Locus coeruleus alpha-adrenergic-mediated activation of cortical astrocytes in vivo

Abstract

The locus coeruleus (LC) provides the sole source of norepinephrine (NE) to the cortex for modulation of cortical synaptic activity in response to salient sensory information. NE has been shown to improve signal-to-noise ratios, sharpen receptive fields and function in learning, memory, and cognitive performance. Although LC-mediated effects on neurons have been addressed, involvement of astrocytes has thus far not been demonstrated in these neuromodulatory functions. Here we show for the 1st time in live mice, that astrocytes exhibit rapid Ca(2+) increases in response to electrical stimulation of the LC. Additionally, robust peripheral stimulation known to result in phasic LC activity leads to Ca(2+) responses in astrocytes throughout sensory cortex that are independent of sensory-driven glutamate-dependent pathways. Furthermore, the astrocytic Ca(2+) transients are competitively modulated by alpha(2)-specific agonist/antagonist combinations known to impact LC output, are sensitive to the LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, and are inhibited locally by an alpha-adrenergic antagonist. Future investigations of LC function must therefore consider the possibility that LC neuromodulatory effects are in part derived from activation of astrocytes.

Figure 1.

Noradrenergic agonists elicit Ca²⁺ waves in cortical astrocytes. (A) The image at left illustrates the experimental setup with a cranial window over the somatosensory cortex and a glass electrode containing agonist positioned in the molecular layer (50–80 μm deep). (B) Series of images demonstrating a Ca²⁺ wave to pressure ejection of 200 μM methoxamine. (C) Changes in Ca²⁺ over time corresponding to numbered cells circled in (B) showing the time course of wave propagation. (D) Histogram showing that both α- (34 cells in 5 animals) and β-agonists (200 μM; 27 cells in 4 animals) are capable of eliciting Ca²⁺ responses in cortical astrocytes. meth, methoxamine; iso, isoproterenol.

Figure 2.

Direct LC stimulation elicits Ca²⁺ transients in cortical astrocytes. (A) Schematic illustrating experimental setup. (B) Average fluorescence of the image field in (C) over time showing Ca²⁺ response to LC stimulation. (C) Fluo-4 labeled astrocytes taken at time points indicated by numbers in (B). Insets are blown-up view of boxed area in pictures. Scale bar 50 μm. (D) Images from coronal sections through somatosensory cortex illustrating the effect of DSP-4 on LC projection neurons as labeled by antibodies to TH and MAP-2 with DAPI labeling of nuclei for orientation. Scale bar 100 μm (20 μm inset). (E) Consistent with the significant reduction in LC projection neurons and despite almost twice the stimulation intensity (216 ± 33 vs. 400 ± 39 μA; P = 0.004, t-test), there is a significant reduction in the cortical Ca²⁺ response to LC stimulation in DSP-4 treated animals (P = 0.013, t-test).

Figure 3.

Diagram outlining neural pathways activated in response to contralateral footshock or direct LC stimulation. Footshock triggers diffuse release of NE via LC activation in large areas of cortex (red), whereas sensory glutamatergic input is restricted to hindlimb sensory cortex. Insets: Astrocytes located in cortical layer I of hindlimb are activated by both NE and glutamate in response to footshock, whereas astrocytes in nonhindlimb areas only receive LC-mediated NE output. Glu, glutamate; SmC, somatosensory cortex; Th, thalamus.

Figure 4.

Footshock stimulation elicits Ca²⁺ responses in cortical astrocytes independent of sensory activity. (A) Significant Ca²⁺ responses to contralateral foot stimulation with image of single cell response (inset) illustrated in top red trace. Images of time points indicated by numbers in top trace. (B) Comparison of bilateral Ca²⁺ responses (P = 0.002, t-test). (C) Contralateral hindlimb Ca²⁺ responses were subdivided into hindlimb and nonhindlimb sensory fields for further comparison (P = 0.140, t-test). Nonhindlimb areas include trunk, forelimb, and barrel sensory fields. (D) Effects of metabotropic glutamate antagonism in hindlimb versus nonhindlimb sensory fields (P = 0.043, paired t-test). MP-HL, MPEP with window over hindlimb; MP-Tr, MPEP over trunk. Scale bar is 100 μm.

Figure 5.

Pharmacology implicates LC–NE in footshock-induced astrocyte Ca²⁺ responses. (A) Schematic illustrating LC pathway to cortex with proposed sites of intervention. (B), Example images of cortical astrocyte Ca²⁺ after contralateral foot stimulation corresponding to footshock (FS) displayed on timeline of fluo-4 fluorescence intensity of the whole field. The 2nd image is of a FS response 20 min after xylazine administration. Yohimbine reverses xylazine block after 20 min (>2 h typically). (C) Same as (B) except the drugs were given in reverse order to further demonstrate their competitive action at the α₂-adrenergic receptor. (D) Histogram illustrating competitive action of the agonist/antagonist combinations on LC output. (E) The LC neurotoxin, DSP-4, dramatically reduces responses to ipsilateral footshock. N, numbers in parentheses. *P < 0.05 by t-test. Scale bar represents 50 μm. α₂-AR, α₂-adrenergic receptor; FS, footshock; Xyl and X, xylazine; Yoh and Y, yohimbine.

Figure 6.

The astrocytic Ca²⁺ response is mediated by α-adrenergic receptors. (A) Diagram illustrating imaging setup for local antagonist effects on footshock over nonhindlimb somatosensory cortex. Drug solution containing Alexafluor 594 is pressure ejected prior to footshock-mediated Ca²⁺ responses. (B) Cortical astrocyte Ca²⁺ responses to contralateral hindlimb stimulation during local drug application. Inset displays Alexafluor 594 from pipette at the same time point as the corresponding fluo-4 image for demonstrating drug delivery. (C) Effect of antagonist application as percentage of the surrounding area increase (P « 0.001, t-test). ACSF, artificial cerebral spinal fluid; CGP, CGP54626; F, fluorescence intensity; Phen, phentolamine; Prop, propranolol. Scale bar is 100 μm.