Norepinephrine elicits both excitatory and responses from Purkinje cells in the in vitro rat cerebellar slice

Brain Research, 296 (1984) 15-25 Elsevier

15

Norepinephrine Elicits both Excitatory and Inhibitory Responses from Purkinje Cells in the In Vitro Rat Cerebellar Slice ANTHONY S. BASILE and THOMAS V. DUNWIDDIE* Department of Pharmacology, Universityof Colorado Health Sciences Center, 4200 E. 9th Ave., C236 Denver, CO 80262 (U.S.A.)

(Accepted July 26th, 1983) Key words: rat - - cerebellum - - Purkinje cells - - in vitro brain slices - - norepinephrine - - a receptors - - f l receptors

Superfusion of Purkinje neurons in the in vitro rat cerebellar slice with norepinephrine caused increases and decreases of spontaneous Purkinje cell firing. Excitations were evoked by low concentrations of norepinephrine (0.5-10/~M) and by the fl receptor agonist isoproterenol (0.1-5/~M). These excitations were reduced by timolol (1-2/~M), a fl receptor antagonist. Perfusion with higher concentrations of norepinephrine (> 16/~M), caused a depression of Purkinje neuron spontaneous activity. This inhibitory response was blocked by the a receptor antagonist phentolamine. The a 1 selective agonist phenylephrine had no effect on spontaneous activity at concentrations up to 100/~M, but the ct2 selec~tiveagonist clonidine (1-50/~M) elicited decreases in firing rate. These responses appeared to be due to a direct action on Purkinje cells, because neither the excitation nor the depression of Purkinje neuron activity elicited by norepinephrine was substantially altered when tested in a medium which substantially blocked synaptic transmission within the slice. Under these in vitro conditions, norepinephrine appears to increase the firing rate of Purkinje neurons via an interaction with fl adrenergic receptors, while norepinephrine induced depressions may be linked to a adrenergic receptor interactions; both receptors appear to be located directly on the Purkinje neurons. INTRODUCTION The Purkinje neurons of the cerebellar cortex receive n o r a d r e n e r g i c fibers that arise from the ipsilateral locus coeruleus 2,12,36. F a l c k - H i l l a r p2,17,28,36 and glyoxylic acid 19 histofluorescence and electron microscopic a u t o r a d i o g r a p h i c techniquesS,31 all show that these fibers synapse primarily on Purkinje neuron dendrites and dendritic spines in the molecular layer, with some fibers synapsing in the Purkinje cell and superficial granule cell layers. Electrophysiologicai studies have shown that Purkinje n e u r o n spontaneous activity can be suppressed by the iontophoretic application of n o r e p i n e p h r i n e ( N E ) 11,15,24 or stimulation of the locus coeruleus in situ 16,23, while excitatory synaptic responses to stimulation of mossy or climbing fibers are facilitated relative to b a c k g r o u n d activity. Because of the difficulty of quantitatively measuring d o s e - r e s p o n s e relationships in nervous tissue,

* To whom correspondence should be addressed. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

the nature of the receptors mediating these effects has not been fully characterized. A s in p e r i p h e r a l organs, radioligand binding studies have suggested the presence of t29,37,38 and fl1,6,27,32 a d r e n o c e p t o r subtypes in various regions of the CNS. In particular, studies by U ' P r i c h a r d et al. 37,38 suggest the presence of t~1 and a 2 receptors in the cerebellum. Specific binding of [3H]WB4101 and [3H]clonidine with a density of 1.7 and 1.4 pmol/g wet tissue was found in the cerebellum indicative of the presence of small numbers of a 1 and a2 receptors. R a d i o l i g a n d binding studies also indicate that the fix and f12 r e c e p t o r subtypes are present in the cerebellum. The density of cerebellar fll and fiE r e c e p t o r subtypes were r e p o r t e d to be 3.3 and 18.4 fmol/mg protein, respectively:l. Although the presence of these adrenergic receptors in the cerebellum seems a p p a r e n t , their distribution and localization on neuronal elements is still not certain. Recent light microscopic a u t o r a d i o g r a p h i c studies 29,43 indicate that fl r e c e p t o r binding is localized in

16 the molecular layer of the cerebellar cortex. Unfortunately, this technique was not sensitive enough to detect the presence of cerebellar a receptors. On the other hand, electrophysiological studies have indicated that the depressions of Purkinje neuron spontaneous activity elicited by NE administered by iontophoresis or pressure ejection 15,24, or by stimulation of the locus coeruleus, with concomitant release of NE 16,23, could be mimicked by the fl receptor agonist isoproterenol and antagonized by the beta adrenoceptor antagonist sotalo115. This suggests that beta adrenoceptors may mediate the noradrenergic depression of Purkinje cell spontaneous activity. In contrast, Yamamoto 42 has found that NE can elicit excitations from neurons in the flocculus of the cat cerebellum. The nature of the receptors mediating these responses was not determined. Given the variety of neuronal responses elicited by NE in various regions of the CNS (refs. 20, 24, 34, 35, 41) it would clearly be of interest to determine whether there is a pharmacological basis for the response diversity which is observed. In view of the limited number of pharmacological studies involving the adrenergic receptors which mediate Purkinje neuron responses to NE, and the evidence which suggests that both a and fl adrenergic receptors are found in the cerebellum, we have attempted to characterize the functional responses of Purkinje neurons to NE in the in vitro cerebellar slice. Our primary goal was to determine the specific adrenergic receptor types or subtypes mediating the Purkinje neuron responses elicited by NE. The in vitro slice preparation is well suited for the study of dose response relationships, due in part to the ability to superfuse the slice with known concentrations of drugs. This approach permits quantification of relative agonist potency, a critical factor in defining which receptors are involved in a particular response. In addition, the isolation of the slice from its extrinsic afferent pathways also eliminates any indirect drug effects that may be caused by changes induced in other brain regions, or in peripheral organ physiology. This is particular important with drugs like the catecholamines which give rise to profound physiological changes in the cardiovascular system. In the present communication, we use these techniques to describe the responses of in vitro cerebellar Purkinje neurons to NE and related adrenergic drugs.

METHODS Male Sprague-Dawley rats, 125-225 g were decapitated and the cerebellum quickly removed and placed in an ice cold modified Ringer's medium containing 124 mM NaCl, 4 mM KC1, 1.2 mM MgSo 4, 1.2 mM KHzPO4, 2 mM CaCI2, 25 mM N a H C O 3 and 10 mM D-glucose 8. The cerebe!iar vermis was cut into 350/~m thick slices on a Sorvall TC-2 tissue slicer and placed into an incubation chamber 10 filled with the same medium presaturated with 95% 02/5% COz and containing 0.001% H202. Slices were maintained in this medium at 37 °C for at least 45 min, at which time they were transferred to a separate chamber t3 for recording. Slices were perfused with a medium similar to that used for incubation (without H202), at a rate of 2.5-3.5 ml/min. Drugs were made up in physiological saline or distilled water at 100-1000 times the final concentration and added to the flow of control medium via a calibrated Sage model 355 syringe pump connected to a port in the media delivery tubing. Recording electrodes were either Parylene coated tungsten of 1.1-1.2 MQ resistance or 25/~m Teflon coated platinum-iridium wire of 60-90 kf~ resistance. These were lowered into the Purkinje cell layer of the slice under visual guidance. Extracellularly recorded action potentials from single Purkinje neurons were separated from background activity and converted to constant voltage pulses using a window discriminator. Prior to a drug treatment, a Gould digital oscilloscope was used to store and display individual spikes. These were monitored to insure that only action potentials from a single neuron were triggering the discriminator. The output of the discriminator was integrated over one second intervals by a ratemeter and displayed on a Gould strip chart recorder. These constant voltage pulses were also fed into a Neurograph STA-1 (Medical Systems Co.) for construction of interspike interval histograms (IIH) to aid in the identification of single Purkinje neurons. Window discriminator settings were generally not altered during treatment. However, activation of 'silent' neurons during drug treatment or changes in the spike amplitude of the primary neuron occasionally necessitated minor changes in the window settings to allow counting of the original cell. If the recordings were contaminated by the un-

17 controllable appearance of additional neuron discharges, the experiment was terminated. In all experiments only one Purkinje cell was recorded from each slice. This protocol allowed us to minimize the variation in responses that can be caused by desensitization of the neuron to the applied drugs. This was occasionally observed with repeated administration of some catecholamines, and particularly with isoproterenol. In addition, since some of the drugs used are very lipophilic, resulting in slow washout from the slice, this protocol allowed us better control over the true concentration of drug in the slice. Drugs were generally superfused onto the slice for 5-15 min intervals. For antagonist studies~ slices were preincubated with the desired concentration of the antagonist in the medium. When the slice was transferred to the recording chamber, it was then superfused with the antagonist at the same concentration. Agonist responses under these conditions typically showed somewhat longer latencies than without the antagonist present. After a drug induced response was recorded from a neuron and recovery achieved, the slice was discarded. Control experiments in which the drug vehicles (either distilled water or physiological saline) were infused showed no effect on Purkinje cell spontaneous activity, demonstrating that the changes in temperature, flow rate, and osmolarity (0.1-1%) which might be associated with drug administration were without effect by themselves. The data from the ratemeter displays were digitized using a Tektronix graphics tablet and fed into a Data General Nova 3/12 computer. The neuronal activity over sequential 1 s intervals was stored and displayed on a Tektronix C R T computer terminal. Vertical cursors could then be superimposed on the C R T display and used to analyze neuronal activity before, during and after drug applications. Drug induced responses were analyzed as a percent change from the average firing rate observed during a control epoch. In examining the results of the present experiments, we found that the percent change in firing rate showed less variability than the absolute change in spikes/s. Furthermore, there was no significant correlation between these percent values and the initial spontaneous discharge rates for any of the drugs tested. In general, drug responses were determined using the averages of the spontaneous firing rates over

pre-drug control periods and time periods when drug effects had become maximal. When biphasic responses to a drug were encountered the excitatory and inhibitory responses were quantified separately, then averaged together for a 'net' response to obtain values for dose-response curves. Although this arbitrary method might make strong biphasic responses appear as 'no effect', we have made clear in the text where this is the case. RESULTS

Cerebellar neuron identification During these experiments, two types of spontaneously active neurons were encountered while recording from the Purkinje neuron layer of the cerebellar slice. These were primarily Purkinje neurons, which we studied exclusively in our experiments. However, Golgi neurons were also encountered, albeit less frequently. Although Golgi neurons in the slice fire spontaneously at about the same rate as Pur-

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Fig. 1. Action potentials recorded extracellularly from a spontaneously active Purkinje neuron in the in vitro rat cerebellar slice are shown in A (filtered record). Average spike amplitude is 280/~V, and overall time period is 1 s. Note the clear single unit activity and consistent firing pattern. An interspike interval histogram of the Purkinje neuron activity in A averaged over a longer time period is displayed in B; the single modal peak is indicative of a single, regularly firing neuron. In this case, the average firing rate was 20.7 spikes/s for a total of 2765 counts.

18 kinje neurons, they are easily discriminated from Purkinje neurons by their triphasic action potential waveform and extremely regular firing rates. The Purkinje neurons in these experiments maintained firing rates between 10 and 40 Hz (average firing rate 19.4 + 0.8 Hz, n = 151), which is somewhat slower than their in situ firing rates (34 + 2 H z ) 17 but faster than the firing rates which we have reported for mouse Purkinje neurons in vitro (11.6 + 0.6 Hz) 4. The action potentials (Figs. 1 and 2) had the biphasic waveform characteristic of extracellularly recorded Purkinje neurons. The identity of Purkinje neurons was also ascertained by driving the cells antidromically (Fig. 2). Since the Purkinje neurons are the only cerebellar neurons giving rise to efferent fibers, the ability to drive cerebellar neurons via antidromic stimulation of their axons is an appropriate test of Purkinje neuron identity. Another index of the identity and viability of the Purkinje neuron, and

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Fig. 2. Photograph of a Purkinje neuron (solid arrow) driven by antidromic activation of its axon by a bipolar stimulating electrode which was placed in the infragranular white matter at the base of the folium. The recording electrode was placed in the Purkinje cell layer of the s a m e folium. Biphasic square wave pulses of 0.02 ms duration were applied to the Purkinje neuron axons through the stimulating electrode every 30 s. At a stimulus intensity of 8.6 V this Purkinje neuron fired about 50% of the time, as illustrated here. The light arrow indicates the beginning of the stimulus artifact. A second cell with a smaller spike amplitude can be seen to fire in every case at a latency of ~ 2 ms, consistent with synaptic activation of this neuron.

of the number of neurons being recorded is indicated by the regularity of the discharge rate, which is apparent both in the filtered record (Fig. 1A) and in the interspike interval histogram (IIH) (Fig. 1B); note the relatively constant spike amplitudes, and the single modal peak in the IIH characteristic of single, regularly firing neurons. Some cells had llHs with multiple peaks, indicative of erratic firing or possible multiple units, and were not used in subsequent experimental analysis of drug actions.

Responses to norepinephrine Superfusion of rat cerebellar slices with L-norepinephrine (NE) in concentrations of 0.5-10 ~M elicited increases in Purkinje neuron firing of somewhat variable amplitude. These effects were elicited within 3 rain of application, with maximal responses usually seen within 5 min (Fig. 3A). At 16ktM NE the nature of the response became more variable. Of 7 Purkinje neurons tested, one showed a pure depression, two showed only increases in rate and 4 responded biphasically, with excitations followed by depressions of activity (Fig. 3C). The type of response which was observed did not depend in any predictable fashion on the baseline firing rate. The 'net' effect of NE at this concentration was a slight, non-significant increase in spontaneous activity (mean 13.5 + 17.8%). At concentrations between 25 and 100 ktM, NE caused very consistent depressions of Purkinje neuron spontaneous activity. These excitatory and inhibitory effects of NE on Purkinje neuron spontaneous activity are summarized as a doseresponse curve in Fig. 4. Consistent effects of NE (at any concentration) on the amplitude or waveform of Purkinje neuron action potentials were not observed. In view of the two distinctly different responses that were elicited by NE within a fairly narrow concentration range, we sought to determine if these responses were mediated by distinct adrenergic receptor subtypes. When the relatively specific fl receptor antagonist timolol was applied to the slices at a concentration of 1 uM, subsequent perfusion with 16 #M NE caused a significant (49 _+ 2.3%) inhibition of Purkinje neuron spontaneous activity (Figs. 3D and 4). Timolol alone (1 tiM) had no significant effect on Purkinje neuron firing rates, as evidenced by their mean discharge frequency of 19.1 + 0.9 Hz. On the other hand, application of 16 #M NE following prein-

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Fig. 3. Ratemeter records of Purkinje neuron responses to NE (A-C), NE and 1/~M timolol (D), and NE and 50/~M phentolamine (E). Norepinephrine elicited a 54% excitation above control levels at a concentration of 5/tM in A, a 56% depression of activity at a concentration of 50/~M in B, and at 16/~M, a biphasic response consisting of a 90% excitation followed by a 47% depression of spontaneous activity. Following pretreatment with 1 ~ M timolol, 16/~M norepinephrine caused a 47% depression of spontaneous activity (D), whereas norepinephrine superfused with phentolamine induced a 200% excitation (E). Note relatively consistent, high control period Purkinje neuron firing rates and recovery from drug effects. Firing rates represent Purkinje neuron activity averaged over the time period indicated by the solid lines. Horizontal bars in all panels represent 30 s time intervals. The vertical bars in A - C represent a ratemeter line height equivalent to 32 spikes, and in D-E, 16 spikes.

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elicited by isoproterenol in a statistically significant manner. As illustrated in Figs. 5B and 6, 1 p M timo1ol significantly decreased isoproterenol induced excitations at 0.25, 0.5, 1 and 5/~M (P < 0.025). Although 2 p M timolol reduced the 0.25 p M isoproterenol induced excitation by 76%, 0 . 5 p M timolol had no significant effect (Fig. 6).

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Fig. 4. Log dose-response curve for the changes in Purkinje , neuron spontaneous activity during perfusion with various concentrations of norepinephrine (triangles). Note the shift from

excitatory to inhibitory effects on spontaneous activity with doses above 10 ~tM. Data points represent the mean of responses from 4 to 9 cells, Responses were recorded from one neuron per slice. In this and subsequent figures, the vertical bar through each point represents the S.E.M. Prior incubation with 50 pM phentolamine converted the response to 16 pM norepinephrine into a consistent excitation (136 + 30%, diamond), while 1 #M timolol pretreatment had the opposite effect (average depression: 49 +_ 2.3%, square). In slices where synaptic transmission had been blocked (see text), superfusion with 10 pM norepinephrine resulted in a slight increase in excitation over the response in control medium (104 + 11 vs 79 _+ 15%, circle). Perfusion with 50/~M NE under the same conditions caused a depression of activity similar to that seen in controls (mean 50 _+3.2%, circle).

cubation with the a receptor antagonist phentolamine at a concentration of 50 ~M caused a large increase in firing rate. For the cell shown in Fig. 3E, the increase reached a maximum of 300%, the mean increase for all cells under these conditions being 136 + 30% (Fig. 4). Unlike timolol, phentolamine alone caused a slight decrease in spontaneous activity to an average rate of 17.3 _+ 0.8 Hz.

Effects of fl adrenergic drugs The fl receptor agonist isoproterenol was used to further investigate the nature of the receptors mediating these responses. Isoproterenol in concentrations greater than 0.1 p M caused increases in spontaneous discharge rates, similar to low doses of NE (see Fig. 5A). At concentrations greater than 0.5/~M the amplitude of the excitations were reduced (Fig. 6), but outright depressions of firing rate were never observed at any concentration. Again, no major alterations in the action potential waveform or amplitude were observed. As with NE, 1/~M timolol reduced the excitations

Responses of Purkinje neurons to several different a adrenergic drugs were examined. The a~ selective adrenoceptor agonist phenylephrine, at concentrations of 25-100 p M , had no significant effect on the discharge rates of 8 neurons tested. However, the a: selective agonist clonidine had consistent, dose-dependent depressant effects on the spontaneous activity of Purkinje neurons (Figs. 5C, D and 7). The maximal response to clonidine was generally an 80% depression of spontaneous discharge, similar to the maximal decrease seen with NE. The concentration of clonidine required to elicit a half-maximal depression was calculated to be approximately 3.8 pM. These depressions could be readily antagonized in a competitive fashion by the a receptor antagonist phentolamine (Figs. 5 and 7). This concentration of phentolamine (50/~M) produced a statistically significant change in the response to clonidine at every concentration tested (P < 0.025). While these experiments demonstrate well defined physiological responses to N E and to a and fl adrenergic agonists, it is unclear whether these are direct effects on Purkinje neurons, or whether they might be mediated indirectly via actions on other local circuit neurons. To determine whether the latter was the case, we superfused the cerebellar slices with medium containing 1 mM Ca2+/3.9 mM Mg 2+, which we have shown to greatly reduce the amplitude of synaptic responses in both cerebellar and hippocampal brain slices (by 77 and 89%, respectively) 4, This medium caused the baseline firing rate of the Purkinje neurons to increase slightly to 24.8 + 3.4 Hz. Under these conditions, 10 pM N E elicited a mean increase in spontaneous activity of 104 _+ 11% (Fig. 4), a 32% increase over the response in normal medium at this concentration of NE. Conversely, 50/~M NE caused an average 50 +_ 3.2% depression of spontaneous activity, slightly less than that caused by 10/~M NE in normal medium. Thus, both these responses were

21 slices responded in a variety of ways, primarily with excitations followed by depressions. The effects of NE were confined to changes in the Purkinje neuron firing rate; action potential amplitude or waveform were not appreciably altered at any concentration of NE used. The dual nature of these responses suggests that several receptors or receptor subtypes might underlie the different neuronal responses to NE. This hypothesis was supported by the observation that specific fl and a receptor agonists consistently elicited

largely unaffected by the alteration of divalent cation concentrations in the perfusion fluid. DISCUSSION

The preceding experiments demonstrate that NE has two major effects on spontaneous Purkinje neuron activity in the cerebellar slice: excitations which predominate at concentrations less than 16 ~M and depressions that are observed at higher NE concentrations. At intermediate concentrations, individual

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Fig. 5. Ratemeter records of the responses of Purkinje neurons to isoproterenol (A, B) and clonidine (C, D). In A, 250 nM isoproterenol caused an 81% increase in spontaneous activity. Isoproterenol induced excitations could be decreased by 1 #M timolol, as in B, where 250 nM isoproterenol in the presence of timolol elicited only a 36% increase in spontaneous activity. Clonidine at a concentration of 5/~M caused a 56% depression of spontaneous discharge in the Purkinje neuron recorded from in C. Addition of 5 #M clonidine to a slice that has been pretreated with 50pM phentolamine results in only a 31% depression of activity (D). In all illustrations, the firing rates represent Purkinje neuron activity averaged over the time period indicated by the solid lines.

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Fig. 6, Log dose-response curves for the changes in Purkinje neuron discharge rates during superfusion with isoproterenol (triangles) and isoproterenol in the presence of I/~M timolol (circles). lsoproterenol caused excitations at all concentrations greater than 100 nM, even in the presence of 1/~M timolol. The effect of isoproterenol was maximal at 500 riM, with the excitations being attenuated at greater concentrations. Timolol (1 /~M) reduced isoproterenol induced excitations by approximately 50%. At higher concentrations (2 ~M) timolol (square) reduced isoproterenol effects by 76%, while at lower concentrations (0.5 ~M, diamond) timolol had no effect. Data points represent the mean response recorded from 3 to 18 neurons. Responses were recorded from one Purkinje neuron per slice.

excitations and depressions of Purkinje neuron spontaneous activity, respectively. The fl receptor agonist isoproterenol caused excitations in concentrations of 0.1-5/~M, and these excitations were blocked by fl receptor antagonist timolol. Although the a I receptor agonist phenylephrine had no effect on sponta-

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Fig. 7. Log dose-response curve illustrating changes in Purkinje neuron spontaneous activity during superfusion with clonidine alone (triangles) and following pretreatment with 50 #M phentolamine (circles). Clonidine depressed Purkinje neuron activity at all concentrations tested (0.5-50/~M). Phentolamine reduced the response to clonidine at all concentrations tested by approximately 50%. Each point represents the mean of responses from 4 to 12 neurons. Responses were recorded from one Purkinje neuron per cerebellar slice.

neous activity even at high doses, the a 2 agonist clonidine caused consistent depressions over the 0.5-50 /~M range. These depressions were effectively antagonized by the a receptor blocker phentolamine. Similarly, NE could elicit either depressions or excitations when applied to slices preincubated with either timolol or phentolamine, respectively. Thus, the available evidence would suggest that NE-induced depressions of spontaneous Purkinje neuron activity are mediated via a adrenergic receptors, while excitations appear linked to activation of a fl receptor. Increases in Purkinje neuron spontaneous activity were observed in response to low concentrations of NE (starting at 500 nM), and the fl receptor agonist isoproterenol (threshold - 5 0 nM). The excitations elicited by these substances were effectively blocked by low micromolar concentrations of the specific fl receptor antagonist timolol. These results strongly suggest that this effect is a fl adrenergic receptor mediated response. The relationship between this in vitro fl receptor mediated excitation and the responses to NE as previously reported in the cerebellum in vivo is still unclear. However, the identification of the ct adrenergic receptor responsible for the depressant effects of NE must remain somewhat tentative. Clonidine, which has been shown to be a very specific and potent ct2 agonist in physiological experiments (EDs0 = 10.4-20 nM) 33,39,4°, and in in vitro receptor binding systems ( K d = 5.8 nM) 38 was the most potent a agonist tested. Paradoxically, the ECs0 for clonidine (3.8/~M) in our system is about 1000 times greater than the in vitro Kd, and approximately 100 times greater than the concentrations which elicit responses in other brain slice preparations (e.g., locus coeruleus, 4-8 nM) TM thought to contain ct2 receptors. The EDs0 we observed for the depressant effect of clonidine on in vitro Purkinje neuron activity is more consistent with an interaction of this drug with a 1 receptor sites. For example, Wikberg 4° has shown that clonidine appears to act at an al receptor in the guinea pig aorta with an ED50 of 4 pM. In in vitro binding studies, U'Prichard et al. 3~ found that clonidine displaces [3H]WB4101 binding to rat brain membranes with a K i of 430 nM. In a similar vein, the concentration of phentolamine required to antagonize the effects of clonidine is relatively high. The K i for phentolamine displacement of clonidine's high affinity binding to a2

23 receptors on rat brain membranes is 22 nM, in contrast to an IDs0 in the cerebellar slice of approximately 50/~M. Thus, while the evidence tentatively suggests that clonidine might inhibit Purkinje neuron firing by actions at an ct (and perhaps an al) receptor, other possible receptor types cannot be ruled out. More experiments utilizing additional specific ct receptor agonists and antagonists will be required to determine the nature of the receptor mediating this depression with greater certainty. The localization of the receptors mediating these effects in the cerebellum is still an open question. Although previous research has suggested fl receptors may be localized on Purkinje neurons 14,22, no unequivocal evidence has been presented with regards to either ct or fl receptor localization on specific neuronal elements in the cerebellum. The present finding that responses were unaffected under conditions where synaptic transmission is largely blocked suggests that both the excitatory and inhibitory receptors may reside directly on the Purkinje neuron. Had these effects been indirect, the observed responses should have been reduced or blocked by perfusion with low calcium medium. Previous reports on the actions of NE on spontaneous unit firing have suggested that this neurotransmitter has multiple and complex actions. Excitations, inhibitions, complex excitatory-inhibitory responses and modulatory neurotransmitter interactions have been reported for NE itself in different" brain regions 7,25,26,30,34.35.41.42. However, the preponderance of evidence to date would suggest that locally applied NE has purely inhibitory actions on cerebellar Purkinje neuron spontaneous activity in situ, and that this response is mediated via a fl adrenergic receptor. In only one case have neuronal excitations in response to NE been reported in the in situ cerebeilum4L This of course stands in direct contrast to the results of the present study, in which the responses to activation of fl adrenergic receptors appears purely excitatory. Several hypotheses might be advanced to account for the apparent discrepancy. (1) Differences between local drug applkation and bath perfusion of drugs. The various subpopulations of adrenergic receptors activated by local NE application in the vicinity of Purkinje neuron somata may differ from those activated by NE administration to the entire slice. Preliminary experiments (data not

shown) using microdrop applications of NE. and isoproterenol to the cerebellar slice have indicated that the types of responses which are observed may differ depending upon the locus of drug application. (2) Functional differences between Purkin]e neurons in situ and in vitro. Considerable evidence has accumulated suggesting that NE can modulate the efficacy of synaptic inputs to Purkinje neurons 11.23,24 and a physiological mechanism to account for this has recently been proposed20. Deprived of their cell bodies by the cutting procedure, many of the fiber afferents to the Purkinje neurons under study in these experiments are probably not active. Hence, an important 'substrate' for modulation has been removed from the in vitro brain slice, and may account for some of the differences between the in vitro and in situ situations. (3) Differences in the external milieu between slices and the intact brain. Most electrophysiological responses depend upon the opening and/or closing of specific ion channels, and hence the nature of the response can be affected by the concentrations of ions in the extracellular space. If the conditions under which we maintain the cerebellum in vitro do not adequately mimic those in situ, corresponding differences in physiological responses could arise. We have previously reported that while excitatory responses to NE predominate in the hippocampus in vitro, the opposite appears to be the case in situ25,26. The same finding in the present experiments suggests that a similar factor may be working in the cerebellum as well. In summary, the present results suggest that noradrenergic responses in the cerebellum involve fl and possibly ct adrenergic receptors. Further experiments will be required to establish the mechanisms which underlie these responses, and to relate these results to phenomena which have previously been described following local drug application in the in situ cerebellum. ACKNOWLEDGEMENTS This research was supported by Grants DA02702 and VA 394463116 to T.V.D.

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