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1
Department of Medical Physiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark
2
Department of Clinical Neurophysiology, Glostrup Hospital, Copenhagen, Denmark
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(Received 12 May 2004;
accepted after revision 19 July 2004;
first published online 22 July 2004)
Corresponding author K. Thomsen: Department of Medical Physiology, Panum Institute 12.5, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Email: kthomsen{at}mfi.ku.dk
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Introduction |
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In neurons with no intrinsic pace-maker capabilities, synaptic input and processing are prerequisites for the generation of spikes. In consequence, the separate contributions of synaptic versus spike activities to stimulation-evoked blood flow or metabolic responses are not directly distinguishable. We have previously shown that topical stimulation of cerebellar parallel fibres caused Purkinje cell spike rate to decrease while synaptic activity increased with increasing stimulation frequencies (Mathiesen et al. 1998). This study was the first to demonstrate a dissociation of synaptic and spiking activities, which occurred in response to increased GABAAergic tone. In the present study, the opposite condition was studied: intrinsic, inhibitory GABAAergic tone was abolished inducing disinhibition, allowing Purkinje cell spike activity to increase without altering excitatory synaptic activity. The aim of this study was to examine the effect of increased spike activity, separate from synaptic activity, on cortical blood flow in the rat cerebellum.
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Methods |
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This study was performed in full compliance with the guidelines of the European Council's Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and was approved by the Danish National Ethics Committee. Seventeen male Wistar rats (270–370 g) with free access to tap water and Altromin 1314 rat chow and acclimatized to a 12 h–12 h light–dark cycle were included.
Surgery
During anaesthesia with isoflurane (5% induction, 2% during surgery and maintainance), the left femoral artery and vein were cannulated for continuous arterial blood pressure monitoring and hourly arterial blood gas measurements, and for I.V. administration of drugs, respectively. The trachea was cannulated for mechanical ventilation with oxygen and nitrous oxide ensuring arterial PO2 values > 100 mmHg and arterial PCO2 values of 37.4 ± 3.5 mmHg. The animals were placed in a head holder with lidocaine gel (2%) applied to the contact spots of the ear bars. An open cranial window surrounded by an agar well containing artificial cerebrospinal fluid (aCSF; volume 
0.5 ml) was placed over the cerebellar vermis and caudal part of the medulla oblongata. The underlying dura was removed. The brain was continuously superfused with aCSF (composition: NaCl 126 mM, NaHCO3 22 mM, Na2HPO4 1.00 mM, KCl 2.80 mM, MgCl2 0.88 mM, CaCl2 1.45 mM, glucose 3.00 mM, bubbled with 5% CO2–95% O2) After positioning the electrodes and the laser Doppler flowmetry probe (see below), anaesthesia was switched to intravenous 
-chloralose (bolus: 45 mg kg–1, supplement: 15 mg kg–1 (20 min)–1). Extra supplements of -chloralose were given upon pilo-erection, increased blood pressure (> 10%) or positive corneal reflex.
Electrophysiological stimulation and recordings
A coated bipolar stainless steel electrode (NE-200X, Rhodes Medical Instruments, Inc., Woodland Hills, CA, USA, contact separation: 0.5 mm) was positioned in the caudal part of the inferior olive nucleus. Stimulation was given as 0.2 ms square wave pulses at 0.15 mA with increasing frequencies (0.5, 2, 5, 7, 10, 15 and 20 Hz). Using a single-barrelled glass microelectrode filled with 2 M saline (impedance 2–3 M
, tip diameter 2 µm), both single unit activity (spikes) and local field potentials of Purkinje cells were measured at a depth of 200–500 µm in folia 5–6 of the vermis. Single unit spike activity was amplified 2000 times and filtered at 300 Hz–6 kHz, while field potential signals were amplified 200 times and filtered at 0.1–2400 Hz using a CyberAmp 380 amplifier (Axon Instruments, Union City, CA, USA). Data acquisition and off-line analysis were performed using Spike 2 software with a 1401+ interface (Cambridge Electronics Design (CED), Cambridge, UK). Digital sampling rates of 20 and 4 kHz were used for spike activity and field potentials, respectively.
Cerebellar cortical blood flow recordings
Cerebellar cortical blood flow (CBF) was monitored continuously using laser Doppler flowmetry (LDF). An optic probe (wavelength 780 nm, 250 µm between the transmitting and receiving fibres; PeriMed, Järfälla, Sweden) measuring blood flow in the upper 1 mm of the cortex was positioned close to and in the same folia as the microelectrode in a region devoid of visible arteries and veins. Once in place, the LDF probe, the microelectrode, and the bipolar stimulation electrode were kept in the same position throughout the experiment. The LDF signal was smoothed with a time constant of 0.2 s (PeriFlux 4001 monitors, PeriMed), then A/D converted and sampled at 10 Hz using Spike 2 software with the 1401+ interface (CED). LDF measures CBF in arbitrary, not absolute, units; nonetheless, LDF is valid for determining and comparing relative flow changes during states of moderate CBF increases (Fabricius & Lauritzen, 1996).
Protocol
In the present study, Purkinje cell spike activity was enhanced without increasing the excitatory synaptic input to these cells. This dissociation of Purkinje cell spike and synaptic activities was achieved by abolishing intrinsic, inhibitory GABAAergic tone using either bicuculline, a GABAA receptor inhibitor, or picrotoxin, a blocker of the chloride channel gated by GABAA. Two GABAA antagonists with different sites of action were employed to ascertain that our findings were due to GABAA-mediated mechanisms. By dissociating spike from synaptic activity, we were able to evaluate the effect of spike activity per se on CBF. To ensure that our results were not due to an altered relationship between neuronal activity and CBF, the effect of disinhibition on the coupling between synaptic activity and evoked CBF responses was examined during inferior olive stimulation. Finally, the effect of disinhibition on vascular reactivity was also examined.
Basal CBF and spontaneous spike rate were registered under control conditions and 30 min after changing superfusion from aCSF alone to aCSF containing either GABAA antagonist. As the agar well surrounding the cranial window was not first emptied of its aCSF content, the GABAA antagonist concentration within the well was not abruptly altered, but increased gradually with time to a final concentration of 0.5 mM. Inferior olive (climbing fibre) stimulation took place under control conditions and in the presence of the GABAA antagonists before and after the basal CBF and spontaneous spike rate measurements. The stimulation protocol used increasing stimulation frequencies from 0.5 to 20 Hz, where each frequency was given for 60 s during which CBF responses and field potentials were registered. CBF and spike rate were allowed to return to baseline in the intervening interstimulus periods lasting at least 150 s. In a separate series of animals, vascular reactivity was tested by the topical application of adenosine (0.5 mM) for 4 min before and after 30 min superfusion with bicuculline. In animals receiving picrotoxin, vascular reactivity was tested with adenosine before and upon completion of the stimulation protocol. Upon completion of all protocols, the animals were killed by intravenous injections of air.
Drugs
Bicuculline methiodide, picrotoxin, and adenosine were obtained from Sigma Chemicals, Denmark. Fresh solutions using aCSF were made each day. Picrotoxin and adenosine, which were not readily soluble in aCSF, were ultrasonicated for 2–5 min.
Calculations and statistics
Field potentials represent spatially weighted averages of synaptic signals evoked by the activation of presynaptic neurons (Llinas & Nicholson, 1974; Logothetis, 2002). Field potential amplitudes, measured from pre-stimulus baseline to nadir (Mathiesen et al. 1998), decreased in magnitude with increasing stimulus frequency while maintaining their general profile. For any given stimulation period, evoked synaptic activity was defined as the sum of the amplitudes of all field potentials induced during that period and was calculated as the average field potential amplitude multiplied by stimulus frequency (Mathiesen et al. 1998). In contrast to field potential amplitude, evoked synaptic activity increased with increasing stimulus frequency. Area under the curve (AUC) was measured for each CBF response and its immediately preceding baseline during the 60 s stimulation period and the preceding 60 s, respectively. CBF responses were then calculated as percentages of their corresponding baselines. CBF responses, field potential amplitudes, and spike rates were transformed into natural logarithms to ensure normal distribution of the residuals. All data are therefore given as means and 95% confidence intervals unless stated otherwise. Statistical analysis was carried out using two- and three-way ANOVA and when significance was found, the paired t test was applied within groups. Multiple regression was used to examine the effect of disinhibition on neurovascular coupling. P < 0.05 was considered significant.
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Results |
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FP; P = 0.8002, 3-way ANOVA; Fig. 2A, C and E). Using the bicuculline salt, bicuculline methiode (n = 9), we found slight, but statistically significant reductions in CBF responses (P = 0.0077), LFP amplitudes (P = 0.0266), and synaptic activities (P = 0.0446, 3-way ANOVA; Fig. 3A, C and E), although the difference between control and bicuculline values at any one frequency was never significant. In contrast to picrotoxin and bicuculline free base, bicuculline methiodide blocks a Ca2+-dependent K+-current (Johnson & Seutin, 1997; Pflieger et al. 2002), which may explain the attenuation of these electrophysiological and vascular responses (Bear et al. 1992; Zhou & Poon, 2000; Gerrits et al. 2002). Importantly, however, the neurovascular coupling between synaptic activity and blood flow responses evoked by inferior olive stimulation remained intact both in the presence of picrotoxin (P = 0.5748, multiple regression, n = 5) and bicuculline methiode (P = 0.2860, multiple regression, n = 9; Figs 2F and 3F).
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Discussion |
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Disinhibition was induced using either bicuculline, a GABAA receptor inhibitor, or picrotoxin, a blocker of the chloride channel gated by the GABAA receptor. In the presence of either GABAA antagonist, spontaneous PC spiking rate increased 200–300%. Conversely, we have recently shown that muscimol, a GABAA agonist, abolishes spontaneous PC spike activity (Caesar et al. 2003). In both studies, the altered PC spiking rate was not accompanied by any change in CBF. Thus in the presence of varying levels of GABAAergic tone, we found no coupling between spontaneous PC spiking rate and basal CBF. GABAAergic tone also modifies stimulation-induced increases in PC spike activity. In a previous study, direct stimulation of cerebellar parallel fibres (PF) inhibited Purkinje cell spiking by 84% due to disynaptic release of GABA from interneurones activated by the PFs (Mathiesen et al. 1998). Subsequently, bicuculline partially alleviated the decrease in PC spike activity without affecting the evoked CBF response (Mathiesen et al. 1998). Thus, PC spiking rate, both in basal conditions and during stimulation, was modified by the prevailing GABAAergic tone while CBF was not; therefore, CBF levels did not parallel PC spiking.
Field potential amplitudes reflect both presynaptic glutamate release (Hashimoto & Kano, 1998; Silver et al. 1998) and postsynaptic transmembrane currents triggered by neurotransmission (Llinas & Nicholson, 1974). Neither field potential amplitude nor synaptic activity was found to be affected by picrotoxin, which may be explained by climbing fibre–Purkinje cell synapses being monosynaptic glutamatergic synapses without presynaptic GABAA receptors to inhibit glutamate release (Vigot et al. 1993). CBF responses are dependent upon glutamate transmission, as AMPA receptor blockade abolishes CBF responses to inferior olive stimulation (Mathiesen et al. 1998; Yang & Iadecola, 1998). Like field potentials, CBF responses were also unaffected by picrotoxin. Thus, disinhibition with picrotoxin did not affect FP amplitude, synaptic activity or CBF responses during inferior olive stimulation. In fact, the coupling between synaptic activity and CBF found under control conditions was preserved in the presence of both GABAA antagonists.
We also examined the possibility that GABAA antagonists might have a direct, inhibitory effect upon cerebral blood vessel dilatation. GABA has been suggested to play a role in regulating cortical blood flow, as GABAergic terminals have been found in close association with both cerebral (Vaucher et al. 2000) and cerebellar (Benagiano et al. 2001) microvessels. However, the maximal vascular response to the endothelium-independent vasodilator adenosine was not different under control conditions from the maximal response found in the presence of either bicuculline or picrotoxin, clearly demonstrating that GABAA antagonists do not affect the ability of cerebellar blood vessels to dilate.
As GABAA antagonists increase spiking activity of Purkinje cells and interneurons (Hausser & Clark, 1997), they conceivably increase the spiking rate of all cerebellar neurons, including granular cells. We cannot exclude the possibility that the observed increase in PC spike activity was partially due to increased granular cell spiking in our preparation. Increased granular cell spiking would be expected to increase glutamate release at the parallel fibre–Purkinje cell synapses thereby raising CBF. However, exposure to the GABAA antagonists did not affect CBF levels, suggesting that granular cell input to Purkinje cells may be minimal in basal conditions. Another point for brief consideration is spike activity in interneurons, which has been shown to increase in the presence of GABAA antagonists in vitro (Hausser & Clark, 1997). In the presence of GABAA receptor blockade in our preparation, increases in interneuron spiking resulting in increased GABA release would be expected to activate GABAB receptors. Activation of GABAB receptors reduces spontaneous PC spike activity by approximately 50% without affecting CBF levels (Caesar et al. 2003). As PC spike rate increased 200–300% in our preparation, we conclude that the GABAA receptor blockade was more massive than the putative GABAB receptor activation.
In conclusion, we found that disinhibition dramatically enhanced spontaneous Purkinje cell spike rate with no concomitant increase of basal cerebellar blood flow, demonstrating that increased PC spike activity per se is not sufficient to raise CBF. We have previously shown that parallel fibre stimulation abolishes PC spike activity while evoking CBF responses, demonstrating that PC spike activity is also not necessary to increase CBF (Mathiesen et al. 1998). Thus, in the rat cerebellum, principal cell spiking is neither necessary nor sufficient to evoke CBF responses. As PC spike activity varied with GABAAergic tone, no coupling was found between spike activity and CBF during disinhibition, while the coupling between synaptic activity and CBF remained intact. The present study supports the view that areas of increased blood flow found in functional neuroimaging represent neuronal populations with increased synaptic input as opposed to increased axonal output.
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CBF) 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. 
