In vivo electrophysiological effects of insulin in the rat brain

Abstract

Brain insulin has widespread metabolic, neurotrophic, and neuromodulatory functions and is involved in the central regulation of food intake and body weight, learning and memory, neuronal development, neuronal apoptosis, and aging. To understand the neuromodulatory role of insulin, we aimed to characterize its yet undefined in vivo electrophysiological effects. We elected to record from the cerebellar cortex because this region has average insulin concentration and insulin receptor content in relation to the whole brain, and has been previously shown to be a target for insulin signaling. We used in vivo microiontophoresis to apply insulin juxtaneuronally while simultaneously recording changes in spontaneous neuronal activity. The analysis included 553 significant neuronal responses to insulin and other related agents recorded from 47 cerebellar neurons of the rat. We found that (1) insulin stimulation produced instant and reversible electrophysiological effects on all of the recorded neurons, and that (2) these effects were mostly dependent on prior or simultaneous GABA application (94-96%). Specifically, (a) inhibitory responses to insulin were the most common (58-62%), and were dose-dependent with respect to GABA pretreatments and blocked by co-administration of the insulin receptor inhibitor HNMPA. (b) In the second largest set of neurons (32-38%) insulin decreased the magnitude of GABA inhibitions when co-applied. (c) In contrast, only a small number of neurons showed GABA-independent responses to insulin application (4-6%), which were exclusively neuronal excitations. The present findings demonstrate that insulin has direct electrophysiological effects on central neurons in vivo and these effects are highly influenced by GABA-ergic inputs.

Fig. 1

GABA pretreatment dose-dependently regulates the magnitude of the subsequent insulin inhibition – single neuronal recordings of typical neurons. A/ Dependence on GABA application time: as the GABA-inhibitions are getting longer in time, the magnitude of the following insulin inhibitions are becoming larger; B/ Dependence on current intensity of GABA: as the GABA-inhibitions are getting more intense, the magnitude of the following insulin inhibitions are becoming larger. Abbreviations: nA, nanoamperes (current intensity); s, seconds (time); insulin application: white arrow, between dashed lines; GABA application: black arrow, between dotted lines.

Fig. 2

GABA pretreatment dose-dependently regulates the magnitude of the subsequent insulin inhibition – neuronal response magnitude analysis to microiontophoretic insulin as a function of GABA pretreatment ejection charge or insulin current intensity. A, Neuronal response magnitudes to insulin are significantly correlating with the GABA pretreatment ejection charge (148 insulin applications recorded from 29 neurons); B, Neuronal responses of 12 neurons with individual GABA-dose-dependence tests and their linear trendlines show the same correlation; C, Neuronal response magnitudes to insulin are not correlating with the current intensity of insulin application (148 insulin applications recorded from 29 neurons). Abbreviations: nC, nanoCoulombs; nA, nanoAmperes; s, seconds.

Fig. 3

Treatment with insulin receptor inhibitor HNMPA blocks the GABA pretreatment dependent insulin inhibitions, when co-applied – single neuronal recording of a typical neuron. Abbreviations: nA, nanoamperes (current intensity); s, seconds (time); insulin application: white arrow, between dashed lines; GABA application: black arrow, between dotted lines, HNMPA application: striped arrow, between rare dotted lines.

Fig. 4

Insulin decreases the magnitude of GABA inhibitions, when co-applied – single neuronal recording of a typical neuron‥ Abbreviations: nA, nanoamperes (current intensity); s, seconds (time); insulin application: white arrow, between dashed lines; GABA application: black arrow, between rare dotted lines.

Fig. 5

Insulin is able to exert excitatory effects on neurons, irrespectively to GABA-application – single neuronal recording of a typical neuron. Abbreviations: nA, nanoamperes (current intensity); s, seconds (time); insulin application: white arrow, between dashed lines; GABA application: black arrow, between dotted lines, glutamate application: striped arrow, between rare dotted lines.

Fig. 6

Histological localization of the recording areas. Left panel: sample image showing the electrode track (arrowheads) and a PSB-labeled recording area next to it. PSB was picked up by granule cells which were located nearby the recording site (arrows). The background is stained with cresyl violet; the insert at the left bottom is 100 µm. Abbreviations: W, white matter; G, granule cell layer; P, Purkinje cell layer; M, molecular layer. Right panel: schematic reconstruction of the placement of 21 recording areas and the effects of insulin recorded in them. Symbols: square, GABA-dependent insulin inhibition; star, HNMPA blocks GABA-dependent insulin inhibition; circle, insulin decreases GABA inhibition; dollar sign, insulin excitation. Numbers 1–5 refer to the rostral lobules 1–5 of the anterior lobe of the cerebellar vermis.

Fig. 7

Summary of the main findings. A, The distribution of the in vivo electrophysiological effects of insulin. A/left, The distribution of the GABA-dependent and GABA-independent effects (the cross-sections with transversal stripes show bi-functional neurons that belong to both groups); A/right, The distribution of the three main electrophysiological effects of insulin. B, Effects of insulin on the in vivo neuronal activity. The “Effects of control drugs” were detected as (1) GABA inhibition and (2) glutamate excitation; the “GABA-dependent effects of insulin” had been described as (1) GABA-dependent insulin inhibition, (2) HNMPA blocks GABA-dependent insulin inhibition, and (3) insulin decreases GABA inhibition; the “GABA-independent effects of insulin” were all (1) GABA-independent insulin excitations. Neuronal responses are displayed as mean (± S.E.M.) poststimulus 10 s firing rates (spikes / seconds), corrected to the prestimulus 10 s spontaneous firing rates (y = 0). * = p < 0.05 paired t-tests comparing mean pre- and poststimulus neuronal activity; # = p < 0.05 two-sample unequal variance t-tests between mean poststimulus firing rates.