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bracketing the injection test day. Required frequency was calculated for each current in both the second and third
passes through the current set on the day before and the day after the injection day. These data were averaged for
each current and pass, yielding six values: mean non- injection required frequency at 200, 400 and 800 mA in the
second pass, and the same means for the third pass. These means were subtracted from the corresponding required
frequency values obtained on the injection test day, yielding another six data points: shift in required frequency
at each of the three currents, for both passes. Pilot testing and others’ work [2,45] indicate a roughly 20 min time
course of effectiveness for the lidocaine injection parame- ters used here. Because each pass through a current set
took 15–20 min, data from the second pass assessed the immediate effects of the lidocaine injection. Data from the
third pass showed whether the rat returned to non-injection day levels of required frequency when lidocaine’s effects
dissipated.
3. Results
Of the 25 rats tested, 11 had both injection sites in or within 0.25 mm of the border of the retrorubral fields. Six
rats had only ipsilateral sites on target and one rat had only the contralateral site on target. For these rats we analyzed
only the data from injection conditions in which the injection site was on target. For example, data from the rat
whose contralateral site alone was on target were included only in the analysis of the effects of contralateral — not
ipsi- or bilateral — lidocaine infusions. In the remaining seven rats neither injection site was in or within 0.25 mm
of the border of the retrorubral fields; these rats comprised a control group against which the results of the other 18
rats were compared.
Figs. 1 and 2 show the stimulation sites and injection sites, respectively, for all 25 rats. Injection sites for the
seven control rats are indicated by putting the rat’s identification number in italic print. The shaded areas in
the second plate in Fig. 2 show the relative location of the
Fig. 1. Stimulation electrode tip locations for all 25 rats in the study. The number in the lower left hand corner of each plate gives the distance of
retrorubral fields, which begin approximately 0.25 mm
the plate from bregma, in mm. All plates in this figure and Fig. 2 are
behind that plate. Therefore, injection sites in that coronal
adapted from the Paxinos and Watson [36] atlas of the rat brain, used with
plane that fall within or adjacent to the shaded area were
permission. Abbreviations: 3V, third ventricle; cp, cerebral peduncle; f,
considered to be retrorubral injection sites. Although it
fornix; fr, fasciculus retroflexus; mfb, medial forebrain bundle; ml, medial
appears that rat A19 should be included in the retrorubral
lemniscus; mp, mammillary peduncle; opt, optic tract; scp, superior cerebellar peduncle; VTA, ventral tegmental area.
group as a ‘contralateral only’ rat, the asterisk indicating this rat’s injection site is a rough estimate of where
injections may have gone. Upon postmortem examination this rat was found to have a large region of heavy gliosis
asterisks indicating these injection sites are also rough surrounding the injection track and the end of the guide
estimates. It is possible that one or several of the injection cannula. The tissue was more suggestive of a large lesion
conditions may have introduced infection into these rats’ than a well-localized injection site. Because of the am-
brains, producing the extensive tissue damage, although biguity of this rat’s injection site, it was included in the
their behavioral data were remarkably stable throughout control group. Similarly, large regions of heavy gliosis
the experiment. surrounded the injection areas in rats A24 and 25; the
The immediate effects of lidocaine and saline infusions
158 M
Fig. 2. Asterisks indicate estimated injection sites for all 25 rats. Injection sites for the 7 rats in the control group are indicated by placing the rat’s
identification number in italic print. The number in the lower left hand corner of each plate indicates the distance from bregma, in mm. The
shaded area in the second plate indicates the relative location of the retrorubral fields which begin approximately 0.25 mm behind this plane.
Abbreviations: A8, A8 dopaminergic cell bodies; CLi, caudal linear nucleus; cp, cerebral peduncle; DpMe, deep mesencephalic nucleus; dtgx,
Fig. 3. Mean shifts in required frequency at each of three currents, dorsal tegmental decussation; ml, medial lemniscus; mlf, medial longi-
relative to bracketing non-injection days, immediately following lidocaine tudinal fasciculus; mtg, mammillotegmental tract; PaR, pararubral nu-
and saline injections in 18 rats with injection sites in the retrorubral fields. cleus; RPC, red nucleus, parvicellular; RPM, red nucleus, magnocellular;
Error bars51 S.E.M. Asterisks indicate lidocaine-induced shifts that were RRF, retrorubral field; rs, rubrospinal tracts; scp, superior cerebellar
significantly larger than the shifts induced by comparable volumes of peduncle; SNCD, substantia nigra, pars compacta; SNR, substantia nigra,
saline see text for discussion of the criterion for significant. The number pars reticulata; VTA, ventral tegmental area; vtgx, ventral tegmental
of observations entering into each mean are indicated within or immedi- decussation; xscp, decussation of the superior cerebellar peduncle.
ately above below each bar; n518 for all saline conditions. All saline injections were bilateral.
in the 18 rats in the retrorubral injection group are shown in Fig. 3. These graphs show the mean shift in required
neither repeated measures analysis of variance nor multiple frequency relative to bracketing non-injection days induced
dependent t-tests nor their non-parametric alternatives by each infusion condition in the second pass through the
could be used validly because of many instances of ‘drop current set, i.e. in the first 15–20 min immediately
out’ following lidocaine infusions. Especially following following the injection. Ideally, one would test the signifi-
bilateral lidocaine injections, several rats did not yield cance of the difference between mean shifts following
valid rate–frequency curves during the second pass. The lidocaine infusions and mean shifts following comparable
number of observations going into each mean in this figure saline infusions using inferential statistical tests. However,
is shown within each bar; n518 for all saline injection
M . Waraczynski, M. Perkins Brain Research 885 2000 154 –165
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conditions. The relatively small number of observations in frequently as large as 0.2–0.4 log units at a particular
many test conditions accounts for the relatively large current.
S.E.M. values around those conditions’ means. In both averaged and individual data, 1.0-ml infusions
As an alternative to traditional significance testing, we did not generally produce notably greater effects than
evaluated the significance of the difference between ob- 0.5-ml infusions, when valid curves were collected. The
served shifts produced by lidocaine and shifts produced rats were, however, more likely to fail to produce valid
by saline by constructing a modified confidence interval curves following 1.0-ml infusions than they were following
around the mean saline-induced shift. To do so, we added comparable 0.5-ml infusions. Note that the number of
2.53 saline S.E.M. to the relevant mean saline-induced observations entering into each mean lidocaine-induced
shift. Most standard tests of significance require differ- required frequency shift is frequently lower in 1.0-ml
ences between means of 2.0–2.5 error units for those infusion conditions versus comparable 0.5-ml conditions.
differences to be significant at the P50.05 level. Using our In this group of 18 rats, six were tested at the LH
2.53 S.E.M. criterion is equivalent to constructing a 98 stimulation site and 12 at the VTA site. There was some
confidence interval, which is equivalent to using a con- indication that the effects of 0.5-ml infusions were some-
servative P50.02 significance level. Any mean lidocaine- what stronger in the rats stimulating at the LH site, but this
induced shift that exceeded that value was considered pattern was not consistent and was not observed following
significantly larger than the mean shift induced by the 1.0-ml infusions. The differences in average required
comparable saline injection. These significant differences frequencies between the two groups were not statistically
are indicated by asterisks over the appropriate bars in Fig. significant using the confidence interval analysis explained
3. above for any laterality current condition except the
In general, lidocaine-induced shifts were more substan- contralateral 0.5-ml infusion at 800 mA.
tial at 400 and 800 mA than at 200 mA. At all three Fig. 5 presents third pass data from the 18 rats with
currents, bilateral 0.5 and 1.0 ml lidocaine injections retrorubral injection sites. By the third pass, lidocaine
produced the largest shifts in required frequency. These infusions did not produce any average shifts that were
shifts were significantly larger than the shifts produced by significantly different from shifts following saline infusion.
comparable volumes of saline in all but one case. Shifts With only a few exceptions, third pass required frequency
following ipsilateral lidocaine infusions were generally on injection test days did not, on average, vary from
smaller than shifts following bilateral infusion, and were required frequency on non-injection test days by more than
significantly greater than bilateral saline-induced shifts in 0.05 log units. Also, in these conditions virtually all rats
four out of six conditions. Contralateral infusions produced produced valid rate–frequency curves; at most, only 1 or 2
the smallest shifts, which were not significantly different rats failed to do so in any given condition. That is, the
from shifts induced by comparable volume bilateral saline majority of rats that did not produce valid rate–frequency
injections. However, in most test conditions the magnitude curves in the second pass were producing valid curves by
of the average shift following bilateral lidocaine injection the third pass, which started 15–20 min after lidocaine
is roughly equivalent to the sum of the average shifts infusion.
produced by ipsilateral and contralateral infusions under In contrast to the effectiveness of lidocaine inactivation
the same conditions. This suggests that the effectiveness of shown in Figs. 3 and 4, Fig. 6 presents second pass shift
bilateral infusions relied at least partly on the contribution data from the seven rats whose injection sites were not on
made by inactivation of the contralateral RRF. target. In only two of the eighteen lidocaine test conditions
Although averaged data present a useful picture, averag- were lidocaine-induced shifts significantly greater than
ing across rats obscures some particularly large shifts shifts induced by comparable volumes of saline 1.0 ml
produced in individual rats. The lidocaine’s effects mani- bilateral lidocaine at 200 mA and 0.5 ml ipsilateral
fested differently at the three currents for different rats. For lidocaine at 400 mA. In eight conditions lidocaine pro-
example, where one rat might not yield a valid curve at duced required frequency values that were on average
200 mA then show a large shift at 400 mA and a moderate lower, though not significantly so, than required frequen-
shift at 800 mA, another rat might show a small shift at cies obtained in the bracketing non-injection days.
200 mA, a larger shift at 400 mA, and not yield a valid Again, averaging obscured some potentially interesting
curve at 800 mA. Thus, the means presented in Fig. 3 show individual results. For example, in all but one condition rat
only a general summary of the effects of lidocaine A8 did not produce valid second pass rate frequency
infusions. Fig. 4 illustrates this intersubject variability. curves at any current following 0.5- and 1.0-ml ipsilateral
These graphs present required frequency shifts relative to lidocaine infusion and consistently showed marked rapid
surrounding non-injection days for two rats whose in- rotation following these infusions. This rat’s ipsilateral
jection sites were all on target. Missing bars indicate test injection site was in the substantia nigra, pars reticulata
conditions that did not yield valid rate–frequency curves. plate 1, Fig. 2. Although 0.5-ml lidocaine infusion
Although average shifts Fig. 1 were generally on the contralateral to the stimulated hemisphere did not produce
order of 0.10–0.15 log units, shifts in individual rats were substantial upward shifts in required frequency in fact, at
160 M
Fig. 4. Second pass shifts in required frequency, relative to bracketing non-injection days, from two individual rats. Conditions for which bars are missing are conditions in which the rat did not produce a valid rate–frequency curve.
800 mA, required frequency shifted downward by over shifted upward by 0.30 log units at 200 mA and downward
0.20 log units, 1.0 ml produced shifts of 0.20 log units at by approximately 0.15 log units at the other two currents.
400 mA and 0.40 log units at 200 mA. No valid curve was Of the three rats in the control group that had ambiguous
collected in the second pass at 800 mA. This rat’s injection sites with extensive gliosis, one, rat A19, often
contralateral injection site was approximately 5.2 mm but not consistently showed upward shifts in required
caudal to bregma, at the ventral edge of the medial frequency. These were more commonly observed follow-
lemniscus and midway between the VTA and substantia ing 1.0 ml bilateral and ipsilateral infusions, particularly at
nigra pars compacta not shown in Fig. 2. Bilateral 200 and 400 mA. On the other hand, rats A24 and 25 had
infusions produced mixed results: following the 0.5-ml lesion-like injection tracks situated dorsal to the RRF
infusion, valid curves could not be collected at 200 or 800 targets. These rats often but not consistently showed
m A, but there was no substantial change in required
substantial downward shifts in required frequency as great frequency relative to non-injection days at 400 mA.
as 0.30 log units. Downward shifts were most often Following the 1.0-ml infusion valid curves could be
observed following either unilateral infusion, but not collected at all three currents, but required frequency
following bilateral infusions.
M . Waraczynski, M. Perkins Brain Research 885 2000 154 –165
161
Fig. 5. Mean third pass shifts in required frequency for all 18 rats Fig. 6. Mean second pass shifts in required frequency for seven rats
receiving retrorubral lidocaine and saline injections; n518 for all saline whose injection sites were outside of the retrorubral fields; n57 for all
conditions. saline conditions.
4. Discussion play a role in the rewarding effect of MFB stimulation,
