Synaptic organisation and behaviour-dependent activity of mGluR8a-innervated GABAergic trilaminar cells projecting from the hippocampus to the subiculum.

In the hippocampal CA1 area, the GABAergic trilaminar cells have their axon distributed locally in three layers and also innervate the subiculum. Trilaminar cells have a high level of somato-dendritic muscarinic M2 acetylcholine receptor, lack somatostatin expression and their presynaptic inputs are enriched in mGluR8a. But the origin of their inputs and their behaviour-dependent activity remain to be characterised. Here we demonstrate that (1) GABAergic neurons with the molecular features of trilaminar cells are present in CA1 and CA3 in both rats and mice. (2) Trilaminar cells receive mGluR8a-enriched GABAergic inputs, e.g. from the medial septum, which are probably susceptible to hetero-synaptic modulation of neurotransmitter release by group III mGluRs. (3) An electron microscopic analysis identifies trilaminar cell output synapses with specialised postsynaptic densities and a strong bias towards interneurons as targets, including parvalbumin-expressing cells in the CA1 area. (4) Recordings in freely moving rats revealed the network state-dependent segregation of trilaminar cell activity, with reduced firing during movement, but substantial increase in activity with prolonged burst firing (> 200 Hz) during slow wave sleep. We predict that the behaviour-dependent temporal dynamics of trilaminar cell firing are regulated by their specialised inhibitory inputs. Trilaminar cells might support glutamatergic principal cells by disinhibition and mediate the binding of neuronal assemblies between the hippocampus and the subiculum via the transient inhibition of local interneurons.

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.

Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling.

Hippocampal CA1 pyramidal cells, which receive γ-aminobutyric acid (GABA)ergic input from at least 18 types of presynaptic neuron, express 14 subunits of the pentameric GABA(A) receptor. The relative contribution of any subunit to synaptic and extrasynaptic receptors influences the dynamics of GABA and drug actions. Synaptic receptors mediate phasic GABA-evoked conductance and extrasynaptic receptors contribute to a tonic conductance. We used freeze-fracture replica-immunogold labelling, a sensitive quantitative immunocytochemical method, to detect synaptic and extrasynaptic pools of the alpha1, alpha2 and beta3 subunits. Antibodies to the cytoplasmic loop of the subunits showed immunogold particles concentrated on distinct clusters of intramembrane particles (IMPs) on the cytoplasmic face of the plasma membrane on the somata, dendrites and axon initial segments, with an abrupt decrease in labelling at the edge of the IMP cluster. Neuroligin-2, a GABAergic synapse-specific adhesion molecule, co-labels all beta3 subunit-rich IMP clusters, therefore we considered them synapses. Double-labelling for two subunits showed that virtually all somatic synapses contain the alpha1, alpha2 and beta3 subunits. The extrasynaptic plasma membrane of the somata, dendrites and dendritic spines showed low-density immunolabelling. Synaptic labelling densities on somata for the alpha1, alpha2 and beta3 subunits were 78-132, 94 and 79 times higher than on the extrasynaptic membranes, respectively. As GABAergic synapses occupy 0.72% of the soma surface, the fraction of synaptic labelling was 33-48 (alpha1), 40 (alpha2) and 36 (beta3)% of the total somatic surface immunolabelling. Assuming similar antibody access to all receptors, about 60% of these subunits are in extrasynaptic receptors.

Interneurons hyperpolarize pyramidal cells along their entire somatodendritic axis.

Although GABAergic interneurons are the main source of synaptic inhibition in the cortex, activation of GABA(A) receptors has been shown to depolarize specific neuronal compartments, resulting in excitation. By using a noninvasive approach to monitor the effect of individual interneurons on the pyramidal cell population, we found that rat hippocampal interneurons hyperpolarized pyramidal cells irrespective of the location of their synapses along the somato-dendritic axis.

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 mGluR1alpha-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.

Climbing fiber innervation of NG2-expressing glia in the mammalian cerebellum.

The molecular layer of the cerebellar cortex is populated by glial progenitors that express ionotropic glutamate receptors and extend numerous processes among Purkinje cell dendrites. Here, we show that release of glutamate from climbing fiber (CF) axons produces AMPA receptor currents with rapid kinetics in these NG2-immunoreactive glial cells (NG2+ cells) in cerebellar slices. NG2+ cells may receive up to 70 discrete inputs from one CF and, unlike mature Purkinje cells, are often innervated by multiple CFs. Paired Purkinje cell-NG2+ cell recordings show that one CF can innervate both cell types. CF boutons make direct synaptic junctions with NG2+ cell processes, indicating that this rapid neuron-glia signaling occurs at discrete sites rather than through ectopic release at CF-Purkinje cell synapses. This robust activation of Ca2+-permeable AMPA receptors in NG2+ cells expands the influence of the olivocerebellar projection to this abundant class of glial progenitors.

Spike timing of dendrite-targeting bistratified cells during hippocampal network oscillations in vivo.

Behavior-contingent network oscillations bring about transient, functionally coherent neuronal assemblies in the cerebral cortex, including the hippocampus. Inhibitory input on and close to the soma is believed to phase intrinsic oscillations and output of pyramidal cells, but the function of GABA release to pyramidal cell dendrites remains unknown. We recorded the oscillation-locked spike timing of identified bistratified interneurons in rats. These cells mainly innervated small dendritic shafts of pyramidal cells co-aligned with the glutamatergic Schaffer collateral/commissural input. During theta oscillations, bistratified cells fired at a phase when, on average, pyramidal cell dendrites are most hyperpolarized. Interneurons targeting the perisomatic domain discharge at an earlier phase. During sharp wave-associated ripples, bistratified cells fired with high frequency and in-phase with basket cells, on average 1-2 ms after the discharges in pyramidal cell somata and dendrites. Our results indicate that bistratified cells rhythmically modulate glutamatergic input to the dendrites of pyramidal cells to actively promote the precise input/output transformation during network oscillations.

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 mGluR1alpha-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 type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I 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.

Brain-state- and cell-type-specific firing of hippocampal interneurons in vivo.

Neural-network oscillations at distinct frequencies have been implicated in the encoding, consolidation and retrieval of information in the hippocampus. Some GABA (gamma-aminobutyric acid)-containing interneurons fire phase-locked to theta oscillations (4-8 Hz) or to sharp-wave-associated ripple oscillations (120-200 Hz), which represent different behavioural states. Interneurons also entrain pyramidal cells in vitro. The large diversity of interneurons poses the question of whether they have specific roles in shaping distinct network activities in vivo. Here we report that three distinct interneuron types--basket, axo-axonic and oriens-lacunosum-moleculare cells--visualized and defined by synaptic connectivity as well as by neurochemical markers, contribute differentially to theta and ripple oscillations in anaesthetized rats. The firing patterns of individual cells of the same class are remarkably stereotyped and provide unique signatures for each class. We conclude that the diversity of interneurons, innervating distinct domains of pyramidal cells, emerged to coordinate the activity of pyramidal cells in a temporally distinct and brain-state-dependent manner.

Enrichment of mGluR7a in the presynaptic active zones of GABAergic and non-GABAergic terminals on interneurons in the rat somatosensory cortex.

The release of glutamate and GABA is modulated by presynaptic metabotropic glutamate receptors (mGluRs). We used immunocytochemical methods to define the location of the group III receptor mGluR7a in glutamatergic and GABAergic terminals innervating GABAergic interneurons and pyramidal cells. Immunoreactivity for mGluR7a was localized in the presynaptic active zone of both identified GABAergic and presumed glutamatergic terminals. Terminals innervating dendritic spines showed a variable level of receptor immunoreactivity, ranging from immunonegative to strongly immunopositive. The frequency of strongly mGluR7a positive terminals innervating the soma and dendrites of mGluR1 alpha/somatostatin-expressing interneurons was very high relative to other neurons. On dendrites that received mGluR7a-enriched glutamatergic innervation, at least 80% of GABAergic terminals were immunopositive for mGluR7a. On such dendrites virtually all (95%) vasoactive intestinal polypeptide (VIP) positive (GABAergic) terminals were enriched in mGluR7a. The targets of VIP/mGluR7a-expressing terminals were mainly (88%) mGluR1 alpha-expressing interneurons, which were mostly somatostatin immunopositive. Parvalbumin positive terminals were immunonegative for mGluR7a. Some parvalbumin immunoreactive dendrites received strongly mGluR7a positive terminals. The subcellular location, as well as the cell type and synapse-specific distribution of mGluR7a in isocortical neuronal circuits, is homologous to its distribution in the hippocampus. The specific location of mGluR7a in the presynaptic active zone of both glutamatergic and GABAergic synapses may be related to the proximity of calcium channels and the vesicle fusion machinery. The enrichment of mGluR7a in the main GABAergic, as well as in the glutamatergic, innervation of mGluR1 alpha/somatostatin-expressing interneurons suggests that their activation is under unique regulation by extracellular glutamate.

Cell type- and input-specific differences in the number and subtypes of synaptic GABA(A) receptors in the hippocampus.

Networks of parvalbumin (PV)-expressing basket cells are implicated in synchronizing cortical neurons at various frequencies, through GABA(A) receptor-mediated synaptic action. These cells are interconnected by GABAergic synapses and gap junctions, and converge with a different class of cholecystokinin-expressing, PV-negative basket cells onto pyramidal cells. To define the molecular specializations in the synapses of the two basket cell populations, we used quantitative electron microscopic immunogold localization of GABA(A) receptors. Synapses formed by PV-positive basket cells on the somata of pyramidal cells had several-fold higher density of alpha1 subunit-containing receptors than synapses made by PV-negative basket cells, most of which were immunonegative. The density of the beta2/3 subunits was similar in the two populations of synapse, indicating similar overall receptor density. Synapses interconnecting parvalbumin-expressing basket cells contained a 3.6 times higher overall density of GABA(A) receptor (beta2/3 subunits) and 3.2 times higher density of alpha1 subunit labeling compared with synapses formed by boutons of PV-positive basket cells on pyramidal cells. Thus, PV-positive basket cells mainly act through alpha1 subunit-containing GABA(A) receptors, but the receptor density depends on the postsynaptic cell type. These observations, together with previously reported enrichment of the alpha2 subunit-containing receptors in synapses made by PV-negative basket cells, indicate that the number and subtypes of GABA(A) receptors present in different synapse populations are regulated by both presynaptic and postsynaptic influences. The high number of GABA(A) receptors in synapses on basket cells might contribute to the precisely timed phasing of basket cell activity.