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. 2010 Dec 15;30(50):16796-808.
doi: 10.1523/JNEUROSCI.1869-10.2010.

The largest group of superficial neocortical GABAergic interneurons expresses ionotropic serotonin receptors

SooHyun Lee  1 , Jens Hjerling-Leffler, Edward Zagha, Gord Fishell, Bernardo Rudy
Affiliations

The largest group of superficial neocortical GABAergic interneurons expresses ionotropic serotonin receptors

SooHyun Lee et al. J Neurosci. .

Abstract

A highly diverse population of neocortical GABAergic inhibitory interneurons has been implicated in multiple functions in information processing within cortical circuits. The diversity of cortical interneurons is determined during development and primarily depends on their embryonic origins either from the medial (MGE) or the caudal (CGE) ganglionic eminences. Although MGE-derived parvalbumin (PV)- or somatostatin (SST)-expressing interneurons are well characterized, less is known about the other types of cortical GABAergic interneurons, especially those of CGE lineage, because of the lack of specific neuronal markers for these interneuron subtypes. Using a bacterial artificial chromosome transgenic mouse line, we show that, in the somatosensory cortex of the mouse, the serotonin 5-hydroxytryptamine 3A (5-HT(3A)) receptor, the only ionotropic serotonergic receptor, is expressed in most, if not all, neocortical GABAergic interneurons that do not express PV or SST. Genetic fate mapping and neurochemical profile demonstrate that 5-HT(3A)R-expressing neurons include the entire spectrum of CGE-derived interneurons. We report that, in addition to serotonergic responsiveness via 5-HT(3A)Rs, acetylcholine also depolarizes 5-HT(3A)R-expressing neurons via nicotinic receptors. 5-HT(3A)R-expressing neurons in thalamocortical (TC) recipient areas receive weak but direct monosynaptic inputs from the thalamus. TC input depolarizes a subset of TC-recipient 5-HT(3A)R neurons as strongly as fast-spiking cells, in part because of their high input resistance. Hence, fast modulation of serotonergic and cholinergic transmission may influence cortical activity through an enhancement of GABAergic synaptic transmission from 5-HT(3A)R-expressing neurons during sensory process depending on different behavioral states.

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Figures

Figure 1.
Figure 1.
5HT3aR-BACEGFP cells of the somatosensory cortex are 5-HT3AR-expressing GABAergic interneurons. A, B, Double in situ hybridization for 5HT3aR and EGFP/GAD67. A, Double in situ for 5HT3aR and EGFP show complete overlap in the adult somatosensory cortex. B, All 5HT3aR-positive cells are also GAD67 positive. C, D, Immunohistochemistry for GFP and Satb2/Olig2. C, No overlap with pyramidal cell marker Satb2 confirms the interneuron identity. D, No EGFP-positive cells express oligodendrocyte marker Olig2. Scale bars, 100 μm. dISH, Double in situ hybridization; IHC, immunohistochemistry.
Figure 2.
Figure 2.
5-HT3AR-expressing cells are the third major group of cortical interneurons. A–C, Double in situ hybridization (dISH) for the three markers PV, SST, and 5HT3aR with pan-GABAergic marker Gad67. D, Cumulative graph showing the percentage of Gad67 cells expressing each marker. Note that 100 ± 5% of interneurons of all layers can be accounted for using these three markers and that 5-HT3AR-expressing neurons predominantly occupy superficial layers. Notably we observed some overlap in the in situ signal between SST and PV even though this cannot be seen at the protein level. Error bars represent SEM.
Figure 3.
Figure 3.
A–D, 5HT3aR-BACEGFP mice have labeled neurons in the CGE (as well as more ventral structures) but not the LGE or MGE. This EGFP expression reflects true 5HT3aR mRNA expression as shown by ISH for 5HT3aR (C). E–H, Genetic fate mapping confirms that 5-HT3AR-expressing cells in mature cortex are CGE derived. We used Nkx2.1-BACCre and Mash1-BACCreER, mouse lines in combination with the R26RtdRFP-reporter crossed onto the 5HT3aR-BACEGFP to label cells embryonically and follow their fate into the mature cortex. G, H, The complete lack of Nkx2.1-BACCre;R26RtdRFP and 5HT3aR-BACEGFP overlap shows that the cells are not of MGE origin, whereas the almost complete overlap with Mash1-BACCreER;R26RtdRFP-labeled interneurons confirms the CGE origin. CC1 was used as a marker to exclude RFP-expressing oligodendrocytes from quantization (H). Scale bars: B, D, 250 μm; E, 100 μm; F, 50 μm. Error bars represent SD.
Figure 4.
Figure 4.
Neurochemical marker expression profiles of 5-HT3AR-expressing cortical interneurons. A, Immunofluorescent histochemistry show the relationship of seven neuronal markers (CR, CCK, VIP, SST, PV, Rln, and NPY) and 5-HT3AR-positive neurons in somatosensory cortex (P21–P25) of 5HT3aR-BACEGFP mice (n = 3). B, The percentage of specific neuronal markers that is also positive for EGFP across all layers (EGFP/marker). C, Same as in B but analyzed per layer. D, The percentage of 5-HT3AR-expressing neurons expressing specific neuronal markers per individual layers (marker/EGFP). E, The proportion of 5-HT3AR-expressing neurons expressing specific neuronal markers across all layers (marker/EGFP), individual bars are displaced to illustrate the overlap caused by colocalization of markers. F, Same as E but analyzed per layer. Note that >85% of 5-HT3AR-expressing neurons were found to express at least one of the seven neuronal markers. Rln, Reelin; NPY, neuropeptide Y; CR, calretinin; VIP, vasoactive intestinal peptide; CCK, cholecystokinin; SOM, somatostatin; PV, parvalbumin. Scale bar, 100 μm. Error bars represent SEM.
Figure 5.
Figure 5.
Intrinsic electrophysiological properties and morphologies of 5-HT3AR-expressing cortical interneurons. A, Representative traces of voltage responses to 500 ms step current injection in current-clamp whole-cell configuration of six major subtypes of 5-HT3AR interneurons. B, Examples of morphologies of different subtypes reconstructed using Neurolucida tracing. Dendrite and soma are shown in blue and axon in red. LS1, Late spiking 1; LS2, late spiking 2; bNA1, burst nonadapting 1; bNA2, burst nonadapting 2; IS, irregular spiking; fAD, fast adapting; POA, preoptic area. Scale bar, 100 μm.
Figure 6.
Figure 6.
Functional expression of 5-HT3ARs and nAChRs in 5-HT3AR-expressing interneurons. Intrinsic firing patterns to step current injections recorded from three different types of cortical interneurons from 5HT3aR-BACEGFP (A1), B13 (B1), and GIN (C1) transgenic mice. Application of a 5-HT3AR selective agonist (mCPBG; 100 μm; 30 ms puffing) causes strong depolarization and evokes a burst of spikes in 5-HT3AR cells (A2) but not in FS (B2) or SST-expressing cells (C2) (30 ms puff indicated by red line below). D, Functional expression of nACh receptors in EGFP-positive neurons of 5HT3aR-BACEGFP mouse. D1, Carbachol application (1 mm) strongly depolarizes and evokes burst of spikes in a 5-HT3AR neuron recorded in whole-cell configuration in current clamp. The inset shows intrinsic firing pattern of recorded cell to step current injections. D2, Functional expression of nicotinic ACh receptors in EGFP-positive neurons of 5HT3aR-BACEGFP mouse. Nicotine puffing (100 μm for 30 ms) depolarizes the 5-HT3AR neuron (D2), but muscarine application (D3) to the same neuron did not change membrane potential, indicating 5-HT3AR interneurons express nicotinic AChRs (30 ms puff indicated by red line below). The insets in A1 and D1–D3 show schematics of the recording configuration.
Figure 7.
Figure 7.
Monosynaptic thalamocortical input to 5-HT3AR-expressing neurons. A, Voltage responses of 5-HT3AR (A1) and FS (A2) neurons to 500 ms step current injection. B, Weak monosynaptic TC input to 5-HT3AR-expressing neurons. Stimulation of TC afferents evokes smaller EPSCs onto a 5-HT3AR (B1) compared with a FS neuron (B2) recorded in the whole-cell voltage-clamp configuration (note difference in scale). C, TC stimulation depolarized a 5-HT3AR neuron (C1) to a similar degree as an FS neuron (C2). Under current-clamp configuration (V m = −65 mV), voltage responses to TC stimulation were recorded from the same example 5-HT3AR and FS neurons shown in B1 and B2, respectively. D, TC stimulation can elicit spikes in a 5-HT3AR neuron. Increased intensity of TC stimulation evoked spikes in a 5-HT3AR (D1) and a FS neuron (D2). Summary data comparing input resistance (E), evoked EPSC (F), paired pulse ratio (G), and evoked EPSP (H) of 5-HT3AR and FS neurons to 20 Hz TC stimulation. The open and filled symbols indicate 5-HT3AR and FS neurons, respectively. The bars indicate mean values. I, Relationship between input resistance (R m) and evoked membrane depolarization (eEPSP) normalized to EPSC in response to TC stimulation.
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