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

Abstract

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 β 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.

Figure 1.

Cell type-dependent insulin mRNA expression in the cerebral cortex. Aa, Typical responses of a neurogliaform cell (NGFC), pyramidal cell (PC), fast spiking cell (FS), and a glial cell (Glia) to hyperpolarizing and depolarizing current pulses recorded before harvesting their cytoplasm. Ab, Anatomical reconstructions of the cells shown in Aa, colors of dendrites correspond to firing patterns in Aa, axons are black. B, Single-cell qRT-PCR results of the ins2 gene in neurogliaform cells (top) with negative controls (RT-, bottom). Ca, Representative raw data from a single-cell digital PCR array showing the rps18 housekeeping gene and ins2 under high (10 mm) or low (0.5 mm; asterisks) extracellular glucose concentrations in neurogliaform cells (NGFCs). Results of negative controls for both genes (rps18- and ins2-; RT-) are also shown. Color coding indicates the cycle number at which reactions crossed threshold for detecting ins2 or rps18 in each nanowell. Cb, The number of ins2 mRNAs in neurogliaform cells (green) increased significantly (asterisks) together with the extracellular glucose concentration from hypoglycaemic to euglycemic and further to hypergylcemic extracellular conditions. In contrast, the number of ins2 mRNAs remained stable, thus significantly lower in pyramidal (red) and fast spiking (blue) cells regardless of changes in glucose concentration. Copy numbers of ins2 in glial cells were smaller compared with other cell types tested.

Figure 2.

Neurogliaform cells mimic the action of external insulin via insulin receptors. A, The frequency of spontaneous EPSCs arriving to neocortical neurons was decreased in response to physiological concentrations of insulin (100 nm), and the specific insulin receptor antagonist S961 (20 nm) reversed the effect. B, Mimicking the effect of insulin shown in A, local application of hyperglycemic extracellular solution containing 10 mm glucose (gluc) to neurogliaform cells (NGFCs) identified electrophysiologically and anatomically decreased the frequency of spontaneous EPSCs arriving to neighboring neurons recorded in hypoglycemic (0.5 mm) conditions and S961 (20 nm) also reversed the effect. Top, Individual experiment. Bottom, Population data. C, The effect of hyperglycemic puffs to neurogliaform cells on spontaneous EPSCs in neighboring pyramidal cells was blocked by lavendustin (5 μm) intracellularly applied in the pyramidal cells. D, Insulin suppresses the amplitude of unitary EPSCs between layer 2/3 pyramidal cells while leaving the paired pulse ratio unchanged.

Figure 3.

K_ATP channels and intracellular Ca²⁺ contribute to insulin receptor-mediated action of neurogliaform cells. A, Current–voltage (I–V) relationship of the glibenclamide-sensitive component of currents recorded in a late spiking (inset) neurogliaform cell in response to ramping membrane potential from −145 to −65 mV with and without glibenclamide (20 μm). B, A neurogliaform cell identified by its firing pattern (top) responds to bath-applied glibenclamide (20 μm) with an increase of the intracellular Ca²⁺ concentration detected by changes in OGB-1 fluorescence (right) in one of the dendrites (bottom, red border indicates imaged area). C, Whole-cell recordings performed in hypoglycemia (0.5 mm) show that glibenclamide (20 μm) delivered to neurogliaform cells (NGFC) significantly decreased the frequency of EPSCs in simultaneously monitored neighboring neurons, and this effect was blocked by the insulin receptor blocker S961 applied extracellularly and also by intracellular application of BAPTA (B) in the neurogliaform cell. In contrast, glibenclamide applied to pyramidal cells (PC) and fast spiking cells (FSC) caused no significant changes in the frequency of EPSCs in neighboring neurons. D, Time course of sEPSC amplitude (top) and frequency (bottom) changes in a representative experiment as shown in C (top left). E, Radioimmunoassay measurements in homogenates of neocortical slices showed significantly increased insulin levels relative to hypoglycemia during glibenclamide (glib) application and normoglycemia or hyperglycemia.