ATP-mediated glucosensing by hypothalamic tanycytes

Abstract

The brain plays a vital role in the regulation of food intake, appetite and ultimately body weight.Neurons in the hypothalamic arcuate nucleus, the ventromedial hypothalamic nuclei(VMH)and the lateral hypothalamus are sensitive to a number of circulating signals such as leptin, grehlin, insulin and glucose. These neurons are part of a network that integrates this information to regulate feeding and appetite. Hypothalamic tanycytes contact the cerebral spinal fluid of the third ventricle and send processes into the parenchyma. A subset of tanycytes are located close to, and send processes towards, the hypothalamic nuclei that contain neurons that are glucosensitive and are involved in the regulation of feeding. Nevertheless the signalling properties of tanycytes remain largely unstudied. We now demonstrate that tanycytes signal via waves of intracellular Ca2+; they respond strongly to ATP, histamine and acetylcholine – transmitters associated with the drive to feed. Selective stimulation by glucose of tanycyte cell bodies evokes robust ATP-mediated Ca2+ responses. Tanycytes release ATP in response to glucose. Furthermore tanycytes also respond to non-metabolisable analogues of glucose. Although tanycytes have been proposed as glucosensors, our study provides the first direct demonstration of this hypothesis.Tanycytes must therefore now be considered as active signalling cells within the brain that can respond to a number of neuronally derived and circulating transmitters and metabolites.

Figure 1. The dependence concentration of glucose delivered via puffer pipettes on the duration of the puff

A, the traces are recordings from a 125 μm disk biosensor for glucose aligned opposite and close to the puffer pipette. The timing pulse of the puffer is shown together with the signal recorded by the glucose biosensor. The concentration of glucose delivered is proportional to pulse with from 0.1 to 0.5 s and depends upon the concentration of glucose within the pipette. The graph shows results from two different pipettes loaded with 150 mm and 300 mm glucose. The traces were obtained with the 150 mm pipette. Both pipettes were from the same series pulled with identical settings on the puller. B, repeated puffs of glucose lead to sustained rises in glucose rather than higher glucose concentrations. Top traces show glucose biosensor responses to repeated 0.1 s puffs leading to a steady state concentration of glucose of around 5 mm. The bottom traces show glucose biosensor responses to single and repeated 0.5 s puffs. Glucose at 300 mm in pipette.

Figure 2. Identification of hypothalamic tanycytes

A, schematic diagram of recording area (bounded by dashed rectangle). VMH, ventromedial hypothalamus; ARC, arcuate nucleus; ME, median eminence; 3v, third ventricle. B, low power image (equivalent to dashed rectangle in A showing the strip of tanycytes (arrow) and a puffer pipette poised for action. C, high power image from a different slice – processes of the tanycytes are visible (arrowheads) and some cilia can be seen at the bottom right hand side of the image. D, image at 340 nm of fura-2 loaded tanycytes (from a different slice); their processes are also faintly evident.

Figure 3. Tanycytes respond to ATP with travelling Ca²⁺ waves via P2Y1 receptors

A, set of fura-2 images (each 6.4 s apart) obtained at 380 nm excitation of the tanycyte layer of the third ventricle (3v). A 0.5 s puff from pipette containing 10 mm ATP (and 1 μm carboxyfluorescein for visualisation) was used to stimulate the tanycytes. The extent of the puff can be seen in the first image (*); the dashed line marks the upstream limit of the puff and 8 ROIs (4 above and 4 below this limit) are marked on the images. The changes in intracellular Ca²⁺ wave at this excitation wavelength are seen as a loss of fluorescence. This loss travelled along the layer of cells and beyond the dashed line (limit of ATP application). The direction of bath flow is indicated by arrow. Thus tanycytes upstream of the pipette exhibited an increase in intracellular Ca²⁺ (ROIs 5–8). B, graph of responses in the 8 ROIs shown in A calculated as ratios of fluorescence at 340 nm and 380 nm excitation (F₃₄₀/F₃₈₀). The top 4 traces (ROIs 1–4) clearly exhibit a fluorescence change (downward deflection) resulting from detection of the carboxyfluorescein in the puffing solution and show a fast and near simultaneous increase in intracellular Ca²⁺. The bottom 4 traces (ROIs 5–8) show no evidence of the fluorescence change associated with the puff and exhibit a delayed, travelling and decrementing wave of Ca²⁺ increase (speed 8 μm s⁻¹, black circles indicate peak of wave). C, fura-2 ratio measurements of the response to bath application of 10 μm 2-methylthio-ADP (2MeSADP, measured with multiple ROIs represented by different coloured lines). The actions of 10 μm 2MeSADP were blocked by 100 nm MRS2500, indicating that the response is mediated by P2Y1 receptors.

Figure 4. Tanycytes respond to transmitters associated with wakefulness and feeding

A, fura-2 ratio images of the response to bath application of 10 μm histamine. Note the response clearly starts in the tanycyte cell layer (visible at 12 s). B and C, graphs of the responses (multiple ROIs around tanycytes – coloured lines) to bath applied histamine (above experiment in A) and 10 μm acetylcholine.

Figure 5. Bath applied glucose can evoke only small Ca²⁺ signals in tanycytes

A, multiple ROI measurements from tanycytes during the application of 5 mm glucose in control aCSF – no responses to glucose are evident. B, multiple ROI measurements from tanycytes in a different slice that had been primed with 1 μm acetylcholine and 1 μm 5HT (present throughout the recording) – a small transient response to 5 mm glucose was seen.

Figure 6. Tanycytes respond strongly to brief puffs of glucose

Fura-2 ratio images showing the response to a puff from a glucose-containing patch pipette (position indicated, 150 mm glucose in pipette, 0.2 s pulse, approximately 3 mm glucose at tanycytes). This evoked a long lasting Ca²⁺ wave in the tanycyte layer; note that the response starts in the tanycyte layer and spreads into the slice.

Figure 7. Controls for the specificity of glucose puffs

Multiple ROI measurements (coloured lines) of fura-2 fluorescence from the same slice during a series of puffs of aCSF (arrows), followed by 300 ms puffs from a 300 mm glucose pipette (arrow, approximately 8 mm at tanycytes), a series of 300 ms puffs from a 300 mm sucrose pipette (arrows, approximately 8 mm at tanycytes), followed once more by 300 ms puffs from a 300 mm glucose pipette. All puffer pipettes were pulled from the same series pulled with identical settings on the puller. The inset shows a comparison in the same slice of puffing glucose directly at the tanycyte cell bodies versus puffing, with the same pipette towards the interior of the slice. A single puff (300 ms from a 300 mm glucose pipette, effective dose approximately 8 mm) evoked a clear response, whereas multiple 300 ms puffs from the same pipette moved to the interior did not evoke a response.

Figure 8. Response from a small group of tanycytes to glucose puffs

Fura-2 ratio images (top) and plot of F₃₄₀/F₃₈₀ from 6 ROIs (bottom) around 6 distinct tanycytes (ovals, top). The timings on each image relate it to the graph below. The dashed white line indicates the ventricular border of the slice. The individual tanycyte cell bodies exhibit an increase in intracellular Ca²⁺. Note that the Ca²⁺ signal appears to spread down the tanycyte process. (Response to series of 0.5 s puffs from 150 mm glucose pipette, approximately 5 mm glucose.)

Figure 9. Non-metabolisable glucose analogues evoke Ca²⁺ waves in tanycytes

A and B, deoxyglucose (A) and methyl-α-d-glucopyranoside (B) were effective in evoking substantial Ca²⁺ signals measured by multiple ROIs from tanycytes. C, the glucose-evoked Ca²⁺ wave was reversibly blocked by the P2Y1 antagonist MRS2179 (5 μm, bath applied). Arrows indicate timing of puffs from pipettes (A, 150 mm deoxyglucose 0.4 s, approximately 4 mm at tanycytes; B, 300 mm methyl-α-d-glucopyranoside 0.3 s, approximately 8 mm at tanycytes; and C, 300 mm glucose, 0.3 s, approximately 8 mm at tanycytes). Coloured lines represent different ROIs. D, tanycytes release ATP in response to glucose. Inset, recording arrangement showing ATP biosensor and glucose pipette; the tanycyte layer can be seen as a translucent strip at the edge of the slice. The numbers indicate the positions of the glucose pipette. Main figure: ATP release evoked by the three positions of the puffer pipette, indicated in the inset, relative to the timing of the puff. Glucose in pipette, 300 mm; 0.3 s puff, approximately 8 mm at the tissue.