M . Waraczynski, M. Perkins Brain Research 885 2000 154 –165
155
focused on the relationship between MFB self-stimulation cannulae were blocked with stylets made from 30 gauge
and the A9 and especially A10 cells of the substantia nigra tubing. The rats were housed individually with food and
and ventral tegmental area, respectively. Relatively little water continuously available in a day night reversed
attention has been paid to the A8 cells of the retrorubral colony. All testing was conducted during the dark phase of
fields, possible because these cells only comprise about the cycle.
10 of mammalian midbrain dopamine cells [33]. These cells have efferent control over both the A9 and A10
2.2. Procedures groups [6] and can be considered contiguous with the A9
and A10 groups [17]. A8 efferent projections often parallel 2.2.1. Rate–frequency testing
those of the A9 [19,31,46,51] and to some degree the A10 Rate–frequency testing was performed with the rat
e.g. [22] groups. A8 afference from the amygdala also placed in an operant chamber with a lever protruding from
parallels amygdaloid afference to the lateral A9 group one wall. The rat was connected to a Stimtek ST1200
[23,47]. Thus, the links between midbrain dopamine and stimulation generator San Diego Instruments via a flex-
self-stimulation also implicate the RRF as a target of ible cable and commutator Plastics One. All experimental
interest. events were controlled by Stimtek ST1000 CPU and
We have previously suggested that temporary inactiva- ST1100 I O boards in communication with a master PC.
tion of targets thought to be relevant to MFB stimulation After 3–5 days postsurgical recovery, the rats were
reward may be more effective, and more consistently trained to press a lever for a 0.5 s train of 0.1 ms cathodal
effective, than permanent lesions [1,48,49]. In part, this pulses delivered by a constant current generator. The rats
may be true because postlesion hyperexcitability in cells were trained using stimulation of either the LH or VTA
surrounding a lesion, and or synaptic plasticity in synapses site, whichever supported the more robust responding.
surviving the lesion, compensate for the loss of lesioned Once the lever press response was reliably established the
cells. Neither would be a factor if, instead of lesioning, rats would press without coaching for stimulation delivered
target tissue were rendered temporarily inactive via the on a VI 3-second schedule, the rat was tested at several
injection of an anesthetizing agent. In this report, we use pulse frequencies at 200, 400 and 800 mA at both sites.
lidocaine to temporarily inactivate cells in the RRF, both The site that yielded the lower and most consistent values
ipsi- and contralateral to MFB self-stimulation sites. of the frequency required to maintain half maximal per-
Changes in stimulation reward value are measured using formance at all three currents was chosen for all further
the rate–frequency curve shift method. If the RRF are testing.
important to the stimulation’s reward effect, then their Each point in a single rate–frequency curve was de-
inactivation should temporarily render MFB stimulation termined as follows: for 30 s, the rat was allowed to press
less rewarding. for a 0.5-s train of pulses of a given frequency, delivered
on a VI 3 s schedule. At the start of each 30-s trial, the rat received three non-contingent trains of the stimulation that
2. Methods would be available during that trial. Data from the first 10
s of the trial were discarded to allow response rate to 2.1. Subjects and surgery
adjust to the presented frequency, and response rate over the last 20 s was recorded.
Twenty-five male Long–Evans rats, 330–450 g at time At each current, an entire rate–frequency sweep con-
of surgery, received MFB stimulation electrodes and sisted of several usually 7–9 trials separated by 1 s.
bilateral injection guide cannulae aimed at the retrorubral Between trials, frequency values were incremented in 0.1
fields. Coordinates for the LH electrodes were 2.8 mm log
unit steps. The frequencies presented were selected
10
caudal to bregma, 1.7 mm from the midline, and 7.7 mm so as to yield a rate–frequency curve that began at low
ventral to the dura. Coordinates for the VTA electrodes frequency values with a response rate under ten presses per
were 4.5 mm caudal to bregma, 0.9 mm from the midline, minute and rose to an upper asymptote, defined as at least
and 7.8 mm ventral to the dura. Coordinates for the guide two adjacent points at maximal rate. Maximal rates were
cannulae were 6.6 mm caudal to bregma, 1.5 mm from the defined as high rates that did not vary from each other by
midline, and 5.4 mm ventral to dura for half of the rats, more than 10. To maximize the chances of collecting
and 7.0 mm caudal to bregma, 1.5 mm from the midline, valid rate–frequency curves should the lidocaine infusions
and 5.0 mm ventral to the dura for the other half. All degrade
the stimulation’s
reward value,
the upper
electrodes were made from 0.25 mm diameter stainless asymptote of these curves was ‘overdetermined’. That is,
steel wire coated with insulation to within 0.25 mm of the the rat was tested at 3–5 frequencies beyond the point at
tip. A machine screw in the skull served as the stimulation which the curve reached asymptote in baseline conditions,
anode. The cannulae were made from 23-gauge stainless so that should injection day curves shift upward right-
steel tubing 11 mm long and were implanted such that the ward along the frequency axis, the rat should still receive
tip was placed 1 mm above the injection target. The frequencies that would support some responding.
156 M
For each rate–frequency curve, the frequency required positioning the tip at the desired target. Because the 11 mm
to maintain half-maximal responding, called the ‘required cannulae were implanted such that the tips were 1 mm
frequency’, was calculated by fitting a broken-line function dorsal to the injection target, the injectors had a collar
to the curve. The broken line function fits three line placed 12 mm above their tips. Thus, the injector tip
segments to the curve: two horizontal segments for the protruded exactly 1 mm below the end of the cannula and
lower and upper asymptotes, respectively, and a third was prevented by the collar from sinking further ventral.
segment connecting those two. The required frequency was Injectors were connected via flexible polyethylene tub-
computed by interpolating the frequency corresponding to ing to the needle of a 10-ml Hamilton microsyringe which
the midpoint of the connecting segment. Required fre- was mounted in a Harvard Apparatus dual programmable
quency was averaged across curves at each current, syringe pump. Bilateral infusions were made simultaneous-
yielding a daily average required frequency for each ly, using two syringes. Lidocaine or saline was infused at
current tested. a rate of 0.5 ml min. Once the infusion was complete the
Stable baseline was defined as 5 consecutive test days injector remained in place for 1 min to prevent diffusion
with no apparent trend in required frequency at any back up the guide cannula. The injector was then removed
current. Once this was reached, required frequency was no and replaced with the stylet, and behavioral testing re-
longer averaged across currents each day. Instead, a daily sumed as soon as the rat was replaced in the operant
session was divided into three separate passes through the chamber, within 1 min after the stylets were replaced.
rat’s set of currents. The first pass was treated as a warm-up condition and its data were discarded from
2.2.3. Histology further analyses. On injection days, testing was paused
At the end of postlesion testing the rats were euthanized after the first pass and the rat was removed for injection.
using an overdose of sodium pentobarbital. They were Testing resumed immediately after the injection with a
perfused transcardially with saline followed by 10 formal second and third pass through the current sets. The order of
saline and the brains were harvested. The brains were fixed current presentation within a pass was randomized for each
in 10 formal saline for at least 3 days followed by 1 day rat, but that order remained constant across passes.
in 20 sucrose formalin. The brains were then quick Lidocaine and saline test days were always bracketed by
frozen and cut in the coronal plane on a microtome non-injection test days. The effects of a given injection
mounted in a cryostat. Alternate sections through the condition were assessed relative to the two injection days
stimulation and lesion sites were saved for staining with that bracketed that condition. The order of test conditions
formal thionin to visualize cell bodies and hematoxylin to was randomized for each rat.
visualize fiber tracts. The tissue was examined via a tissue projector and the location of electrode tips and injection
2.2.2. Lidocaine injection sites were plotted on coronal plates from the Paxinos and
The lidocaine testing phase of the experiment began Watson [36] atlas of the rat brain. Electrode tip sites were
with an initial 0.5 ml bilateral saline injection to condition taken to be at the bottom of the electrode track left in the
the target tissue. In preliminary work we found that the tissue after the implant was removed. The injector track
initial bolus of any injectate often affected required typically appeared as a narrow, dark length of gliosis
frequency, regardless of whether that injectate was saline extending approximately 1 mm below the end of the
or lidocaine. Additional 0.5 and 1.0 ml bilateral saline cannula track. The infusion site was inferred to be at the
injections were included in the randomly determined end of the injector track. Sites within 0.25 mm of the
sequence of test conditions and served as control con- border of the retrorubral fields were considered on target
ditions against which lidocaine’s effects were compared. In based on estimates of lidocaine’s spread of effectiveness as
addition to saline, all rats received 0.5 and 1.0 ml of a function of infusion volume obtained by Tehovnik and
lidocaine injected ipsi- and contralateral to the stimulation Summer [45]: 0.5 mm radius for 0.5 ml and 0.65 mm for
site and bilaterally, yielding a total of eight test injection 1.0 ml. These estimates were obtained using different
conditions. Contralateral-only and ipsilateral-only saline injection parameters 2 lidocaine injected into cortex,
injections were not tested in order to minimize the number but are consistent with other estimates of the spread of
of times the injector tip was introduced into the tissue. effectiveness of 0.5 and 1.0 ml injections of 2 and 4
Thus, the effects of contra- and ipsilateral lidocaine lidocaine
infused into
subcortical structures
infusions were compared with the effects of bilateral [2,14,30,37,39]. Thus, we use a conservative estimate in
injections of saline of comparable volume. assuming that infusions made within 0.25 mm of the
During the infusion the rat was loosely restrained in a retrorubral field border affected the retrorubral field.
small towel and held in the experimenter’s hand. The stylets were removed from the guide cannulae and replaced
2.2.4. Data analysis with injectors constructed of 30-gauge stainless steel
Shifts in required frequency following lidocaine or tubing. The injectors had a collar of 23-gauge tubing at a
saline infusions were calculated relative to required fre- set distance from the tip that acted as a means for precisely
quency values obtained on the two non-injection days
M . Waraczynski, M. Perkins Brain Research 885 2000 154 –165
157
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
