Enhanced dendritic activity in awake rats

Abstract

Almost nothing is known about dendritic activity in awake animals and even less about its relationship to behavior. The tuft dendrites of layer 5 (L5) pyramidal neurons lie in layer 1, where long-range axons from secondary thalamic nuclei and higher cortical areas arrive. This class of input is very dependent on active thalamo-cortical loops and activity in higher brain areas and so is likely to be heavily influenced by the conscious state of the animal. If, as has been suggested, the dendrites of pyramidal neurons actively participate in this process, dendritic activity should greatly increase in the awake state. Here, we measured calcium activity in L5 pyramidal neuron dendrites using the "periscope" fiberoptic system. Recordings were made in the sensorimotor cortex of awake and anesthetized rats following sensory stimulation of the hindlimb. Bi-phasic dendritic responses evoked by hindlimb stimulation were extremely dependent on brain state. In the awake state, there was a prominent slow, delayed response whose integral was on average 14-fold larger than in the anesthetized state. Moreover, the dramatic increases in dendritic activity closely correlated to the strength of subsequent hindlimb movement. These changes were confined to L5 pyramidal dendrites and were not reflected in the response of layer 2/3 (L2/3) neurons to air-puff stimuli in general (which actually decreased in the awake state). The results demonstrate that the activity of L5 pyramidal dendrites is a neural correlate of awake behavior.

Fig. 1.

Dramatic increase in dendritic calcium in awake rats. (A) Schematic diagram of experimental design. Left, “periscope” fiberoptic imaging device attached to rat cortex. Tension and EMG of upper hindlimb muscles were measured in response to an air-puff to the hindlimb. Middle, head mount allowing application of drugs with simultaneous imaging of the somatosensory cortex. Right, schematic diagram showing the light path for collecting fluorescence changes from populations of L5 pyramidal neuron dendrites (blue arrow, excitation at 475 nm; green arrow, emission at 520 nm). (B) Dendritic Ca²⁺ fluorescence changes recorded under anesthesia (AN, black), in awake state (AW) with hindlimb movement (red) and without movement (blue). Signals were evoked by air-puff stimulation (50-ms, dashed line) to contralateral hindlimb. The data are shown as averages (20 trials for AN, 11 trials for AW with movement, 16 trials for AW without movement). The areas of the slow components are highlighted. (C) Summary of dendritic Ca²⁺ signals in anesthetized (AN) and awake (AW) states (n = 6 rats). pFC, peak amplitude of fast component and aSC, area of slow component (see also Fig. S1 for pSC, peak amplitude of slow component; dSC, duration of slow component). All statistics show significant differences (P < 0.05), except pFC between movement and no movement in the awake state (NS, no significant difference). Dashed lines show SEM. (D) Example of single, simultaneous recording of dendritic Ca²⁺ signals, electromyogram (EMG) and tension of hindlimb in awake state.

Fig. 2.

No increase in L2/3 evoked response in awake rats. (A) Schematic diagram showing the experimental setup with bolus loading of L2/3 and imaging vertically from the cortical surface with an optical fiber. (B) Average L2/3 Ca²⁺ fluorescence changes recorded under anesthesia (black), in awake state with hindlimb movement (red) and without movement (blue, n = 6 rats). Signals were evoked by air-puff stimulation to contralateral hindlimb. (C) Summary of L2/3 Ca²⁺ activity in anesthetized (AN) and awake (AW) states (n = 6 rats). pFC, peak amplitude of fast component and aSC, area of slow component (see also Fig. S2 for pSC, peak amplitude of slow component; dSC, duration of slow component). Dashed lines show SEM.

Fig. 3.

Dendritic slow Ca²⁺ activity correlates with the strength of hindlimb movement. (A) Single trials of simultaneous recordings of dendritic Ca²⁺ signals (colored) and tension of movement (gray) in the same rat. Area of slow component is highlighted. AN, anesthetized; AW, awake; HLS, hindlimb stimulus. (B and C) Strength of movement versus peak amplitude of fast component (B) and area of slow component (C) evoked by hindlimb stimulation (n = 6 rats). Blue and orange circles indicate the data corresponding to the traces shown in A. Filled squares (gray) show trials with movement. Open squares show those without movement. The linear fits (red) were applied to all movement data points (85 trials in 6 rats). Statistics of the Pearson's r correlation were performed from the 85 points. Black, blue and red points show binned averages (bin size, 20%) of awake data. Anesthetized data are averaged as a single value (i.e., no movement).