Expression of GLP-1 receptors in insulin-containing interneurons of rat cerebral cortex.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptors are expressed by pancreatic beta cells and GLP-1 receptor signalling promotes insulin secretion. GLP-1 receptor agonists have neural effects and are therapeutically promising for mild cognitive impairment and Alzheimer's disease. Our previous results showed that insulin is released by neurogliaform neurons in the cerebral cortex, but the expression of GLP-1 receptors on insulin-producing neocortical neurons has not been tested. In this study, we aimed to determine whether GLP-1 receptors are present in insulin-containing neurons. METHODS: We harvested the cytoplasm of electrophysiologically and anatomically identified neurogliaform interneurons during patch-clamp recordings performed in slices of rat neocortex. Using single-cell digital PCR, we determined copy numbers of Glp1r mRNA and other key genes in neurogliaform cells harvested in conditions corresponding to hypoglycaemia (0.5 mmol/l glucose) and hyperglycaemia (10 mmol/l glucose). In addition, we performed whole-cell patch-clamp recordings on neurogliaform cells to test the effects of GLP-1 receptor agonists for functional validation of single-cell digital PCR results. RESULTS: Single-cell digital PCR revealed GLP-1 receptor expression in neurogliaform cells and showed that copy numbers of mRNA of the Glp1r gene in hyperglycaemia exceeded those in hypoglycaemia by 9.6 times (p < 0.008). Moreover, single-cell digital PCR confirmed co-expression of Glp1r and Ins2 mRNA in neurogliaform cells. Functional expression of GLP-1 receptors was confirmed with whole-cell patch-clamp electrophysiology, showing a reversible effect of GLP-1 on neurogliaform cells. This effect was prevented by pre-treatment with the GLP-1 receptor-specific antagonist exendin-3(9-39) and was absent in hypoglycaemia. In addition, single-cell digital PCR of neurogliaform cells revealed that the expression of transcription factors (Pdx1, Isl1, Mafb) are important in beta cell development. CONCLUSIONS/INTERPRETATION: Our results provide evidence for the functional expression of GLP-1 receptors in neurons known to release insulin in the cerebral cortex. Hyperglycaemia increases the expression of GLP-1 receptors in neurogliaform cells, suggesting that endogenous incretins and therapeutic GLP-1 receptor agonists might have effects on these neurons, similar to those in pancreatic beta cells.

Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type.

We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.

GABAergic neurogliaform cells represent local sources of insulin in the cerebral cortex.

Concentrations of insulin in the brain are severalfold higher than blood plasma levels. Insulin in the brain regulates the metabolism, molecular composition, and cognitive performance of microcircuits and reduces food intake; cerebral insulin levels are altered in diabetes, aging, obesity, and Alzheimer's disease. Released by pancreatic ß cells, insulin passes the blood-brain barrier, but sources of locally released insulin still remain unclear. We find that insulin is strongly expressed in GABAergic neurogliaform cells in the cerebral cortex of the rat detected by single-cell digital PCR. Focal application of glucose or glibenclamide to neurogliaform cells mimics the excitation suppressing effect of external insulin on local microcircuits via insulin receptors. Thus, neurogliaform cells might link GABAergic and insulinergic action in cortical microcircuits.

Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording.

Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.