Classification of fusiform neocortical interneurons based on unsupervised clustering

Abstract

A classification of fusiform neocortical interneurons (n ‫؍‬ 60) was performed with an unsupervised cluster analysis based on the comparison of multiple electrophysiological and molecular parameters studied by patch-clamp and single-cell multiplex reverse transcription-PCR in rat neocortical acute slices. The multiplex reverse transcription-PCR protocol was designed to detect simultaneously the expression of GAD65, GAD67, calbindin, parvalbumin, calretinin, neuropeptide Y, vasoactive intestinal peptide (VIP), somatostatin (SS), cholecystokinin, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, N-methyl-D-aspartate, and metabotropic glutamate receptor subtypes. Three groups of fusiform interneurons with distinctive features were disclosed by the cluster analysis. The first type of fusiform neuron (n ‫؍‬ 12), termed regular spiking nonpyramidal (RSNP)-SS cluster, was characterized by a firing pattern of RSNP cells and by a high occurrence of SS. The second type of fusiform neuron (n ‫؍‬ 32), termed RSNP-VIP cluster, predominantly expressed VIP and also showed firing properties of RSNP neurons with accommodation profiles different from those of RSNP-SS cells. Finally, the last type of fusiform neuron (n ‫؍‬ 16) contained a majority of irregular spiking-VIPergic neurons. In addition, the analysis of glutamate receptors revealed cell-type-specific expression profiles. This study shows that combinations of multiple independent criteria define distinct neocortical populations of interneurons potentially involved in specific functions. B ecause, in part, of their diversity, the function of neuron subtypes in the physiology of the neocortex is still poorly understood. A better knowledge of the different neuronal populations that compose this heterogeneous brain structure may therefore contribute to elucidating their specific role.Attempts to classify neurons rely on several independent criteria (morphological, physiological, and molecular). In the neocortex, neurons are classified as pyramidal cells or nonpyramidal cells according to their morphology. Pyramidal cells accumulate glutamate and constitute the main class of excitatory projecting neurons (1). In contrast, nonpyramidal cells, also termed interneurons, are mainly inhibitory ␥-aminobutyric acid-ergic neurons with a short axon involved in local circuits (2) and have a large diversity of morphology (3). The expression of biochemical markers has been used to define different classes of nonpyramidal cells. The distribution of three calcium binding proteins, calbindin (CB), parvalbumin (PV), and calretinin (CR), and four neuropeptides, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), somatostatin (SS), and cholecystokinin (CCK), defines partially overlapping groups of interneurons (4 -12). In addition to this morphological and molecular diversity, nonpyramidal cells also have a large repertoire of firing behaviors (13-18), such as fast spiking (FS), regular spiking nonpyramidal (RSNP), or irregular spiking (IS).Some types of interneurons have b...

Selective, State-Dependent Activation of Somatostatin-Expressing Inhibitory Interneurons in Mouse Neocortex

“…These values-all larger than those reported by Beierlein et al (2003)-could be due to differences between layers 2/3 and 4. GIN cells share many characteristics with inhibitory interneurons identified in other studies as bitufted cells (Reyes et al 1998), regular-spiking nonpyramidal somatostatin-expressing neurons (Cauli et al 2000), and LTS (low-threshold-spiking) cells (Amitai et al 2002;Beierlein et al 2000Beierlein et al , 2003Gibson et al 1999;Kawaguchi and Kubota 1996).…”

Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis

“…These methods have recently been used to more objectively characterize neuronal populations in several brain areas such as neocortex and suprachiasmatic nucleus (Azouz et al, 1997, Pennartz et al, 1998, Cauli et al, 2000, Halabisky et al, 2004. A key element of cluster analysis is the selection of variables (electrophysiological parameters) to be considered in creating different subgroups.…”

“…The neurons spatially closest to each other are grouped together as a cluster. The analysis was performed using Ward's method which is based on the sum of squares between the clusters (Euclidean distances, summed over all variables) (Anderson et al, 1998) allowing an objective segregation of cell groups as previously reported (Pennartz et al, 1998, Cauli et al, 2000, Nowak et al, 2003. Electrophysiological variables were normalized to a common range (0-1) before performing the multidimentional analysis.…”

“…This technique can assist in uncovering data structure and providing a scheme for classifying neuronal subtypes (Pennartz et al, 1998, Nowak et al, 2003, Halabisky et al, 2004. This method consists of plotting individual objects (neurons) in multidimensional space according to similarities or dissimilarities of input Pennartz et al, 1998, Cauli et al, 2000, Nowak et al, 2003. Electrophysiological variables were normalized to a common range (0-1) before performing the multidimentional analysis.…”

A Subpopulation of Serotonergic Neurons That Do Not Express the 5-HT1A Autoreceptor

“…The methodology involving harvesting of cytoplasmic content and subsequent single-cell PCR amplification has been described previously. 37 Briefly, borosilicate glass pipettes (3−5 MΩ) were made in a HEKA PIP5 puller and filled with 8 μL of autoclaved RT-PCR internal solution (in mM): 144 K-gluconate, 3 MgCl 2 , 0.5 EGTA, 10 HEPES, pH 7.2 (285/295 mOsm). Single EGFP-positive neurons were approached with a pipet, and whole-cell recordings were obtained using a Multiclamp 700B instrument (Molecular Devices, Sunnyvale, CA).…”