bursts, presumably causing peptide release. The ENK-expressing interneurons innervating parvalbumin-positive interneurons might contribute to the organization of the sharp-wave/ripple episodes by decreased firing during and rebound activity after the ripple episodes, as well as to the coordination of activity between the CA1 and subicular areas during network oscillations.

Neurogliaform cells of amygdala: a source of slow phasic inhibition in the basolateral complex

•u2002 The amygdala is a key brain structure implicated in processing emotions and is thought to underlie anxiety‐related behaviours. •u2002 Interneurons of the basolateral amygdala provide constant silencing of principal cells, and are proven to be active cellular elements during emotional processing. •u2002 We report here a novel interneuron type of the amygdala, termed neurogliaform cell (NGFC), and study its function by a combination of in vivo and in vitro techniques. •u2002 NGFCs provide a peculiar cell‐to‐cell form of communication via volume transmission of GABA. •u2002 In‐depth analysis of specific neuron type is likely to improve the understanding of amygdala circuits in health and disease.