Modification of activity-dependent increases of cerebral blood flow by excitatory synaptic activity and spikes in rat cerebellar cortex

Abstract

1. Mechanisms of activity-dependent increases in cerebral blood flow (CBF) were examined in rat cerebellar cortex using the laser Doppler flow technique and extracellular recordings of single unit activity and field potentials. 2. Stimulation of the monosynaptic climbing fibre system evoked long-lasting complex spikes in Purkinje cells, and extracellular field potentials with a characteristic profile that indicated contributions from both passive and active membrane mechanisms. The concomitant CBF increases were reproducible at fairly short intervals, and suggest that both synaptic activity and spikes may contribute to increased CBF. 3. Stimulation of the disynaptic parallel fibre system inhibited the spiking activity in Purkinje cells, while the postsynaptic activity increased as indicated by the simultaneously recorded field potential. Nevertheless, CBF always increased. The inhibition of spike firing activity was partly dependent on GABAergic transmission, but may also relate to the intrinsic membrane properties of Purkinje cells. 4. The CBF increases evoked by parallel or climbing fibre stimulation were highly correlated to the sum of neural activities, i.e. the negativity of field potentials multiplied by the stimulus frequency. This suggests a robust link between extracellular current flow and activity-dependent increases in CBF. 5. AMPA receptor blockade attenuated CBF increases and field potential amplitudes, while NMDA receptor antagonism did not. This is consistent with the idea that the CBF responses are of neuronal origin. 6. This study has shown that activity-dependent CBF increases evoked by stimulation of cerebellar parallel fibres are dependent on synaptic excitation, including excitation of inhibitory interneurones, whereas the net activity of Purkinje cells, the principal neurones of the cerebellar cortex, is unimportant for the vascular response. For the climbing fibre system, not only synaptic activity but also the generation of complex spikes from Purkinje cells contribute to the increases in CBF. The strong correlation between CBF and field potential amplitudes suggests that extracellular ion fluxes contribute to the coupling of brain activity to blood flow.

Figure 1. Schematic three-dimensional drawing of experimental set-up, including neurones of interest and position of laser Doppler probe, stimulating and recording electrodes

The positions of the three cerebellar layers, molecular (Mol, with a thickness of 400 μm), Purkinje cell (PcL, about 100 μm) and granular (GrL, 400–500 μm), are indicated. The molecular layer contains granule cell axons, called parallel fibres, the dendrites of Purkinje cells, stellate cells (S) and basket cells (B). The granule cell layer contains granule cells (Gr) and Golgi cells (GC). The superficial parallel fibres were stimulated by a bipolar stimulating electrode, while climbing fibres (CF) were stimulated by a monopolar electrode lowered into the caudal part of the inferior olive (IO). Field potentials and single unit spike activity were recorded with a glass microelectrode. CBF was recorded by a laser Doppler flowmetry (LDF) probe located 0.3–0.5 mm above the pial surface (Pia).

Figure 2. Neuronal signals recorded from Purkinje cells at a depth of 300 μm with a glass microelectrode

A, spontaneous simple spike (average of 15 spikes). B, spontaneous complex spike, which is characterized by a variable waveform. C, averaged complex spike (average of 10 sweeps) evoked by climbing fibre stimulation. Neuronal signals in A, B and C were recorded with a bandpass filter of 0.3–5 kHz, emphasizing fast components of sodium spikes and the early part of the climbing fibre response. The vertical bar in C indicates 0.1 mV for A, B and C. D, single evoked field potential recorded under the same conditions as in C, but with a filter that allows passage of the slow component of the signal (0.5 Hz to 3 kHz). Sodium spikes are seen as small volleys indicated by arrows. E, laminar analysis of field potentials in response to climbing fibre stimulation. The potential shown is an average of 100 sweeps of the signal shown in D. The depth profile shows a characteristic source-sink profile with a net positive current passing into the cell at the top of the molecular layer giving rise to a negative extracellular potential, with potential reversal at 300–500 μm, corresponding to the Purkinje cell body layer. F, laminar analysis of field potentials in response to parallel fibre stimulation. The parallel fibre response consisted of a presynaptic component (N1) due to action potentials in the fibres, and a postsynaptic component (N2) due to activation of AMPA receptors. The parallel fibre response was largest close to the cerebellar surface and decreased as a function of depth.

Figure 3. Activity-dependent CBF increases and spike activity in response to parallel fibre stimulation recorded along the activated parallel fibre beam at a stimulation frequency of 30 Hz and stimulus duration of 30 s

A, Purkinje cell spike firing activity almost vanished after 1–3 s of stimulation, and spontaneous firing did not return to basal levels until 19–25 s after the end of stimulation. CBF increased during stimulation, continued to increase for 5–10 s after the end of stimulation, and reversed to baseline after 40–50 s. B, example of inhibition during stimulation followed by post-inhibitory rebound excitation. The Purkinje cell spike firing rate was inhibited during the first part of stimulation, but gradually returned during the stimulation period. Following the stimulus train the spontaneous activity increased for 25–30 s. CBF started to increase 1–2 s after the start of stimulation, but did not decrease until 1–2 s after the end stimulation. C, bicuculline methiodide (0.5 mm, topical application, horizontal bar) did not affect the evoked CBF increase but attenuated the inhibition of Purkinje cell spike activity during stimulation. Stimulation periods are indicated by horizontal bars below the CBF trace.

Figure 4. Frequency-dependent CBF increases in response to parallel fibre stimulation were correlated to the summed field potentials

A, typical example of CBF increases evoked by 10 Hz for 60 s. B, enlarged evoked field potential response to indicate how the field potential amplitude was calculated. The first negativity (N1) was associated with the presynaptic action potential, while the second negativity (N2) represents postsynaptic excitation. The amplitude was measured as the voltage difference between the two dashed lines. C, in the frequency range of 2–30 Hz the amplitude of evoked field potentials decreased at high stimulus frequencies due to the short recovery time between stimuli. D, correlation analysis of CBF increases (ordinate) versus summed field potentials (abscissa) from one rat suggested a sigmoidal relationship.

Figure 5. Frequency-dependent CBF increases in response to climbing fibre stimulation were directly correlated to the sum of active and passive postsynaptic activity

A, typical example of CBF increases evoked by 1, 2.5, 5, 10 and 20 Hz for 60 s (bars indicate time of stimulation). B, CBF (n = 8) increases reached a maximum of around 141% corresponding to a stimulation frequency of 10 Hz. C, profile of evoked field potentials as a function of stimulus frequency. The amplitude of the field potential per stimulus decreased with increasing stimulus rate due to the short recovery time between stimuli. The evoked field potentials were recorded at a depth of 400 μm. D, scatter plot of CBF increases versus summed field potentials, i.e. the product of field potential amplitudes (indicated by arrows in C) and stimulus rate. The relationship was best fitted to a straight line (r =−0.985, P = 0.0022).

Figure 6. AMPA receptor blockade inhibited climbing fibre-evoked CBF increases and the major part of the evoked field potential

A, data points indicate maximal CBF increase and field potential amplitude evoked during successive periods of climbing fibre stimulation (10 Hz for 60 s) before, during and after topical application of CNQX. Topical application of CNQX (500 μm, horizontal bar) reversibly inhibited climbing fibre-evoked CBF increases (•) and summed field potentials (▪). Original data for CBF increases and field potentials are shown in B and C, respectively. B, CBF trace before (control, 1) and after application of CNQX (2), corresponding to the same numbers in A. C, control trace (a) of field potential, and trace after application of CNQX (b), corresponding to same letters in A. D, correlation analysis of maximal CBF increase versus summed field potentials fitted to a straight line (r = 0.784, P < 0.0001).

Figure 7. Activity-dependent CBF increases in response to climbing fibre stimulations (10 Hz for 60 s) were dependent on activation of AMPA receptors

A, example of reversible inhibition of CBF increases and field potential amplitudes by an AMPA receptor antagonist (GYKI 52466; slow bolus of 20 mg kg⁻¹ i.v. (arrow) followed by 10 min continuous infusion of 4 mg kg⁻¹ min⁻¹ (horizontal line)). The figure demonstrates the clear correlation between CBF increases and summed field potentials (▪) during inhibition and recovery from treatment. Blood pressure (BP, upper trace) was unaffected by the slow bolus and subsequent continuous injection of GYKI 52466. The filled bars (bottom) indicate duration of climbing fibre activation. B, analysis of CBF increases versus summed field potential suggested a direct correlation (r =−0.956, P < 0.0001). C, summary of effect of glutamate antagonists on CBF increases after administration of CNQX (500 μm, topical application, n = 4), GYKI 52466 (20 mg kg⁻¹ plus continuous i.v., n = 6), APH (10 mg kg⁻¹ i.v., n = 5) and MK-801 (2 mg kg⁻¹ i.v., n = 5). The AMPA antagonists CNQX and GYKI 52466 significantly inhibited climbing fibre-evoked CBF increases, whereas the NMDA receptor antagonists APH and MK-801 had no effect on climbing fibre-evoked CBF increases.