Distinct temporal expression of 5-HT(1A) and 5-HT(2A) receptors on cerebellar granule cells in mice.

Serotonin plays an important role of controlling the physiology of the cerebellum. However, serotonin receptor expression has not been fully studied in the developing cerebellum. We have recently shown that cerebellar granule cells transiently express 5-HT3 receptors. In the present study, we investigate expression of 5-HT1 and 5-HT2 receptors in the mouse cerebellum both during postnatal development and in juvenile mice. Here, we show for the first time that 5-HT1A and 5-HT2A receptors are present on cerebellar granule cells with a distinct temporal expression pattern: 5-HT1A receptors are expressed only during the first 2 weeks, while 5-HT2A receptor expression persists until at least 8 weeks after birth. Because of its prolonged expression pattern, we investigated the electrophysiological properties of the 5-HT2A receptor. 5-HT2A receptors expressed by cerebellar granule cells promote stability by reducing variability of the synaptic response, and they modulate the paired-pulse ratio of the parallel fibre-Purkinje cell synapse. Furthermore, pharmacological block of 5-HT2A receptors enhances short-term synaptic plasticity at the parallel fibre-Purkinje cell synapse. We thus show a novel role for serotonin in controlling function of the cerebellum via 5-HT2A receptors expressed by cerebellar granule cells.

Serotonergic control of Purkinje cell maturation and climbing fibre elimination by 5-HT3 receptors in the juvenile mouse cerebellum.

Functional serotonin 3 (5-HT3) receptors are transiently expressed by cerebellar granule cells during early postnatal development, where they modulate short-term synaptic plasticity at the parallel fibre-Purkinje cell synapse. Here, we show that serotonin controls maturation of Purkinje cells in the mouse cerebellum. The 5-HT3 receptors regulate morphological maturation of Purkinje cells during early postnatal development, and this effect is mediated by the glycoprotein reelin. Using whole-cell patch-clamp recordings we also investigated physiological development of Purkinje cells in 5-HT3A receptor knockout mice during early postnatal development, and found abnormal physiological maturation, characterized by a more depolarized resting membrane potential, an increased input resistance and the ability to fire action potentials upon injection of a depolarizing current at an earlier age. Furthermore, short-term synaptic plasticity was impaired at both the parallel fibre-Purkinje cell and the climbing fibre-Purkinje cell synapses, and both the amplitude and the frequency of spontaneous miniature events recorded from Purkinje cells were increased. The expedited morphological and physiological maturation affects the whole cerebellar cortical network, as indicated by delayed climbing fibre elimination in 5-HT3A receptor knockout mice. There was no difference between wild-type and 5-HT3A receptor knockout mice in any of the morphological or physiological properties described above at later ages, indicating a specific time window during which serotonin regulates postnatal development of the cerebellum via 5-HT3 receptors expressed by granule cells.

Altered dendritic complexity affects firing properties of cortical layer 2/3 pyramidal neurons in mice lacking the 5-HT3A receptor.

We have previously shown that the serotonergic input on Cajal-Retzius cells, mediated by 5-HT(3) receptors, plays an important role in the early postnatal maturation of the apical dendritic trees of layer 2/3 pyramidal neurons. We reported that knockout mice lacking the 5-HT(3A) receptor showed exuberant apical dendrites of these cortical pyramidal neurons. Because model studies have shown the role of dendritic morphology on neuronal firing pattern, we used the 5-HT(3A) knockout mouse to explore the impact of dendritic hypercomplexity on the electrophysiological properties of this specific class of neurons. Our experimental results show that hypercomplexity of the apical dendritic tuft of layer 2/3 pyramidal neurons affects neuronal excitability by reducing the amount of spike frequency adaptation. This difference in firing pattern, related to a higher dendritic complexity, was accompanied by an altered development of the afterhyperpolarization slope with successive action potentials. Our abstract and realistic neuronal models, which allowed manipulation of the dendritic complexity, showed similar effects on neuronal excitability and confirmed the impact of apical dendritic complexity. Alterations of dendritic complexity, as observed in several pathological conditions such as neurodegenerative diseases or neurodevelopmental disorders, may thus not only affect the input to layer 2/3 pyramidal neurons but also shape their firing pattern and consequently alter the information processing in the cortex.

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-HT(3) 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-HT(3A) 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.

Transient expression of functional serotonin 5-HT3 receptors by glutamatergic granule cells in the early postnatal mouse cerebellum.

The serotonin 5-HT(3) receptor is the only ligand-gated ion channel activated by serotonin and is expressed by GABAergic interneurons in many brain regions, including the cortex, amygdala and hippocampus. Furthermore, 5-HT(3) receptors are expressed by glutamatergic Cajal-Retzius cells in the cerebral cortex. We used 5-HT(3A)/enhanced green fluorescent protein (EGFP) transgenic mice to show that 5-HT(3) receptors are also ubiquitously expressed by glutamatergic granule cells in the cerebellum during the first three postnatal weeks. Using whole-cell patch clamp recordings, we show that local application of either serotonin or the selective 5-HT(3) receptor agonist SR57227A to granule cells results in a small inward current, demonstrating a post- and/or extrasynaptic localisation of the 5-HT(3) receptors. Functional 5-HT(3) receptors were also observed presynaptically at the parallel fibre-Purkinje cell synapse. Pharmacological block using the selective 5-HT(3) receptor antagonist tropisetron induced a reduction in the frequency of miniature synaptic events recorded from Purkinje cells. Paired-pulse stimulation of parallel fibres on whole-cell voltage clamped Purkinje cells from 1-week-old mice did not yet show synaptic plasticity. In the presence of tropisetron, the parallel fibre-Purkinje cell synapse showed paired-pulse depression. Taken together, these results show that functional 5-HT(3) receptors are present during early postnatal development in the cerebellum, where they modulate synaptic plasticity.

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-HT(3A) promoter (5-HT(3A):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-HT(3A):GFP mice, we identified 2 populations of 5-HT(3A)-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-HT(3A) mRNA detection showed that cortical 5-HT(3A) interneurons are generated around embryonic day 14.5. Although at this stage the 5-HT(3A) receptor subunit is expressed in both the caudal ganglionic eminence and the entopeduncular area, homochronic in utero grafts experiments revealed that cortical 5-HT(3A) interneurons are mainly generated in the caudal ganglionic eminence. This protracted expression of the 5-HT(3A) subunit allowed us to study specific cortical interneuron populations from their birth to their final functional phenotype.

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-HT(3)-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.

On the voltage-dependent Ca2+ block of serotonin 5-HT3 receptors: a critical role of intracellular phosphates.

Natively expressed serotonin 5-HT(3) receptors typically possess a negative-slope conductance region in their I-V curve, due to a voltage-dependent block by external Ca(2+) ions. However, in almost all studies performed with heterologously expressed 5-HT(3) receptors, this feature was not observed. Here we show that mere addition of ATP to the pipette solution is sufficient to reliably observe a voltage-dependent block in homomeric (h5-HT(3A)) and heteromeric (h5-HT(3AB)) receptors expressed in HEK293 cells. A similar block was observed with a plethora of molecules containing a phosphate moiety, thus excluding a role of phosphorylation. A substitution of three arginines in the intracellular vestibule of 5-HT(3A) with their counterpart residues from the 5-HT(3B) subunit (RRR-QDA) was previously shown to dramatically increase single channel conductance. We find this mutant to have a linear I-V curve that is unaffected by the presence of ATP, with a fractional Ca(2+) current (Pf%) that is reduced (1.8 +/- 0.2%) compared to that of the homomeric receptor (4.1 +/- 0.2%), and similar to that of the heteromeric form (2.0 +/- 0.3%). Moreover, whereas ATP decreased the Pf% of the homomeric receptor, this was not observed with the RRR-QDA mutant. Finally, ATP was found to be critical for voltage-dependent channel block also in hippocampal interneurons that natively express 5-HT(3) receptors. Taken together, our results indicate a novel mechanism by which ATP, and similar molecules, modulate 5-HT(3) receptors via interactions with the intracellular vestibule of the receptor.

Mest/Peg1 Is Essential for the Development and Maintenance of a SNc Neuronal Subset.

Mesodiencephalic dopaminergic (mdDA) neurons originate at the floor plate and floor plate-basal plate boundary of the midbrain ventricular zone. During development mdDA neurons are specified by a unique set of transcription factors and signaling cascades, to form the different molecular subsets of the mdDA neuronal population. In a time series micro-array study performed previously, mesoderm specific transcript (Mest) was found to be one of the most upregulated genes during early mdDA neuronal development. Here, we show that Mest transcript is expressed in the midbrain throughout development and becomes restricted to the substantia nigra (SNc) at late stages. In Mest KO animals mdDA neurons are progressively lost in the adult, mostly affecting the SNc, reflected by a 50% decrease of TH protein and DA release in the striatum and a reduction of climbing behavior. Analysis of Lrp6 KO embryos suggest a subtle opposite phenotype to the Mest KO, hinting toward the possibility that specific loss of mdDA neurons in Mest ablated animals could be due to affected WNT-signaling. Interestingly, the mdDA neuronal region affected by the loss of Mest remains relatively unaffected in Pitx3 mutants, suggesting that both genes are essential for the development and/or maintenance of different mdDA neuronal subsets within the SNc. Overall, the neuroanatomical and phenotypical consequences detected upon the loss of Mest, resemble the loss of SNc neurons and loss of movement control as seen in Parkinson's Disease (PD), suggesting that the Mest mouse model may be used as a model-system for PD.

Engrailed 1 shapes the dopaminergic and serotonergic landscape through proper isthmic organizer maintenance and function.

The isthmic organizer (IsO) is a signaling center that specifies the correct and distinct embryonic development of the dopaminergic midbrain and serotonergic hindbrain. The IsO is a linear boundary between the two brain regions, emerging at around embryonic day 7-8 of murine embryonic development, that shapes its surroundings through the expression of instructive signals such as Wnt and growth factors. Homeobox transcription factor engrailed 1 (En1) is present in midbrain and rostral hindbrain (i.e. rhombomere 1, R1). Its expression spans the IsO, and it is known to be an important survival factor for both dopaminergic and serotonergic neurons. Erroneous composition of dopaminergic neurons in the midbrain or serotonergic neurons in the hindbrain is associated with severe pathologies such as Parkinson's disease, depression or autism. Here we investigated the role of En1 in early mid-hindbrain development, using multiple En1-ablated mouse models as well as lineage-tracing techniques, and observed the appearance of ectopic dopaminergic neurons, indistinguishable from midbrain dopaminergic neurons based on molecular profile and intrinsic electrophysiological properties. We propose that this change is the direct result of a caudal relocation of the IsO as represented by ectopic presence of Fgf8, Otx2, Wnt1 and canonical Wnt-signalling. Our work suggests a newly-discovered role for En1: the repression of Otx2, Wnt1 and canonical Wnt-signaling in R1. Overall, our results suggest that En1 is essential for proper IsO maintenance and function.

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.

Low affinity block of native and cloned hyperpolarization-activated Ih channels by Ba2+ ions.

Ba2+ is commonly used to discriminate two classes of ion currents. The classical inward-rectifying K+ current, I(Kir), is blocked by low millimolar concentrations of Ba2+, whereas the hyperpolarization-activated cation current, I(h), is assumed not to be sensitive to Ba2+. Here we investigated the effects of Ba2+ on I(h) currents recorded from rat hippocampal CA1 pyramidal neurons, and on cloned I(h) channels composed of either HCN1 or HCN2 subunits transiently expressed in Human Embryonic Kidney (HEK) 293 cells. The results show that low millimolar concentrations of Ba2+ reduce the maximal I(h) conductance (IC50 approximately 3-5 mM) in both CA1 pyramidal neurons and in HEK 293 cells without specificity for HCN1 or HCN2 subunits. In addition, Ba2+ decreases the rate of activation and increases the rate of deactivation of I(h) currents. Neither the half-maximal voltage of activation, V(h), nor the reversal potential of the I(h) channels were affected by Ba2+. The combined results suggest that B2+, at concentrations commonly used to block I(Kir) currents, also reduces the conductance of I(h) channels without subunit specificity, and affects the kinetics of I(h) channel gating.

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.

Fast Green FCF (Food Green 3) inhibits synaptic activity in rat hippocampal interneurons.

The effects of the certified food dye Fast Green FCF (Food Green 3) on miniature synaptic events in whole-cell voltage clamped hippocampal interneurons were examined. Fast Green FCF reduced the frequency, but did not affect the amplitude or kinetics, of miniature synaptic events in a dose-dependent manner. At 1 mg/ml (1.2 mM), the frequency was reduced to 21% of control. The half-maximum effective concentration was estimated to be 200 microg/ml (250 microM). In contrast, the dye Phenol Red neither affected frequency, amplitude nor kinetics of synaptic events. The results suggest that Fast Green FCF acts at a presynaptic locus, thereby inhibiting the release of neurotransmitter.

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-HT(3A)R) 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-HT(3A)R 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-HT(3A)R 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.

Subset specification of central serotonergic neurons.

The last decade the serotonin (5-hydroxytryptamine; 5-HT) system has received enormous attention due to its role in regulation of behavior, exemplified by the discovery that increased 5-HT tone in the central nervous system is able to alleviate affective disorders. Here, we review the developmental processes, with a special emphasis on subset specification, leading to the formation of the 5-HT system in the brain. Molecular classification of 5-HT neuronal groups leads to the definition of two independent rostral groups positioned in rhombomere 1 and 2/3 and a caudal group in rhombomere 5-8. In addition, more disperse refinement of these subsets is present as shown by the selective expression of the 5-HT1A autoreceptor, indicating functional diversity between 5-HT subsets. The functional significance of the molecular coding differences is not well known and the molecular basis of described specific connectivity patterns remain to be elucidated. Recent developments in genetic lineage tracing models will provide these data and form a major step-up toward the full understanding of the importance of developmental programming and function of 5-HT neuronal subsets.

The serotonin 5-HT3 receptor: a novel neurodevelopmental target.

Serotonin (5-hydroxytryptamine, 5-HT), next to being an important neurotransmitter, recently gained attention as a key-regulator of pre- and postnatal development in the mammalian central nervous system (CNS). Several receptors for 5-HT are expressed in the developing brain including a ligand-gated ion channel, the 5-HT3 receptor. Over the past years, evidence has been accumulating that 5-HT3 receptors are involved in the regulation of neurodevelopment by serotonin. Here, we review the spatial and temporal expression patterns of 5-HT3 receptors in the pre- and early postnatal rodent brain and its functional implications. First, 5-HT3 receptors are expressed on GABAergic interneurons in neocortex and limbic structures derived from the caudal ganglionic eminence. Mature inhibitory GABAergic interneurons fine-tune neuronal excitability and thus are crucial for the physiological function of the brain. Second, 5-HT3 receptors are expressed on specific glutamatergic neurons, Cajal-Retzius cells in the cortex and granule cells in the cerebellum, where they regulate morphology, positioning, and connectivity of the local microcircuitry. Taken together, the 5-HT3 receptor emerges as a potential key-regulator of network formation and function in the CNS, which could have a major impact on our understanding of neurodevelopmental disorders in which 5-HT plays a role.

Prenatal fluoxetine exposure induces life-long serotonin 5-HT3 receptor-dependent cortical abnormalities and anxiety-like behaviour.

Selective serotonin reuptake inhibitors (SSRIs) are the first choice of drugs to treat depression and anxiety during pregnancy. However, there is evidence that in utero exposure to SSRIs leads to adverse effects in offspring. Here we show that in mice, the adverse effects of the widely used antidepressant and SSRI fluoxetine are critically dependent on the 5-HT(3) receptor, the only ligand-gated ion channel in the family of serotonin receptors. In utero exposure to fluoxetine induces anxiety-like behavior in wildtype, but not in mice lacking the 5-HT(3) receptor. In addition to this behavioral phenotype, these mice show life-long abnormalities of cortical cytoarchitecture, which can be reversed in vitro by pharmacological block of 5-HT(3) receptors. Moreover, the effect of fluoxetine on the development of cortical neurons is absent in 5-HT(3) receptor knockout mice. These findings pinpoint the pivotal role of serotonergic signaling during development and provide a novel basis to investigate the adverse effects of the use of fluoxetine during pregnancy. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Alterations in Apical Dendrite Bundling in the Somatosensory Cortex of 5-HT(3A) Receptor Knockout Mice.

In various species and areas of the cerebral cortex, apical dendrites of pyramidal neurons form clusters which extend through several layers of the cortex also known as dendritic bundles. Previously, it has been shown that 5-HT(3A) receptor knockout mice show hypercomplex apical dendrites of cortical layer 2/3 pyramidal neurons, together with a reduction in reelin levels, a glycoprotein involved in cortical development. Other studies showed that in the mouse presubicular cortex, reelin is involved in the formation of modular structures. Here, we compare apical dendrite bundling in the somatosensory cortex of wildtype and 5-HT(3A) receptor knockout mice. Using a microtubule associated protein-2 immunostaining to visualize apical dendrites of pyramidal neurons, we compared dendritic bundle properties of wildtype and 5-HT(3A) receptor knockout mice in tangential sections of the somatosensory cortex. A Voronoi tessellation was performed on immunostained tangential sections to determine the spatial organization of dendrites and to define dendritic bundles. In 5-HT(3A) receptor knockout mice, dendritic bundle surface was larger compared to wildtype mice, while the number and distribution of reelin-secreting Cajal-Retzius cells was similar for both groups. Together with previously observed differences in dendritic complexity of cortical layer 2/3 pyramidal neurons and cortical reelin levels, these results suggest an important role for the 5-HT(3) receptor in determining the spatial organization of cortical connectivity in the mouse somatosensory cortex.

Impaired Social Behavior in 5-HT(3A) Receptor Knockout Mice.

The 5-HT(3) receptor is a ligand-gated ion channel expressed on interneurons throughout the brain. So far, analysis of the 5-HT(3A) knockout mouse revealed changes in nociceptive processing and a reduction in anxiety related behavior. Recently, it was shown that the 5-HT(3) receptor is also expressed on Cajal-Retzius cells which play a key role in cortical development and that knockout mice lacking this receptor showed aberrant growth of the dendritic tree of cortical layer II/III pyramidal neurons. Other mouse models in which serotonergic signaling was disrupted during development showed similar morphological changes in the cortex, and in addition, also deficits in social behavior. Here, we subjected male and female 5-HT(3A) knockout mice and their non-transgenic littermates to several tests of social behavior. We found that 5-HT(3A) knockout mice display impaired social communication in the social transmission of food preference task. Interestingly, we showed that in the social interaction test only female 5-HT(3A) knockout mice spent less time in reciprocal social interaction starting after 5 min of testing. Moreover, we observed differences in preference for social novelty for male and female 5-HT(3A) knockout mice during the social approach test. However, no changes in olfaction, exploratory activity and anxiety were detected. These results indicate that the 5-HT(3A) knockout mouse displays impaired social behavior with specific changes in males and females, reminiscent to other mouse models in which serotonergic signaling is disturbed in the developing brain.