Principal neuron spiking: neither necessary nor sufficient for cerebral blood flow in rat cerebellum

Abstract

Neuronal activity, cerebral blood flow, and metabolic responses are all strongly coupled, although the mechanisms behind the coupling remain unclear. One of the key questions is whether or not increases in spiking activity in the stimulated neurons are sufficient to drive the activity-dependent rises in cerebral blood flow (CBF) that form the basis of the signals used in functional neuroimaging such as the blood oxygen level-dependent (BOLD) signal. To this end the present study examined the effect of enhanced spike activity per se on CBF in rat cerebellar cortex under conditions of disinhibition, achieved by blocking GABA(A) receptors using either bicuculline or picrotoxin. Purkinje cell spiking activity and local field potentials were recorded by glass microelectrodes, and laser Doppler flowmetry was used to monitor CBF. Disinhibition increased Purkinje cell spiking rate to 200-300% of control without incurring any increase in basal CBF. This demonstrates that increased spike activity per se is not sufficient to affect basal CBF. The neurovascular coupling between excitatory synaptic activity and CBF responses evoked by inferior olive (climbing fibre) stimulation was preserved during disinhibition. Thus, the unchanged basal CBF in the presence of the dramatic rise in Purkinje cell spiking rate was not explained by impaired synaptic activity-CBF coupling. On the basis of our previous and the present studies, we conclude that increased spiking activity of principal neurons is neither sufficient nor necessary to elicit CBF responses and in turn BOLD signals, and that activation-dependent vascular signals reflect excitatory synaptic activity.

Figure 1. Effect of disinhibition, i.e. abolishment of GABA_Aergic tone, on spontaneous Purkinje cell spike rate and basal cerebellar blood flow

A, bicuculline topically applied to the cerebellum at gradually increasing concentrations (final concentration 0.5 mm) increased spontaneous Purkinje cell spike rate without affecting basal cerebellar blood flow in the same brain area. For each animal, control values were taken as the average values obtained during 60 s immediately preceding drug application (first set of dotted lines) and compared to the average values obtained during 60 s measured after 30 min of drug exposure (second set of dotted lines). Raw data from one animal on the left; compiled data (n = 9) given as means and 95% confidence intervals on the right; ***P < 0.005. B, same data presentation as in A using picrotoxin (0.5 mm); n = 5; *P < 0.05.

Figure 2. Effect of picrotoxin on evoked cerebellar blood flow responses, local field potentials, synaptic activity, and neurovascular coupling during inferior olive stimulation

A, compiled data (n = 5) showing no effect of picrotoxin on evoked blood flow responses (ΔCBF) to inferior olive stimulation using increasing stimulation frequencies. ▪ and continuous line, control responses; ○ and dashed line, responses measured after 30 min exposure to picrotoxin. Data given as means and 95% confidence intervals. B, raw data showing continuous recordings of cerebellar blood flow responses to increasing stimulation frequencies (0.5–15 Hz) under control conditions and in the presence of picrotoxin. C, compiled data (n = 5) showing no effect of picrotoxin on the amplitude of field potentials (FPs) evoked by inferior olive stimulation. Note that field potential amplitudes decrease with increasing stimulation frequencies. ▪ and continuous line, field potential amplitudes under control conditions; ○ and dashed line, field potential amplitudes after 30 min exposure to picrotoxin. Data given as means and 95% confidence intervals. D, field potentials from the same animal evoked at 5 Hz under control conditions and after 30 min exposure to picrotoxin. E, compiled data (n = 5) showing no effect of picrotoxin on synaptic activity (ΣFP) evoked by inferior olive stimulation. Evoked synaptic activity is defined as the summation of all field potential amplitudes induced during each stimulatory period, i.e. field potential amplitude × stimulation frequency. ▪ and continuous line, synaptic activity under control conditions; ○ and dashed line, synaptic activity after 30 min exposure to picrotoxin. Data given as means and 95% confidence intervals. F, compiled data (n = 5) revealing no effect of picrotoxin on neurovascular coupling between evoked CBF responses and synaptic activity. ▪ and continuous line, control values; ○ and dashed line,values obtained after 30 min exposure to picrotoxin. Data are given as means and 95% confidence intervals.

Figure 3. Effect of bicuculline on evoked cerebellar blood flow responses, local field potentials, synaptic activity and neurovascular coupling during inferior olive stimulation

A, compiled data (n = 9) showing a slight effect of bicuculline on the development of evoked blood flow responses (ΔCBF) to increasing stimulation frequencies. However, when regarding each frequency separately, no significant differences were found between control responses and those found in the presence of bicuculline; this was the case for all three panels A, C and E. ▪ and continuous line, control responses; ○ and dashed line, responses measured after 30 min exposure to bicuculline. Data given as means and 95% confidence intervals. ***P < 0.005. B, raw data showing continuous recordings of cerebellar blood flow responses to increasing stimulation frequencies (0.5–20 Hz) under control conditions and in the presence of bicuculline. C, compiled data (n = 9) showing slight effect of bicuculline on the amplitude of field potentials (FPs) evoked by inferior olive stimulation. ▪ and continuous line, field potential amplitudes under control conditions; Δ and dashed line, field potential amplitudes after 30 min exposure to bicuculline. Data given as means and 95% confidence intervals *P < 0.05. D, field potentials from the same animal evoked at 0.5 Hz under control conditions and after 30 min exposure to bicuculline. E, compiled data (n = 9) showing slight effect of bicuculline on synaptic activity (ΣFP) evoked by inferior olive stimulation. Evoked synaptic activity is defined as the summation of all field potential amplitudes induced during each stimulatory period, i.e. the product of field potential amplitude and stimulation frequency. ▪ and continuous line, synaptic activity under control conditions; ○ and dashed line, synaptic activity after 30 min exposure to bicuculline. Data given as means and 95% confidence intervals. *P < 0.05. F, compiled data (n = 9) revealing no effect of bicuculline on neurovascular coupling between evoked CBF responses and synaptic activity. ▪ and continuous line, control values; ○ and dashed line, values obtained after 30 min exposure to bicuculline. Data are given as means and 95% confidence intervals.

Figure 4. Vascular reactivity under control conditions and in the presence of picrotoxin and bicuculline

Adenosine induced cerebellar blood flow responses of the same magnitude under control conditions as after 30 min exposure to either picrotoxin (n = 5) or bicuculline (n = 3), indicating that vascular reactivity is maintained in the presence of disinhibition. Data are presented as means and s.e.m.