Dissociation of spikes, synaptic activity, and activity-dependent increments in rat cerebellar blood flow by tonic synaptic inhibition

Abstract

Functional neuroimaging relies on the robust coupling between neuronal activity, metabolism and cerebral blood flow (CBF) to map the brain, but the physiological basis of the neuroimaging signals is still not well understood. Here we applied a pharmacological approach to separate spiking activity, synaptic activity, and the accompanying changes in CBF in rat cerebellar cortex. We report that tonic synaptic inhibition achieved by topical application of gamma-aminobutyric acid type A (GABAA) (muscimol) or GABAB (baclofen) receptor agonists abolished or reduced spontaneous Purkinje cell spiking activity without affecting basal CBF. The magnitude of CBF responses evoked by climbing fiber stimulation decreased gradually over time after exposure to muscimol, being more pronounced in the superficial than in the deep cortical layers. We provide direct evidence in favor of a laminar-specific regulation of CBF in deep cortical layers, independent of dilatation of surface vessels. With prolonged exposure to muscimol, activity-dependent CBF increments disappeared, despite preserved cerebrovascular reactivity to adenosine and preserved local field potentials (LFP). This dissociation of CBF and LFPs suggests that CBF responses are independent of extracellular synaptic currents that generate LFPs. Our work implies that neuronal and vascular signals evoked by glutamatergic pathways are sensitive to synaptic inhibition, and that local mechanisms independent of transmembrane synaptic currents adjust flow to synaptic activity in distinct cortical layers. Our results provide fundamental insights into the functional regulation of blood flow, showing important interference of GABAA receptors in translating excitatory input into blood flow responses.

Fig. 1.

GABA receptor activation decreased spontaneous spike activity in PCs. (a) GABA_A receptor activation by topical application of 0.2 mM muscimol (starting point indicated by arrow) abolished spontaneous spike activity with 93 ± 2% (n = 5). (b) Activation of GABA_B receptors reduced the spontaneous spike rate by 49 ± 6% (n = 4). Horizontal bars indicate periods of PF stimulation. Synchronized stimulation of parallel fibers results in net inhibition of the PCs due to the release of GABA from the inhibitory stellate and basket cells. This inhibition is similar to the inhibition achieved by GABA_A receptor activation (Upper) and can transiently shut down PC spiking on top of the GABA_B receptor activation.

Fig. 2.

Magnitude of evoked increases in CBF was gradually reduced after GABA_A-receptor activation. (a) Diagram showing neural elements including receptors for AMPA and GABA_A in the CF–PC network. (b) Drawing of the two-channel LDF probe (LDF_green and LDF_red) that recorded blood flow (CBF) to depths of 250 μm and 1,000 μm, respectively. (c) Original sample trace of CBF in response to CF stimulation during control stimulations and after topical application of muscimol (starting point indicated by arrow). (Upper) CBF measured with LDF_green. (Lower) CBF measured with LDF_red. Evoked CBF responses at 2, 5, and 10 Hz (indicated by colored bars) were gradually reduced over time after exposure to muscimol. Red boxes to the upper right of each CBF trace depict an expanded superimposition of the marked stimulation sequences from the original traces. Red or green trace represents CBF during control, and blue trace represents CBF after exposure to muscimol.

Fig. 3.

Time course of reduction of evoked CBF increases at 2, 5, and 10 Hz measured with LDF_green (Upper) and LDF_red (Lower). Graphs show that evoked changes in CBF were reduced faster in the upper layer of the cortex as compared to the deeper layer. Each point represents the mean ± SEM. (n = 6).

Fig. 4.

Superimposition of individual traces of CBF responses obtained for each animal. (Left) Measurements obtained with LDF_green. (Right) Measurements obtained with LDF_red. Green trace in Left and red trace in Right represent increases in CBF in response to control stimulations, and blue traces in both panels represent CBF responses to the same stimulations 50 min after muscimol application.

Fig. 5.

GABA_A-receptor activation dissociated LFPs and CBF responses evoked by CF stimulation. (a) Averaged traces of control and conditioned LFPs in response to CF stimulation (n = 6). Red trace represents averaged LFPs during control stimulation; blue traces represent averaged LFPs during stimulation at different time points after drug application. All traces are superimposed below. (b) Comparative time course of normalized LFP amplitudes and normalized changes in CBF evoked at 10 Hz.

Fig. 6.

No effect of muscimol on CBF in the PF–PC pathway. (a) Original traces of CBF during control stimulations and after topical application of muscimol (starting point indicated by arrow) measured with LDF_green (Upper) and LDF_red (Lower) in response to PF stimulation. Horizontal bars indicate stimulation periods. (b) Diagram showing neural elements including receptors for AMPA, GABA_A, and GABA_B in the PF–PC network.