Cerebellar inhibition and ICSS from stimulation in the area of the nucleus locus coeruleus

Brain Research ~u~~et~n,Vol. 3, pp. 193-202.

Printed in the U.S.A.

Cerebellar Inhibition and ICSS from Stimulation in the Area of the Nucleus Locus Coeruleus

(Received 20 December 1977) SINNAMON, H. M., B. SHAW, D. G. AMARAL AND D. J. WOODWARD. Cerebellar inhibition and ICSS from stimulation in the area of the nucleus locus coeruleus. BRAIN RES. BULL, 3(3) 193-202, 1978. - The purpose of this study was to determine the relationship between intracranial self-stimulation (ICSS) and the long-lasting inhibition (LLI) of cerebellar Purkinje cells which are produced by stimulation around the dorsal pontine nucleus locus coeruleus (LC). No strong correlation was found between the dorsal pontine sites which produced LLI and those sites which yielded ICSS. Moreover, ICSS sites were no more effective than non-ICSS sites in producing LLI. LLI of Purkinje celIs was produced most effectively by st~mu~tion of an area dorsolateral to the LC where axons arising from the LC collect to ascend to the cerebellum. The LLI produced by stimulation of this dorso~atera1 region was less often associated with short latency excitations, compared to the LLI produced by stimulation of tfle cerebellar white matter. This characteristic may be useful as an indication of LC-produced LLL Sites yielding ICSS were. scattered around the LC but were most consistent ventrolateral to the LC. These results indicate that ICSS and LLI of Purkinje cells appear to be independent phenomena

which depend on different mechanisms. Locus coerubus

ICSS

Cerebellum

Monoamines

a nucleus of norepinephrineTHE LOCUS coeruleus, containing neurons in the dorsal pons, holds considerable promise as a model site for the study of central noradrenergic systems. The neurobiology of the LC system has recently been reviewed (Amaral and Sinnamon, in press}. The present study was concerned with the reIationsh~p between two facets of LC physiology. One was the inhibitory influence of the LC on cerebeflar Purkinje cells, a profound cessation of ongoing activity lasting up to several seconds. This iong-lasting inhibition (LLI) was described in detail by Hoffer et al. [ 121. The other aspect was the capability of the nucleus to support intracranial selfstimulation (ICSS), a function originally proposed by Crow et al. [ 71 and supported by the reports of others [24,X] _ The present study sought to determine whether LLI and ICSS shared a common mechanisIn that would be revealed by a mutual dependency on site of stimulation. A demonstrable common element in these two functions would have important implications for the understanding of the neural basis of reinforcement. Evidence is consistent with the conclusion that activation of LC axons by electrical stimulation produces LLI of Purkinje cells by means of norepinephrine liberated at the terminals. The sites maximally effective in producing LLI are confined to the area in and near the nucleus [ 12,261. Destruction of noradrenergic terminals in the cerebellum with Ghydroxydopamine substantially reduces the percentage of cells inhibited by LC stimulation while the otherwise similar LLI produced by juxtafastigial stimulation is unaffected. The neurotoxin also changes the

spontaneous activity of Purkinje cells by selectively reducing the occurrences of long interspike intervals f 131. Administration of the phenothiazine, fluphenazine, a blocker of iontophoretical~y applied NE, also decreases the LLI produced by LC stimulation f IO] _ LLI consequent to climbing fiber or parallel fiber activation is not affected [10,12]. Stimulation of the LC in the mutant Weaver mouse which lacks granule cells also produces a similar phenothiazine-sensitive LLI f 261. While there is general agreement that sites around the LC nucleus will support ICSS, some controversy attends the assertion that an actual activation of the LC cell bodies is required for the phenomenon. The principal evidence for a role of the LC comes from studies showing a high density of positive ICSS sites in or near the nucleus 17,241. Additional support comes from the depressing effects of chlorpromazine, a catecholamine blocker, and the facilatdry effects of R-amphetamine, a catecholamine releaser, on ICSS on the region ]7,24]. However, the detection of ICSS sites around the LC requires explicit training procedures that appear not to be necessary, at Ieast to the same degree, in other ICSS areas f 1,271. Considerably more difficulty for the proposed role of the LC in ICSS stems from the finding that ICSS in sites ventrolateral to the central gray, which has been attributed to activation of LC axons [7, 11, 241, survives bilateral lesions of the LC nucleus. Conversely, ICSS in and around the LC nucleus is not attenuated when the LC axons projecting through the dorsal midbrain are damaged by 6-hydroxydopamine f5]. Thus it is possible that activation of the ascending axons

’ Present address of D.G.A. is Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 631 IO. ‘Present address of D.J.W. is apartment of Cell Biology, Southwestern Medical Center, Dallas, TX 75235. 193

Copyright @ 19?8 ANKNO I~te~ati~n~

fnc .-0361-9230178/0303-0193$05.05

194

SINNAMON,

from the dorsal LC which project to the forebrain through the dorsal bundle in the midbrain may not be necessary for ICSS. Some LC cells which contribute axons to the dorsal ascending bundle also give rise to collaterals which innervate the cerebellum [20] and the influence of this projection on Purkinje cells, LLI, is relatively well documented [ 10, 12, 261 Our goal in this study was to study ICSS and LLI in the same preparation to establish the degree of concordance between the two phenomena. Stimulation electrodes were implanted in and around the LC and tested for ICSS. Subsequently, with acute experiments, the same sites were tested for capacity to produce LLI in the cerebellum. Supplemental information was obtained from a series of sites that were tested for inhibitory properties alone. In general, the results indicated an independence between LLI and ICSS elicited from sites in the area of the LC nucleus. METHOD

The principal experment had four phases: implantation of chronic stimulation electrodes, behavioral testing for ICSS, electrophysiological analysis of the effects of LC stimulation on unit activity of cerebellar Purkinje cells, and finally, histological verification of the stimulation sites. Electrode

Implantation

Teflon-insulated stainless-steel wire (130 pm dia.) was twisted into a bipolar configuration, attached to Amphenol pins, and cut to expose only the cross section of the tips. The two tips of the pair were either cut evenly or separated vertically by approximately 1 mm. Bipolar electrodes were implanted usually bilaterally, in rats anesthetized with Nembutal and mounted in a Kopf stereotaxic apparatus. The electrode pins were inserted in a plastic strip that was anchored to the skull by means of small screws and dental cement. The interparietal bone was left clear to allow access to the cerebellum later in the recording phase of the study. Behavioral

Testing

ICSS in 31 electrode sites was assessed primarily by bar pressing tests in a standard Plexiglas operant conditioning chamber. Shaping procedures were used in which brief trains of stimulation were provided to the rat whenever it oriented to, approached, or depressed the bar. Sustained bar pressing at rates above lO/min for 5 min was considered ICSS. Rats failing to self-stimulate were subjected to additional shaping sessions until the electrode site could be classified as an ICSS site or a non-ICSS site (failed to support ICSS or supported escape behavior). Some sites (N = 8) failing to show ICSS in bar pressing sessions were subjected to additional tests in a shuttlebox that has been described previously [28]. Various types of stimulation were tested with this procedure including single 0.5 set trains at 100 Hz, continuous trains at 10 Hz, and repetitive 0.5 set trains at a pulse frequency of 100 Hz and a train frequency of 1 Hz. The 18 sites eventually classified as non-ICSS were tested with a median of two bar pressing sessions, range I- 15 sessions. The length of a test session ranging from approximately 20 min to varied widely, several hours. Generally, sessions continued as long as the rat showed any signs of approach or orientation to the bar or to the side associated with stimulation, or until clear escape behavior was apparent. The various procedures and

SHAW, AMARAL

AND WOODWARD

forms of stimulation used were intended to minimize the possibility of false negatives in the behavioral classification. As an additional precaution against false negative classifications, some sites (N = 12) were tested after administation of d or dl-amphetamine (1.0 mg/kg) injected intraperitoneally 15-30 min before a bar pressing session. Use of the drug was prompted by the observation that some rats during the initial sessions showed sustained orientation to the bar and occasional pressing but at levels below criterion. The drug was successful in facilitating bar pressing to above criterion level in 7 of 12 cases. For these sites which were located in all of the dorsal pontine regions studied, ICSS was found to be sustained when tested at least one day later. The five sites at which amphetamine was not effective were located in the dorsal pons dorsolateral and medial to the LC nucleus. Electrical stimulation was provided by biphasic rectangular pulses, each phase of 0.3 msec duration in tests at 19 sites and by monophasic rectangular pulses, of 0.3 msec duration in tests at 12 sites. For most of the ICSS tests current was varied from 50 MA up to a limit of 300 MA. Movement, primarily of the face, mouth and forelimbs, as described by Crow et al. [7], occurred well below 300 PA in the vast majority of cases. An upper limit of 300 PA was selected to restrict the spread of current to a size appropriate to a target as small as the LC, less than 0.5 mm in the medio-lateral plane and 1 .O mm along the longitudinal axis. This range of current is well within the range used by others [ 7, 24, 251 to demonstrate ICSS in the region. Nevertheless, as an additional precaution against a false negative classification, higher intensities were used with five non-ICSS sites, 500 PA in three cases and 400 PA in two others. These sites were located within, dorsolateral to, or medial to the LC nucleus and none showed ICSS at the higher intensities. A range (50-I 50 Hz) of stimulation pulse frequencies were used in the bar press shaping sessions but most training was attempted at 100 Hz. Animals that failed to bar press and that were tested in the shuttle box were also given the opportunity to self-stimulate with recurrent trains at 50 or 100 Hz or continuous trains at 10 Hz. This latter frequency, which is much lower than typically employed in ICSS studies, was selected because of reports that low frequencies of stimulation in the LC nucleus are optimal for inhibition of Purkinje cell activity [ 121 and neocortical norepinephrine turnover [ 151. Acute

Recording

Procedures

In the recording phase of the study in which sites were tested for the ability to produce LLI of Purkinje cells, rats were anesthetized with 0.5% halothane and mounted in a stereotaxic apparatus. The cerebellum was exposed and covered with warm agar for acute recording. Purkinje cells, identified by their characteristic pattern of bursting discharge, were recorded in the vermal areas of folia 5-9 through glass pipettes filled with 2 M NaCl or through glass-insulated tungsten microelectrodes. The unit activity was amplified and lead into a voltage window and fall time discriminator which produced standardized 5 V pulses. The discriminator allowed for a complete suppression of output for a selectable time after each stimulation pulse. In this way, a precise analysis of spike activity during the train of stimulation was possible without contamination by stimulation artifact. Analysis of spike activity employed either a general purpose computer (PDP- 12) which constructed

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.

Stimulation intensities used in the recording sessions were suprathreshold for barpressing at ICSS sites or motorie effects in non-ICSS sites. In the acutely prepared rats, intensities were selected that produced either field potentials in the cerebellar cortex or head and face movements. Intensity was not varied in these tests since higher intensities often resulted in movements or autonomic responses that disrupted recording of unit activity despite restraint of the rat in the stereotaxic apparatus. All units were tested with 10 set trains of stimulation at a pulse frequency of 10 or 20 Hz, frequencies reported [ 12 1 to be optimal for Purkinje cell inhibition, Single pulses were also tested for possible short latency effects. Generally, when a cell showed LLI, it was tested with trains of higher frequency stimulation (50- 100 Hz). Histology

ME-J ,/.--+

[

”

’

VL

Following the acute recording experiment, the rats were perfused transcardially with normal saline followed by 10% Formalin. After several days of additional fixation in 10% Formaiin, transverse sections (I 6 to 32 Erm were taken on a cryostat microtome. The deepest point of electrode penetration was determined from cresyl violet stained sections.

--.__e’ I

RESULTS Histotogy

\-.

-3

FIG. I. Locations of stimulation sites in the area of the LC nucleus. The upper section is rostra1 and the lowest section is posterior. Symbols: circle, site tested only for LLI and not for ICSS; triangle, ICSS site; square, non-ICSS site. A black symbol indicates that stimulation produced LLI. An open symbol indicates that stimulation did not produce LLI in any of at least four ipsilateral Purkinje cells. Regional analyses were based on sectors delimited by the dashed lines emanating from the LC nucieus. Abbreviations: BC, brachium conjunctivum; CER, cerebellum; DL, dorsolateral region; LC, locus coeruleus nucleus; MED, medial region; nmtV, nucleus of the mesencephalic tract of the trigeminal; nMV, motor nucleus of the trigeminal; VII, root of the facial nerve; VL, ventrolateral region; 4V, fourth ventricle%

persistimulus time histograms or a raster generator and a storage oscilloscope which produced dot displays of discriminated spike activity. Unit activity was also integrated over I-set epochs and displayed on a polygraph operating at a paper speed of 1 mm/set. The polygraph write-out was useful in studying the long lasting changes often elicited by stimulation. The main body of electrophysiol~~ical data was derived from tests of 31 chronic stimulation sites. Supplemental data was provided by tests of another 21 acute stimulation sites in other rats.

A total of 52 stimulation sites in the dorsal pons, midbrain and cerebellar white matter was tested. Of these sites, 39 were sufficiently close to the LC nucleus in the dorsal pons or extreme ventral cerebellum to be of direct concern and are illustrated in Fig. 1. The symbols showing the sites of stimulation are either circles, triangles or squares. A circle indicates a site that was tested in a recording session oniy, i.e., it was not tested for behavioral properties in a chronic preparation. A triangle indicates a site that was tested in a chronic preparation and found to be positive for ICSS. A square indicates a site found to be negative for ICSS. A filled-in symbol of any type indicats that the site was effective in producing LLI. Only these sites tested with at least four ipsilateral Purkinje cells were included in the analysis. Figure 1 shows the LC nucleus in three transverse sections. The upper section shows the LC at approximately 0.45 mm from its anterior pole, as described by Swanson [29] ; the section is at the approximate midpoint of the nucleus. The middle and lowest sections, respectively, are about 0.60 and 0.75 mm from the anterior pole. The dashed lines emanating from the LC nucleus demarcate the regions of the dorsal pons used to study the distribution of inhibitory and ICSS sites. Sites were judged to be within the LC nucleus only when the depest point of the electrode tract contacted the cell bodies of the LC. A totaf of six sites were so judged, two in the rostra1 section, three on the middle, and one on the caudal section. Sites were judged to be dorsolateral (DL) to the LC nucleus (N = 18) when the electrode tract did not contact the cells of the LC and were lateral to the vertical dashed line and dorsal to the horizontal dashed line. The limits of this region were based on descriptions of the course of LC axons projecting to the cerebellum in the rat [ 12, 2 1, 23 ] . The region ventrolateral (VL) to the nucleus contained six sites and the region medial (MED) to the nucleus contained nine sites. Not shown in Fig. I are the midbrain and cerebellar sites remote

196

~~NNAMUN,

SHAW, AMARAL

AND WOU~WAR~

L

2cY sec 20sec 20Hr

20HZ

FIG. 2. Long-lasting inhibition of a Purkinje cell 71R-14 by stimulation in the caudal LC nucleus which did not support ICSS. The continuous polygraph record shows the effects of stimulation at 300 @A, 100 pulse tram length, at frequencies from 10 to 100 Hz. To generate the record, the output of the spike discriminator causes an upward deflection of the pen which resets to baseline each second. The output of the discriminator is suppressed for a selectable period during each stimulation pulse. Thus, the ratemeter does not reflect false counts caused by stimulation artifact. This stimulation elicited no short latency excitation and produced a marked slowing that was apparent for over 1 min at higher freqnencies.

lO6Hr

20 Hz

IO0 Hz

40Hz

1 Hz

i

FIG. 3. LLI of Purkinje cell 71R-13 by stimulation in the LC nucleus wkick also produced a generalized increase in activity during the train. Panel A, 20 and 40 Hz trains produced increased activity during the stimulation and decreases lasting approximately 5 see afterwards. Panet B, marked stowing of activity with 100 Hz stimulation. Panel C, train of 1 Hz pnfses fails to produce LLI but is followed by increase in activity lasting over I min. Panel D, upper part shows raster display of dots produced by spike discriminator output. The dots step down with successive pulses of train. The tower trace shows the accumulated traces of the raw activity. The response to 100 pulses is shown in 1, 2, and 3; 50 pulses were given in 4. Note that the stimulation fails to produce a consistent short latency response despite an overall increase in activity at higher frequencies. Sweep times for D: 1, 20 msec; 2 and 3,lO msec; 4,500 msec.

from the LC nucleus. The midbrain sites (N = 8) were principally in or near the superior cerebellar peduncle and the cerebeIlar sites (N = 5) were in medultary area or juxtafastigiai region.

Long-lasting

Inhibition

(LLI)

o.fPurkinje Cells

The LLI produced by stimulation around the LC nucleus conformed in major respects to the descriptions by Hoffer et al. f 121‘ Generally, the slowing or complete cessation

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FIG 4. Continuous ratemeter record of Purkinje cell 67-14 showing LLI with stimulation in the area dorsolateral to the LC nucleus. Note the variability in the magnitude of the LLI produced by 20 Hz stimulation. All trains were 100 pulses in duration and 300 @Aintensity. This stimulation site did not support ICSS.

FIG. 5. Continuous ratemeter record of LLI of a Purkinje cell, 79-1, produced by stimulation in an ICSS site near the dorsal tegmental nucleus of Gudden. Stimulation of 20 Hz produced relatively less LLI than 50 Hz. Tram duration was 100 pulses for the first five trains and 200 pulses thereafter. Intensity constant at 300 HA. Dot displays at bottom show absence of any excitatory response to either 1 pulse or 3 pulses at 100 Hz. The raw unit activity record represents the last of 12.5 sweeps. Sweep time for dot displays was 500 msec. appeared during the train and persisted beyond the offset of stimulation, sometimes for over 1 min. Figure 2 provides an example of the long-lasting effects that were typically seen. Most cases of LLI observed with stimulation in the LC and DL region were not associated with concurrent signs of excitatory afferent activity (see below). However, some cases were found and Fig. 3 shows an example with the same stimulation site as in Fig. 2. Note that in this case trains of 20, 40 and 100 Hz stimulation all produce an increase in activity that was followed by inhibition at the offset of the train. The excitatory response in this case was peculiar in that no clear-cut, fixed latency response could be detected (Fig. 3D). Stimulation in areas other than the LC nucleus also could produce LLI of Purkinje cells. Figure 4 illustrates a case with stimulation in the DL region. The variability in the response to 20 Hz stimulation shown here was common. Note that in the tower record of Fig. 4, a spontaneous slowing of activity is shown which resembles the slowing produced by the initial 20 Hz stimulation.

Figure 5 shows LLI produced by stimulation in the MED region. As with the DL stimulation in Fig. 4, no excitatory response was evoked. Cases of LLI with short latency excitatory responses (SLEs) are shown in Fig. 6. These excitatory responses had a variety of forms which are represented in Panels A-C. The LLI that followed the train in these cases could not be distinguished from that produced by stimulation that failed to produce SLEs. The relatively weak LLI produced by 50 Hz stimulation was the minima1 level warranting a classification as LLI. Purkinje cells showing LLI were routinely tested with stimulation trains of various frequencies and for responses to single pulses, In these areas the present results deviate from those of Hoffer et al. [ 121 In the first regard, contrary to Hoffer et al.‘s observations, LLI was typically of greater magnitude and duration at frequencies above 20 Hz. Figure 2 which shows the effects of LC stimulation provides a clear example. With the train duration held constant at 100 pulses, the higher frequencies consistently produced a stronger reduction in activity. Similar trends are

SINNAMON, SHAW, AMARAL AND WO~~WAR~

198

20 HZ

300uA

FIG. 6. Examples of LLI associated witk short latency excitatory responses. Panel A, celt 71L-3, stimulation site in the DL region dorsal to the LC nucbus at the junction of the pons and cerebeflum, an ICSS site, see middfe section of Fig. 1. Stimulation at IO Hz, 500 @A, 100 pulses produces a spike at 2.5 msec and LLI after the train. At the left, the spike discriminator output appears as short lines aligned vertically. Note the progressive increase in latency. Calibration for A, left, 400 WA and 3 msec. Panel B, cell 31-10, stimulation at 300 MA in cerebellar white matter produces a complex field respanse with spike discharges and LLI post train. Calibration for B, left, 1 mV and 2 msec for upper and 50 msec for lower trace. Panel C, cell 64-4, stimulation in a non-ICSS site just dorsolateral to the LC produces a spike with a variable latency (2 -3 msec). The response is masked by background activity at 300 @A but is revealed with 500 &A stimulation which suppresses all but the evoked activity. Note the weak LLI produced by 20 Hz, 500 rtA despite the clear short latency excitation. Calibration for lower records, 200 PV and 1 msec. All stimulation trains 300 pulses in duration.

in Figs. 3, 5 and 6. A second point of disagreement between Hoffer et ~1,‘s report [ 121 and the present results relates to our failure to observe consistent signs of a long latency (about 12.5 msec) inhibition to single pulses of stimulation. Figures 3-D and 5 provide examples. These discrepancies may relate to the relatively low intensities used in this present study which were generally under 300 PA, and up to 500 .uA in a few cases. Hoffer et at. f 121 used intensities up to 1S mA. The low intensities used here in order to allow a more precise localization of stimulation sites were also likely responsible for the relatively low occurrence of LLI observed (see below).

seen

An overview of the distribution of sites effective in producing LLI of cerebellar Purkinje cells can be gained from Table 1. First it may be seen that all regions studied, including the midbrain and cerebellum, contained sites at which long trains of stimulation caused a marked, slowing

or cessation of cerebellar activity. Stimulation in the DL region and in the cerebellar white matter were the most effective with 16 of 18 and 4 of 5 inhibitory sites, respectively. These two regions were also the most potent with regard to the percentage of Purkinje cells inhibited. However, as will be seen below, the two regions differed considerably in the degree to which the LLI was associated with short Iatency events. Stimulation of areas containing LC axans appeared to be the most potent in eliciting LLI. Compared to the other pontine areas shown in Fig. 1, including the LC nucleus itself, and the regions medial and ventromed~al to it, the DL region of the pans contained a significantly greater proportion of inhibitory sites (Fishers Exact Test, p = 0.01). Stimulation in the DL region also inhibited a greater percentage of ipsilateral Purkinje cells (26%) than did the other pontine sites combined, 1 l%, x2 = 9.86, p
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the degree to which SLEs were associated with LLI produced by stimulation in the various regions. Overall, of the 373 Purkinje cells tested, LLI was seen in 76 (20%). Of these cases of inhibition, 59 were associated with SLEs. However, the various regions differed in the proportion of cases of LLI associated with SLE. Notice in the bottom row of Table 2 that with stimulation sites in the cerebellum, midbrain, and the regions medial and ventrolateral to the LC nucleus, a high proportion of the inhibitions were associated with SLEs. By contrast, a stimulation in the LC nucleus and in the DL region produced LLI that was in most cases free of the SLEs. The difference between the cerebellum and the DL region was significant (p = 0.03). The cerebellar stimulation sites were interesting also in that no cases were found in which stimulation that produced a SLE did not also produce LLI. Note in the second row of Table 2 that with all other regions, the occurrence of SLEs did not imply the occurrence of LLI. These findings are consistent with the earlier observations of Hoffer et al. 1121 which dissociated the LLI produced by juxtafastigial stimulation from the LLI produced by stimulation of the LC and its axons.

Purkinje cells tested showed LLI with LC nuclear stimulation, a potency which was less than the 26% found with stimulation in the DL region, x2 = 3.00, ~~0.10. Sites medial to the LC nucleus were effective in producing inhibition in some cases. However, compared to the DL region, the medial sites also were less frequently effective (Fishers Exact Test, p = 0.08). As seen in Fig. I and Table 1, sites in the region ventrolateral to the nucleus were the least effective in producing LLI. Compared to the DL region, the VL region showed a lower proportion of effective sites (p - 0.02) and a lower percentage of Purkinje cells inhibited, x2 = 9.52, p
TABLE DISTRIB~~ON

1

AND EFFECTIVENESS OF SITES THAT PRODUCE LONG-LASTING CE~BELLAR PURKINJE CELLS

LC Nucleus Stimulation Sites Tested Inhibitory Sites* F’urkinje Cells Tested Purkinje Cells Inhibited (%)

Medial to Nucleus

Ventrolateral to Nucleus

Dorsolateral to Nucleus

INHIBITION OF

Midbrain

Cerebellum

6

9

6

18

8

5

4

5

2

16

5

4

39

51

51

130

67

35

13

16

6

26

18

40

*Stimulation produced long-lasting inhibition in at least one Purkinje cell. Sites declared negative for inhibition were tested with four or more ipsilateral Furkinje cells.

TABLE 2 REL .ATION BETWEEN LONG-LASTING INHIBI~ON (LLI) AND SHORT LATENCY EXCITATION (SLE) OF CEREBELLAR PURKINJE CELLS WITH VARIOUS STIMULATION SITES

LC Nucleus Total Purkinje Cells Tested Cells with SLE and No LLI Cells with SLE and LLI Cells with LLI and No SLE SLEILLI"

Medial to Nucleus

Ventrolateral to Nucleus

Dorsolateral to Nucleus

Midbrain

Cerebellum

39

51

51

130

67

3.5

2

5

2

8

3

0

2

6

3

15

8

11

2 .75

0 1.0

19 .44

4

3 .40

*Proportion of units showing LLI that also showed SLE.

.67

3 .78

200

SINNAMON,

TABLE

SHAW, AMARAL

AND WOODWARD

3

RELATION BETWEEN PRESENCE OR ABSENCE OF ICSS AND CAPAClTY TO PRODUCE LONG-LASTING INHIBITION WITH AND WITHOUT SHORT LATENCY EXCITATION (SLE) WITH VARIOUS PONTINE STIMULATION SITES

Sites Tested LC Nucleus Medial to Nucleus Ventrolateral to Nucleus Dorsolateral to Nucleus Total

2”( 1I) 4 (21) 3 131) 4 (46) 13 (109)

ICSS Present % Cells LLI & SLE LLI, No SLE 0 14 0 6 5

0 9 0 15 8

Sites Tested 4(28) 4(23) 1(12) 6(38) lS(101)

Note: Number in parenthesis is the total number of ipsilateral Purkinje cells tested. *Stimulation in one positive ICSS site in the LC nucleus inhibited a single contralateral

Distribution

of ICSS Sites

A total of 28 pontine stimulation sites met the requirement of both having behavioral tests for ICSS and electrophysiological tests for LLI on at least four ipsilateral Purkinje cells. The behavioral properties of most stimulation sites in the midbrain and cerebellum were not tested and these areas were not treated in this analysis. As may be seen in Fig. 1 and Table 3, the ICSS sites were widely distributed and there was no indication of a concentration of positive sites around the LC nucleus. As seen in Table 3, all regions showed both positive and negative sites with only the area ventrolateral to the nucleus showing any tendency toward a high proportion of positive sites (three of four cases positive). Only two of the six LC nuclear sites supported ICSS. Positive ICSS sites were also found medial to the LC nucleus (four of eight sites) and dorsolateral to the nucleus (4 of 10 sites).

Stimulation of sites near the LC nucleus or its axonal projections that supported ICSS was no more likely to produce LLI than similar stimulation that was negative for ICSS. Table 3 lists the positive and negative ICSS sites by region and with respect to their capacity to produce LLI. The two types of LLI, with and without SLEs, are treated separately. Note first that for all regions, stimulation in the 13 positive and 15 negative sites produced LLI of either type in a similar percentage of cells (combined LLI, 13% and 19%, respectively). Positive and negative sites did not differ significantly either in proportion of cells showing any type of LLI, (x2 = 1.82, p>O.lO) or in the relative proportion of cells showing LLI with and without SLE (x2 = 0.77, p>O.SO). Further evidence against the association between LLI and ICSS may be seen in a detailed examination for each region. Note that stimulation of the two sites in the LC nucleus that were positive for ICSS failed to inhibit any of the 11 ipsilateral Purkinje cells tested, although it should be noted that stimulation in one of these sites inhibited one contralateral Purkinje cell. Stimulation in the three positive ICSS sites of the ventrolateral area near the LC nucleus also failed to produce LLI. The single ventrolateral site that was negative for ICSS supported LLI only with SLE. In the other areas dorsolateral and medial to the IC, roughly similar percentages of cells were inhibited by stimulation in positive and negative ICSS sites. Note particularly for the dorso-

(LLI)

ICSS Absent % Cells LLI, No SLE LLI & SLE 7 13 16 13 11

10 0 0 15 8

Purkinge cell.

lateral area, positive and negative without LSE in an identical percentage

sites produced of cells (15%).

LLI

DISCUSSION

This study has provided information on two basic issues. First, it has delimited the stimulation sites that are effective for producing LLI in terms of location and physio~ogica1 characteristics. Second, it has shown that the capacity of a stimulation site to support LLI is apparently independent of its capability to support ICSS. Pontine stimulation sites capable of yielding a longlasting cessation of Purkinje cell activity follow an anatomical organization that is consistent with the trajectory of coeruleo-cerebellar projections. The region dorsolateral to the LC nucleus where LC axons begin their ascent to the cerebellar cortex was clearly more effective in eliciting LLI than other dorsal pontine regions. The superiority of the DL region was manifest in measures of both the proportion of inhibitory sites and by the percentage of cells inhibited. Compared to stimulation in the cerebellar white matter, another area where stimulation was effective in producing LLI, the inhibitory DL stimulation produced fewer signs of short latency excitatory afferent activity of Purkinje cells. Previously Hoffer et al. [ 121 distinguished the LLI attributable to activation of LC axons from the LLI produced by the juxtafastigical stimulation on the basis of experiments with catecholaminergic blocking drugs. It appears now that the absence of short latency excitation is a feature characteristic of stimulation in inhibitory sites within the LC system. Thus the present data extend the findings of Hoffer et al. and may provide an additional method by which the LLI produced by activation of coeruiear projections may be distinguished from the apparently similar LLI produced by other afferents to Purkinje cells. The anatomical pattern displayed by pontine sites most effective for producing LLI was not followed by the sites positive for ICSS. Positively reinforcing sites were found not only in the LC nucleus but also medial, dorsolateral and ventrolateral to it. The only region displaying any tendency for a concentration of positive sites was the VL, the area least effective for eliciting LLI. The sparse and diffuse distribution of ICSS sites found in the present study is consistent with several earlier reports [ 5, 6, 19, 27J. However, others 17, 24, 251 have reported not only a heavy concentration of ICSS around the nucleus hut also that

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INHIBITION

sites in the nucleus invariably SUpQOrt ICSS. With the methods used here, four of the six nuclear sites could not be shown to support ICSS. It is difficult to attribute these negative cases to insensitive testing methods since these same methods detected ICSS sites in nearby areas reported to be negative by others [7,25]. Further attesting to the sensitivity of the screening methods if the relatively high yield (13 of 28) of ICSS sites found in the dorsal pans which is at least comparable to other studies of the area. It remains puzzling that some laboratories find a high yield of ICSS sites in the LC nucleus and others do not. Although some of the negative reports can be attributed to a lack of explicit bar pressing training [ 11, the findings of the present study and those of others [S&J cannot be explained in that way. Also inadequate are explanations in terms of stimulation parameters and electrode configuration which were very similar in the present study and the studies of others [ 7, 24, 25). In general, stimulation of ICSS sites was no more effective in inhibiting Purkinje cells than stimulation in non-ICSS sites. Neither type of cerebellar inhibition, with or without associated SLEs, was related to the motivational properties of the site. Indeed several cases were found in which sites positive for ICSS were negative for either type cerebellar inhibition. Two of these cases involved sites in the LC nucleus and three involved sites just ventrolateral to the nucleus. The converse condition was also found and, in fact, was most prevalent - most sites negative for ICSS were positive for inhibition. The overall pattern of these results suggests that the phenomena of ICSS and cerebellar inhibition produced by stimulation in the area of the LC are independent and disassociable phenomena. If, on the other hand, the two phenomena were based on the same substrate, it would be difficult to explain why the ICSS sites failed to conform to the anatomical pattern displayed by the inhibitory sites. It would be similarly inexplicable why ICSS sites thoroughout the area of the LC showed no tendency to be more effective than non-ICSS sites in yielding inhibition. The interpretation of independence may be countered by the possibility, albeit unlikely, that the inhibitory non-ICSS sites would have proved to be ICSS sites with continued testing. Although this possibility can never be dismissed, it does not address the difficulty caused by the several demonstrated ICSS sites that failed to yield LLI. The stimulation parameters used in these cases were certainly effective by behaviora indices and the electrophysiological methods were sufficiently sensitive as indicated by the anatomical pattern uncovered. Since these sites were rested with no fewer cells than other sites, it seems implausible to argue that these sites would have shown substantially more inhibition with extended testing. These results are consistent with the interpretation that ICSS at sites in and near the LC nucleus is not dependent on the coerulear cells that send axons to the cerebellum and forebrain.

The necessary

involvement

of these

LC fibers

in

ICSS has also been questioned by other findings. Clavier and Routtenberg [4] showed that ICSS in sites ventrolateral to the midbrain central gray, which has been attributed to activation of LC axons [7, 11, 241, is not blocked by bilateral lesions of the LC nucleus. Conversely, ICSS in sites in and near the LC nucleus is not blocked by destruction in the midbrain of the LC axons that project to the forebrain [ 8 f . Lesions of the LC nucleus have mixed effects on ICSS in the diencephalon [9,14] and have provided no decisive evidence for the involvement of the LC in ICSS. Another difficulty for the putative role of the LC in ICSS is the long refractory period of the axons, ranging from 4 to 20 msec, which would seem to preclude their important participation in ICSS at sites with behaviorally determined refractory periods of 1.O msec or less I81 * These findings and the present results appear to be at variance with those of Segal and Bloom [24] who reported that stimulation sites that were effective for ICSS in the dorsal pons tended to be closer to the LC nucleus and to be more effective in producing inhibition of hippocampal neuronal activity compared to sites further from the nucleus. They also found that ICSS and hippocampai inhibition showed parallel responses to several catecholaminergic drugs. ICSS at sites near the LC nucleus may depend on the activation of noradrenergic fibers that do not take their origin from the dorsal LC. One possible candidate is the projection from the ventral portion of the LC which has axons of larger caliber and project more ventrally to the diencephalon [ 16, 17, 18, 21, 22, 291. Conceivably, these fibers could be activated by stimulation near the dorsal LC at intensities which would not be effective for activation of the smaller fibers projecting to the cerebellum. Another possibility is the noradrenergic projection from the area around the nucleus of the solitary tract which has been shown to be positive for ICSS [3]. These fibers ascend sufficiently close to the LC nucleus to be a serious candidate. Both of these possibilities are consistent with the present finding that electrode sites ventrolateral to the LC were quite effective in yielding ICSS. The involvement of either of these paths could explain the continued effectiveness of amphetamine in facilitating ICSS in the region of the LC nucleus after destruction of the forebrain projections of the LC at a midbrain level [ 51. Whatever the basis for ICSS, noradrenergic or otherwise, in the region of the LC nucleus, there seems to be ample reason to question the role of at least the dorsal LC and to consider other feasible alternatives. ACKNOWLEDGEMENTS This work was supported by NIH Postdoctoral Fellowship (IF02 NS 52692-01) and Wesleyan Faculty Research Grants to Harry’ M. Sinnamon, and by Grants NIMH lR03 MH-21406 and NSF (X-43301 to Donald .I. Woodward. We thank J. g. Schwartzbaum for use of equipment.

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