Johannes A. van Hooft

Neurogenesis and widespread forebrain migration of distinct GABAergic neurons from the postnatal subventricular zone

Most forebrain GABAergic interneurons in rodents are born during embryonic development in the ganglionic eminences (GE) and migrate tangentially into the cortical plate. A subset, however, continues to be generated postnatally in the subventricular zone (SVZ). These interneurons populate the olfactory bulb (OB) reached via migration in the rostral migratory stream (RMS). Employing transgenic mice expressing EGFP in 5-HT3-positive neurons, we identified additional migratory pathways in the early postnatal brain. Time-lapse imaging experiments revealed massive migration of EGFP-positive cells from the SVZ into numerous forebrain regions, including cortex, striatum, and nucleus accumbens. The neuronal fate of the migratory EGFP-labeled cells was indicated by their doublecortin (DCX) expression. Birthdating experiments, by using 5-bromo-2′-deoxyuridine (BrdU) and retrovirus-based experiments, provided evidence that migrating neuroblasts were born in the SVZ postnatally and developed a distinct GABAergic phenotype. Our results demonstrate that the SVZ is a reservoir of GABAergic interneurons not only for the OB, but also for other cortical and subcortical areas.

Serotonin 3A Receptor Subtype as an Early and Protracted Marker of Cortical Interneuron Subpopulations

To identify neocortical neurons expressing the type 3 serotonergic receptor, here we used transgenic mice expressing the enhanced green fluorescent protein (GFP) under the control of the 5-HT3A promoter (5-HT3A:GFP mice). By means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reversed-transcriptase polymerase chain reaction on acute brain slices of 5-HT3A:GFP mice, we identified 2 populations of 5-HT3A-expressing interneurons within the somatosensory cortex. The first population was characterized by the frequent expression of the vasoactive intestinal peptide and a typical bipolar/bitufted morphology, whereas the second population expressed predominantly the neuropeptide Y and exhibited more complex dendritic arborizations. Most interneurons of this second group appeared very similar to neurogliaform cells according to their electrophysiological, molecular, and morphological properties. The combination of 5-bromo-2-deoxyuridine injections with 5-HT3A mRNA detection showed that cortical 5-HT3A interneurons are generated around embryonic day 14.5. Although at this stage the 5-HT3A receptor subunit is expressed in both the caudal ganglionic eminence and the entopeduncular area, homochronic in utero grafts experiments revealed that cortical 5-HT3A interneurons are mainly generated in the caudal ganglionic eminence. This protracted expression of the 5-HT3A subunit allowed us to study specific cortical interneuron populations from their birth to their final functional phenotype.

5-HT3 receptors and neurotransmitter release in the CNS: a nerve ending story?

Serotonin (5-HT) 5-HT(3) receptors are ligand-gated ion channels, which are generally thought to be involved in the presynaptic modulation of neurotransmitter release. However, analysis of published data reveals that most of the evidence for the alleged presynaptic role of 5-HT(3) receptors in modulating CNS neurotransmitter release is not compelling. Nevertheless, 5-HT(3) receptors are present in nerve terminals from some brain regions. The increased basic knowledge of the cellular physiology of central 5-HT(3) receptor ligand-gated ion channels provides opportunities for a detailed characterization of the specific presynaptic effects of 5-HT(3) receptors. Such reconsideration is required for the full appreciation of the functional role of 5-HT(3) receptors in the CNS.

The N-terminal region of reelin regulates postnatal dendritic maturation of cortical pyramidal neurons

Cajal-Retzius cells, located in layer I of the cortex, synthesize and secrete the glycoprotein reelin, which plays a pivotal role in neuronal migration during embryonic development. Cajal-Retzius cells persist after birth, but their postnatal role is unknown. Here we show that Cajal-Retzius cells receive a major excitatory synaptic input via serotonin 5-HT3 receptors. Blocking this input using pharmacological tools or neutralization of reelin signaling results in hypercomplexity of apical, but not basal, dendrites of cortical layer II/III pyramidal neurons. A similar hypercomplexity is observed in the cortex of the 5-HT3A receptor knockout mouse. The increased dendritic complexity can be rescued by application of recombinant full-length reelin or its N-terminal fragment, but not by the central fragment of reelin, and involves a signal transduction pathway independent of the activation of the canonical reelin receptors. Taken together, our results reveal a novel role of serotonin, Cajal-Retzius cells, and reelin in the postnatal maturation of the cortex.

Serotonin 5-HT 3 receptors in the central nervous system

The 5-HT3 receptor is a ligand-gated ion channel activated by serotonin (5-HT). Although originally identified in the peripheral nervous system, the 5-HT3 receptor is also ubiquitously expressed in the central nervous system. Sites of expression include several brain stem nuclei and higher cortical areas such as the amygdala, hippocampus, and cortex. On the subcellular level, both presynaptic and postsynaptic 5-HT3 receptors can be found. Presynaptic 5-HT3 receptors are involved in mediating or modulating neurotransmitter release. Postsynaptic 5-HT3 receptors are preferentially expressed on interneurons. In view of this specific expression pattern and of the well-established role of 5-HT as a neurotransmitter shaping development, we speculate that 5-HT3 receptors play a role in the formation and function of cortical circuits.

Gabapentin inhibits presynaptic Ca 2+ influx and synaptic transmission in rat hippocampus and neocortex

Gabapentin is a widely used drug with anticonvulsant, antinociceptive and anxiolytic properties. Although it has been previously shown that Gabapentin binds with high affinity to the alpha(2)delta subunit of voltage-operated Ca(2+) channels (VOCC), little is known about the functional consequences of this interaction. Here, we investigated the effect of Gabapentin on VOCCs and synaptic transmission in rat hippocampus and neocortex using whole-cell patch clamp and confocal imaging techniques. Gabapentin (100-300 microM) did not affect the peak amplitude or voltage-dependency of VOCC currents recorded from either dissociated or in situ neocortical and hippocampal pyramidal cells. In contrast, Gabapentin inhibited K(+)-evoked increases in [Ca(2+)] in a subset of synaptosomes isolated from rat hippocampus and neocortex in a dose-dependent manner, with an apparent half-maximal inhibitory effect at approximately 100 nM. In hippocampal slices, Gabapentin (300 microM) inhibited the amplitude of evoked excitatory- and inhibitory postsynaptic currents recorded from CA1 pyramidal cells by 30-40%. Taken together, the results suggest that Gabapentin selectively inhibits Ca(2+) influx by inhibiting VOCCs in a subset of excitatory and inhibitory presynaptic terminals, thereby attenuating synaptic transmission.

Different levels of Ih determine distinct temporal integration in bursting and regular-spiking neurons in rat subiculum.

Pyramidal neurons in the subiculum typically display either bursting or regular‐spiking behaviour. Although this classification into two neuronal classes is well described, it is unknown how these two classes of neurons contribute to the integration of input to the subiculum. Here, we report that bursting neurons posses a hyperpolarization‐activated cation current (Ih) that is two‐fold larger (conductance, 5.3 ± 0.5 nS) than in regular‐spiking neurons (2.2 ± 0.6 nS), whereas Ih exhibits similar voltage‐dependent and kinetic properties in both classes of neurons. Bursting and regular‐spiking neurons display similar morphology. The difference in Ih between the two classes of neurons is not responsible for the distinct firing patterns, as neither pharmacological blockade of Ih nor enhancement of Ih using a dynamic clamp affects the qualitative firing patterns. Instead, the difference in Ih between bursting and regular‐spiking neurons determines the temporal integration of evoked synaptic input from the CA1 area. In response to stimulation at 50 Hz, bursting neurons, with a large Ih, show ∼50% less temporal summation than regular‐spiking neurons. The amount of temporal summation in both neuronal classes is equal after pharmacological blockade of Ih. A computer simulation model of a subicular neuron with the properties of either a bursting or a regular‐spiking neuron confirmed the pivotal role of Ih in temporal integration of synaptic input. These data suggest that in the subicular network, bursting neurons are better suited to discriminate the content of high‐frequency input, such as that occurring during gamma oscillations, than regular‐spiking neurons.

Serotonin 5-HT3 receptors in rat CA1 hippocampal interneurons: functional and molecular characterization

The molecular makeup of the serotonin 5‐HT3 receptor (5‐HT3R) channel was investigated in rat hippocampal CA1 interneurons in slices using single‐cell RT‐PCR and patch‐clamp recording techniques. We tested for the expression of the 5‐HT3A (both short and long splice variants) and 5‐HT3B subunits, as well as the expression of the α4 subunit of the neuronal nicotinic ACh receptors (nAChRs), the latter of which has been shown to co‐assemble with the 5‐HT3A subunit in heterologous expression systems. Both the 5‐HT3A‐short and α4‐nAChR subunits were expressed in these interneurons, but we could not detect any expression of either the 5‐HT3B or the 5‐HT3A‐long subunits. Furthermore, there was a strong tendency for the 5‐HT3A‐short and α4‐nAChR subunits to be co‐expressed in individual interneurons. To assess whether there was any functional evidence for co‐assembly between the 5‐HT3A‐short and α4‐nAChR subunits, we used the sulphydryl agent 2‐aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5‐HT3Rs that contain the α4‐nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5‐HT3R‐mediated responses, which is consistent with the notion that the α4‐nAChR subunit co‐assembles with the 5‐HT3A subunit to form a native heteromeric 5‐HT3R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single‐channel properties of the 5‐HT3R were investigated in outside‐out patches. No resolvable single‐channel currents were observed. Using non‐stationary fluctuation analysis, we obtained an estimate of the single‐channel conductance of 4 pS, which is well below that expected for channels containing both the 5‐HT3A and 5‐HT3B subunits.

5-HT3 receptors in the CNS: 3B or not 3B?

5-HT(3) receptors are widely distributed in the CNS and PNS where they participate in a variety of physiological processes. Native 5-HT(3) receptors in the CNS display functional and pharmacological heterogeneity, suggesting the existence of multiple receptor subunits. However, recent evidence suggests that of the two known subunits only the 5-HT(3A) receptor subunit (and not the 5-HT(3B) receptor subunit) is functionally present in the CNS. The molecular makeup of the 5-HT(3) receptor therefore remains an open question.

Serotonin receptor 3A controls interneuron migration into the neocortex.

Neuronal excitability has been shown to control the migration and cortical integration of reelin-expressing cortical interneurons (INs) arising from the caudal ganglionic eminence (CGE), supporting the possibility that neurotransmitters could regulate this process. Here we show that the ionotropic serotonin receptor 3A (5-HT3AR) is specifically expressed in CGE-derived migrating interneurons and upregulated while they invade the developing cortex. Functional investigations using calcium imaging, electrophysiological recordings and migration assays indicate that CGE-derived INs increase their response to 5-HT3AR activation during the late phase of cortical plate invasion. Using genetic loss-of-function approaches and in vivo grafts, we further demonstrate that the 5-HT3AR is cell autonomously required for the migration and proper positioning of reelin-expressing CGE-derived INs in the neocortex. Our findings reveal a requirement for a serotonin receptor in controlling the migration and laminar positioning of a specific subtype of cortical IN.