Pontine stimulation overcomes developmental limitations in the neural mechanisms of eyeblink conditioning.

Abstract

Pontine neuronal activation during auditory stimuli increases ontogenetically between postnatal days (P) P17 and P24 in rats. Pontine neurons are an essential component of the conditioned stimulus (CS) pathway for eyeblink conditioning, providing mossy fiber input to the cerebellum. Here we examined whether the developmental limitation in pontine responsiveness to a CS in P17 rats could be overcome by direct stimulation of the CS pathway. Eyeblink conditioning was established in infant rats on P17-P18 and P24-P25 using pontine stimulation as a CS. There were no significant age-related differences in the rate or level of conditioning. Eyeblink conditioned responses established with the stimulation CS were abolished by inactivation of the ipsilateral cerebellar nuclei and overlying cortex in both age groups. The findings suggest that developmental changes in the CS pathway play an important role in the ontogeny of eyeblink conditioning.

Full text

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Research
Pontine stimulation overcomes developmental
limitations in the neural mechanisms
of eyeblink conditioning
John H. Freeman Jr.,1Christine A. Rabinak, and Matthew M. Campolattaro
Department of Psychology, University of Iowa, Iowa City, Iowa 52242, USA
Pontine neuronal activation during auditory stimuli increases ontogenetically between postnatal days (P) P17 and P24
in rats. Pontine neurons are an essential component of the conditioned stimulus (CS) pathway for eyeblink
conditioning, providing mossy fiber input to the cerebellum. Here we examined whether the developmental
limitation in pontine responsiveness to a CS in P17 rats could be overcome by direct stimulation of the CS pathway.
Eyeblink conditioning was established in infant rats on P17–P18 and P24–P25 using pontine stimulation as a CS. There
were no significant age-related differences in the rate or level of conditioning. Eyeblink conditioned responses
established with the stimulation CS were abolished by inactivation of the ipsilateral cerebellar nuclei and overlying
cortex in both age groups. The findings suggest that developmental changes in the CS pathway play an important
role in the ontogeny of eyeblink conditioning.
Eyeblink classical conditioning has been used as a model system
for examining developmental changes in the neural mechanisms
underlying motor learning (Freeman Jr. and Nicholson 2004). It
is well established that eyeblink conditioning in adult mammals
depends on cerebellar interactions with its afferent and efferent
brainstem nuclei (Mauk and Donegan 1997; Christian and
Thompson 2003). The cerebellum receives input stimulation
from an auditory conditioned stimulus (CS) through the pontine
mossy fiber projection (Steinmetz et al. 1986, 1987, 1989; Stein-
metz 1990; Steinmetz and Sengelaub 1992; Tracy et al. 1998).
Input stimulation from an air puff or face shock unconditioned
stimulus (US) reaches the cerebellum through the climbing fiber
projection from the inferior olive (McCormick et al. 1985; Mauk
et al. 1986; Steinmetz et al. 1989). The convergence of mossy and
climbing fiber activation of cerebellar neurons is thought to re-
sult in changes in synaptic efficacy that constitute the substrate
of learning and drive the production of the eyeblink conditioned
response (CR). Cerebellar output produces inhibitory feedback to
the inferior olive and excitatory feedback to the pons (Sears and
Steinmetz 1991; Clark et al. 1997; Kim et al. 1998; Bao et al. 2000;
Medina et al. 2002). The feedback connections are thought to be
important for acquisition and maintenance of CRs (Medina et al.
2002).
Eyeblink conditioning emerges ontogenetically between
postnatal days (P) P17 and P24 in rats (Stanton et al. 1992). Neu-
rophysiological, neuropharmacological, and neuroanatomical
analyses of the ontogeny of eyeblink conditioning in rats indi-
cate that the developmental emergence of eyeblink conditioning
is due to developmental changes in the CS and US pathways
(Freeman Jr. and Nicholson 2004). The most substantial devel-
opmental change in the US pathway is an increase in the mag-
nitude of cerebellar inhibitory feedback to the inferior olive
(Nicholson and Freeman Jr. 2003a,b). The developmental change
in inhibitory feedback is due to an increase in inhibitory syn-
apses within the inferior olive (Nicholson and Freeman Jr.
2003a). Developmental addition of inhibitory synapses within
the inferior olive results in a decrease in complex spike activity in
the cerebellar cortex, which influences the maintenance of syn-
aptic plasticity within the cerebellum. Younger rats with less oli-
vary inhibition have higher baseline rates of climbing fiber ac-
tivity and as a consequence, they are less capable than adults of
maintaining learning-specific plasticity in the cerebellum be-
tween training trials and sessions.
Initial analysis of CS pathway development indicated that
there is an ontogenetic change that results in weaker input to the
cerebellum (Nicholson and Freeman Jr. 2004). A subset of neu-
rons in the pontine nuclei exhibits developmental changes in the
latency and magnitude of activation following the onset of a
tone CS (Freeman Jr. and Muckler 2003). Younger rats have a
weaker pontine response to acoustic stimuli, resulting in less ac-
tivation of cerebellar neurons during the CS. The developmental
change in pontine responsiveness is thought to influence eye-
blink conditioning by limiting activity-dependent synaptic plas-
ticity in the cerebellum.
Here we examined whether the developmental limitation in
pontine responsiveness in P17 rats could be overcome by stimu-
lation of the CS pathway. In the first experiment, eyeblink con-
ditioning was established in freely moving infant rats on P17–
P18 or P24–P25 using electrical stimulation of the basilar pontine
nuclei as a CS. Rat pups were given electrical stimulation through
a bipolar electrode implanted in the pontine nuclei paired with a
peripheral unconditioned stimulus (US) or unpaired stimulation.
The second experiment examined whether learning established
with the stimulation CS was cerebellum-dependent. Pups were
given paired conditioning using pontine stimulation as the CS
on P17–P18 or P24–P25 followed by an infusion of muscimol
into the interpositus nucleus, which was expected to inactivate
the cerebellar nuclei and overlying cortex (Freeman Jr. et al. 2005).
Results
Associative learning
Pontine stimulation induced robust conditioning in both paired
groups. Rat pups receiving paired presentations of pontine stimu-
lation and the US showed a significantly higher level of condi-
tioning compared to pups receiving unpaired training during
1Corresponding author.
E-mail john-freeman@uiowa.edu; fax (319) 335-0191.
Article and publication are at http://www.learnmem.org/cgi/doi/10.1101/
lm.91105.
12:255–259 ©2005 by Cold Spring Harbor Laboratory Press ISSN 1072-0502/05; www.learnmem.org
Learning & Memory
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conditioning sessions 2–6 (Fig. 1). Stimulation did not elicit eye-
blink responses in pups that received unpaired training in either
age group (Fig. 1). A repeated measures ANOVA revealed a sig-
nificant main effect of the condition factor (F(1,31)= 135.11,
P < 0.001), which was due to a higher percentage of CRs in the
paired groups relative to the unpaired groups. The difference be-
tween paired and unpaired group performance indicates that the
CRs produced by paired training were established through asso-
ciative learning and were not due to pseudoconditioning or sen-
sitization. No statistically significant differences were found be-
tween age groups. The current intensity used in the age groups
did not differ significantly (P17–P18 paired mean = 73.3 µA,
range = 50–100 µA; P17–P18 unpaired mean = 70.8 µA,
range = 50–85 µA; P24–P25 paired mean = 67.5 µA, range = 25–
100 µA; P24–P25 unpaired mean = 71.7 µA, range = 50–100 µA),
indicating that the levels of conditioning seen in P17–P18 and
P24–P25 rats were comparable because the stimulation bypassed a
developmental limitation in the eyeblink conditioning circuitry
and were not due to the use of more intense stimulation in the
P17–P18 group. The mean current intensity used in the pups with
ineffective electrode placements was 70.7 µA (range 20–100 µA).
Cerebellar inactivation
A second experiment examined whether the associative learning
established using pontine stimulation as a CS depends on the
cerebellum. Muscimol was infused into the cerebellar interposi-
tus nucleus to inactivate the cerebellar nuclei and overlying cor-
tex (Freeman Jr. and Rabinak 2005; Freeman Jr. et al. 2005). The
infusion was not expected to selectively inactivate the interposi-
tus nucleus (Freeman Jr. et al. 2005). Rat pups were given paired
presentations of the pontine stimulation CS and the US for five
sessions, followed by a test session in which muscimol (5 nmol in
0.5 µL) was infused prior to an additional paired training session.
The infusion session was followed by a test session with addi-
tional paired training to assess reversibility of the inactivation
effect. As in the first experiment, pups in both age groups ac-
quired eyeblink CRs with paired training (Fig. 2). Both age groups
also showed loss of CRs following muscimol inactivation of the
cerebellar hemisphere and complete recovery of CRs during the
last session (Fig. 2). Figure 3 displays the development of the
eyelid CR across training and the effects of muscimol infusion on
the CR in a rat trained on P17–P18. An ANOVA revealed a sig-
nificant effect of the sessions factor (F(6,54)= 32.44, P < 0.001) but
no effects related to age. The session ef-
fect was due to an increase in CR per-
centage from session 1 to sessions 3–5, a
decrease in CR percentage from session 5
to the inactivation session, and a subse-
quent increase in CR percentage from
the inactivation session to the recovery
session (all comparisons, P < 0.05).
Electrode and cannula placements
Histological analysis revealed that
stimulation sites for rats exhibiting suc-
cessful conditioning were in or just dor-
sal (<0.3 mm) to the lateral parts of the
basilar pontine nuclei (Fig. 4). Rats that
failed to learn had electrode placements
in the most medial pontine nucleus or
dorsal to the pontine nuclei (Fig. 5). Sev-
eral of the rats with ineffective elec-
trodes that were placed near the cerebral
peduncle exhibited very strong startle-
like responses to stimulation but did not
exhibit eyeblink conditioning. All the pups given muscimol in-
activation had cannula tips placed in or just dorsal to the anterior
interpositus nucleus (Fig. 6).
Discussion
Stimulation of the pontine nuclei as a CS was effective for estab-
lishing eyeblink conditioning in rat pups on P17–P18 and P24–
P25. The conditioning produced in the groups given paired train-
ing was the result of associative learning and not due to pseudo-
conditioning or sensitization, as indicated by very low response
rates in the groups given unpaired training. Conditioning estab-
lished in both age groups was abolished by muscimol inactiva-
tion of the cerebellar nuclei and overlying cortex that are ipsilat-
eral to the conditioned eye.
The conditioning observed in younger rats using pontine
stimulation as the CS is remarkable in that no other experimental
manipulation has been found that produces conditioning in
P17–P18 pups that is equivalent to the conditioning seen in P24–
P25 pups (Stanton and Freeman Jr. 2000). A series of experiments
by Stanton and colleagues found that systematic alterations in CS
Figure 1.
pups trained with pontine stimulation (left) or a 2-kHz tone (Nicholson and Freeman Jr. 2004) (right)
as the conditioned stimulus (CS) on postnatal days (P) P17–P18 (white symbols) or P24–P25 (black
symbols). The pups were given either paired (circles) or unpaired (triangles) presentations of the CS
and a periorbital shock unconditioned stimulus (US). The amount of associative learning in each paired
group was determined by the increase in responding across training sessions and by the difference in
CR percentage between the paired and unpaired conditions in both age groups.
Mean (? standard error of the mean, s.e.m.) conditioned response (CR) percentage for rat
Figure 2.
rat pups trained with pontine stimulation as the CS on postnatal days (P)
P17–P18 (white symbols) or P24–P25 (black symbols). The pups were
given paired presentations of pontine stimulation and a shock US.
Muscimol was infused into the cerebellar nuclei prior to session 6 to
inactivate the cerebellar nuclei and overlying cortex ipsilateral to the
conditioned eye. CRs established by paired training of pontine stimula-
tion and the US were abolished by muscimol inactivation. Response re-
covery was evident in both groups on session 7.
Mean (? s.e.m.) conditioned response (CR) percentage for
Freeman Jr. et al.
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salience, US intensity, interstimulus interval, motivational state,
amount of training, and CS modality did not reduce the age-
related difference in eyeblink conditioning (Stanton and Free-
man Jr. 2000). Most of these manipulations influence the rate
and magnitude of conditioning in rat pups but do not differen-
tially affect younger rats. Pontine stimulation is, therefore, the
only manipulation to date that eliminates the age-related differ-
ence in eyeblink conditioning.
The demonstration of robust eyeblink conditioning in rat
pups trained on P17–P18 with stimulation of the pontine nuclei
as a CS suggests that the developmental emergence of eyeblink
conditioning is in part due to developmental changes in the CS
pathway. A previous study found a developmental change in the
responsiveness of pontine neurons to acoustic stimuli, which
might be the primary ontogenetic change in the CS pathway
(Freeman Jr. and Muckler 2003). The developmental change in
pontine neuronal activity during an acoustic CS is probably not
related to maturation of the peripheral auditory system or co-
chlear nuclei, because behavioral and auditory brainstem re-
sponse studies indicate that basic auditory function is evident by
P15 or P16 in rats (Hyson and Rudy 1984, 1987; Blatchley et al.
1987; Sananes et al. 1988; Stanton et al. 1992). However, we do
not know whether the developmental change in pontine respon-
siveness to acoustic stimuli is due to developmental changes in
the projection of the cochlear nuclei to the pons, intrinsic prop-
erties of pontine neurons, or feedback projections from the cer-
ebellum and red nucleus (Cartford et al. 1997; Clark et al. 1997).
It is also possible that there are developmental changes in the
mossy fiber projection to the cerebellum including myelination
and increased synaptic efficacy. However, the level of current
used to establish conditioning was similar at P17–P18 and P24–
P25, suggesting that the important developmental changes in the
CS pathway occur in the pons or in a pontine afferent.
The developmental changes in pontine neuronal activity
correspond to developmental changes in CS-elicited neuronal ac-
tivity in the cerebellar cortex and nuclei (Freeman Jr. and Nichol-
son 2004; Nicholson and Freeman Jr. 2004). As a result of CS
pathway development, CS input to the cerebellum in younger
rats may not be strong enough to induce synaptic plasticity. Elec-
trical stimulation might increase the number of mossy fibers ac-
tivated in younger rats relative to a peripheral CS and thereby
increase the magnitude of cerebellar neuronal activation, driving
activity-dependent synaptic plasticity. Developmental changes
in the magnitude of input from the pontine nuclei to the cer-
ebellum might interact with ontogenetic changes in synaptic
plasticity mechanisms. However, various forms of synaptic plas-
ticity and changes in excitability have been demonstrated in cul-
tured cerebellar neurons and slices as young as P13 (Aizenman
and Linden 2000; Hansel et al. 2001). In the absence of direct
evidence for an ontogenetic change in synaptic plasticity mecha-
nisms, our current view is that the developmental change in the
magnitude of CS pathway input to the cerebellum accounts for
the role of CS pathway maturation in the ontogeny of eyeblink
conditioning.
Inactivation of the cerebellar hemisphere ipsilateral to the
conditioned eye abolished eyeblink CRs established with pontine
stimulation as the CS, as seen in adult rats (Freeman Jr. et al.
2005). The loss of CRs in infant rats following muscimol infusion
into the cerebellum suggests that the expression of conditioning
established using a pontine stimulation CS requires cerebellar
activity. However, muscimol inactivation of the cerebellum does
not conclusively establish that the memory underlying condi-
tioning with the stimulation CS is stored in the cerebellum. Stud-
ies that use reversible inactivation of the cerebellum and red
nucleus are needed to determine whether the memory underly-
ing eyeblink CRs established with a pontine stimulation CS is
stored in the cerebellum (Krupa et al. 1993).
Previous studies using pontine stimulation as a CS in adult
rats and rabbits found that the acquisition rate with stimulation
is faster than the acquisition rate observed with a peripheral CS
(Steinmetz et al. 1986; Freeman Jr. and Rabinak 2005). In the
present study, the rate of acquisition in infant rats was about the
same as that seen with conditioning to a tone CS. Moreover, the
rate of acquisition in the infant rats was considerably slower than
the rate of acquisition seen in adult rats. These findings suggest
that even the older rat pups (P24–P25), which show rapid con-
ditioning, are still undergoing maturational changes in the CS
and US pathways.
The present findings and those of previous studies suggest
that the ontogeny of eyeblink conditioning is due to develop-
mental changes in the CS and US pathways. The development of
the US pathway is characterized by an ontogenetic increase in
cerebellar inhibitory regulation of climbing fiber activity (Free-
man Jr. and Nicholson 2004). The developmental change in the
responsiveness of pontine neurons to a tone CS results in a de-
velopmental change in the magnitude of CS-driven input to the
Figure 4.
picting the effective stimulating electrode placements. The electrode
placements for groups trained on P17–P18 (left) or P24–P25 (right) in the
paired (black dots) or unpaired conditions (gray dots) are shown. The
numbers indicate the stereotaxic coordinates in the anterior-posterior
dimension relative to lambda.
Coronal sections of the rat basilar pontine nuclei (PN) de-
Figure 3.
on P17 and P18 during test trials of pontine stimulation without the US
on the first (S1), third (S3), fifth (S5), muscimol inactivation (session 6,
MUS), and recovery (session 7, REC) sessions. The arrows indicate the
onset time of pontine stimulation. Scale bar, 100 msec.
Traces of eyelid EMG activity recorded from a rat pup trained
Development of eyeblink conditioning
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cerebellum. The developmental changes in the CS and US path-
ways are thought to influence the induction and maintenance of
synaptic plasticity within the cerebellum and thereby influence
the acquisition and retention of eyeblink conditioning.
Materials and Methods
Subjects
The subjects were 46 Long-Evans rat pups. Thirty-five pups had
effective electrode placements and were trained on P17–P18
(n = 18) or P24–P25 (n = 17). A subset of the pups with effective
electrode placements was implanted with a cannula in the cer-
ebellum and trained on P17–P18 (n = 6) or P24–P25 (n = 5). The
remaining 11 pups had ineffective electrode placements. The rats
were housed in the animal colony in Spence Laboratories of Psy-
chology at the University of Iowa. The rats were maintained on a
12-h light/12-h dark cycles, with light onset at 7 a.m. Training
sessions occurred between 7 a.m. and 7 p.m.
Surgery
The rat pups (P16 and P23) were given i.p. injections of ketamine
(100 mg/kg), xylazine (5 mg/kg), and atropine (0.8 mg/kg). The
rat’s head was positioned and held securely in an infant stereo-
taxic apparatus and the skull surface was aligned in three planes.
Differential electromyographic (EMG) electrodes were implanted
in the left upper eyelid, and a ground electrode was connected to
one of two skull hooks. The electrode and ground wires were
soldered to gold pins in a plastic connector, which was secured to
the skull by the skull hook and dental acrylic. The second skull
hook was secured to the skull slightly anterior to lambda.
A bipolar stimulating electrode was then implanted into or
just dorsal to the right basilar pontine nuclei. The electrode con-
sisted of two insulated stainless steel wires (50 µm) in a plastic
connector. The stereotaxic coordinates for the pontine nuclei
were taken from lambda (P16/P23: +1.6/+2.0 anterior, ?1.0/
?1.0 medial-lateral, ?8.3/?8.5 dorsal-ventral). Once the elec-
trode was in place it was cemented with dental acrylic covering
the entire length of the electrode above the skull surface, includ-
ing the plastic connector. The rat pups that were given muscimol
infusions into the cerebellum had a 23-gauge guide cannula im-
planted 0.5 mm dorsal to the left anterior interpositus nucleus. A
30-gauge stylet was inserted into the guide cannula. The stereo-
taxic coordinates for the cannula were also taken from lambda
(P16/P23: ?2.3/?2.3 posterior, 2.0/2.0 medial-lateral, and
?4.8/?4.9 dorsal-ventral).
A bipolar stimulating electrode used for delivering the US
was implanted subdermally, immediately caudal to the left eye.
This bipolar electrode was also encased by a plastic connector,
which was secured by dental acrylic. Sutures closed the surgical
site on both sides of the head stage. Ketofen (5 mg/kg), an anal-
gesic, was administered at the end of surgery, along with a sub-
cutaneous injection of Ringer solution.
Conditioning apparatus
The conditioning apparatus consisted of a small-animal sound
attenuation chamber (BRS/LVE) with a small-animal operant
chamber (BRS/LVE) contained inside. The rats were kept in the
operant chamber during conditioning. Lightweight cables with
connectors for the EMG, US, and CS electrodes were attached to
a commutator. The back wall of the sound-attenuation chamber
was equipped with a small houselight that stayed on during con-
ditioning sessions. The electrode leads from the rat’s head stage
were connected to peripheral equipment and a desktop com-
puter. Computer software controlled the delivery of stimuli and
the recording of eyelid EMG activity (JSA Designs). The US was
delivered through a stimulus isolator (model no. 365A; World
Precision Instruments). EMG activity was recorded differentially,
filtered (500–5000 Hz), amplified (2000?), and integrated (time
constant = 20 msec). Pontine stimulation was triggered through
a programmable stimulator (Master 8, A.M.P.I.), which con-
trolled signal input to a stimulus isolator (model no. 365A; World
Precision Instruments) that delivered the electrical stimulation.
Pontine stimulation
Electrical stimulation of the basilar pontine nuclei functioned as
the CS, which was administered in a 200 Hz train of 0.1-msec
biphasic pulses for 300 msec. The stimulation threshold for the
CS was found before training by setting the stimulating current
to 50 µA, and either increasing or decreasing the current in 5-µA
increments until a slight movement was detected (Tracy et al.
1998). Observable movements included, but were not limited to,
eye blinks, eyeball retractions, and head movements. The level of
stimulation during training was set to half the threshold inten-
sity.
Muscimol infusion
Prior to muscimol infusions, the stylet was removed from the
guide cannula and replaced with a 30-gauge infusion cannula.
The infusion cannula was connected to polyethylene tubing (PE
10, 110–120 cm) that was connected to a 10-µL gas-tight syringe
(Hamilton). The syringe was placed in an infusion pump (Har-
vard Apparatus), and 0.5 µL muscimol (5 nmol in saline, pH 7.4)
was infused at a rate of 30 µL/h. The tubing connected to the
infusion cannula was cut and sealed with candle wax. The infu-
Figure 5.
picting the ineffective stimulating electrode placements. The electrode
placements for groups trained on P17–P18 (gray dots) or P24–P25 (black
dots) are shown. The numbers indicate the stereotaxic coordinates in the
anterior-posterior dimension relative to lambda for rats trained on P17–
P18 and P24–P25.
Coronal sections of the rat basilar pontine nuclei (PN) de-
Figure 6.
placements. The cannula placements for the groups trained on P17–P18
(gray dots) or P24–P25 (black dots) were in or just dorsal to the anterior
interpositus nucleus (IP). D, dentate nucleus; F, fastigial nucleus.
Coronal sections of the rat cerebellum depicting cannula
Freeman Jr. et al.
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sion cannula remained in place for the duration of the experi-
mental session.
Paired training
The rat pups in both age groups were given six paired training
sessions, three sessions per day. The paired training sessions con-
sisted of 100 trials, each with 90 trials of the stimulation CS
paired with the shock US (10 msec, 3.0 mA) and 10 stimulation
CS-alone trials, occurring on every tenth trial. The CS-alone trials
were included in order to assess behavioral responses (integrated
EMG activity) uncontaminated by URs. The interstimulus inter-
val for paired trials was 290 msec. Trials were separated by an
intertrial interval that averaged 30 sec. Behavioral data were ex-
amined from computer records of EMG responses. CRs were de-
fined as responses that crossed a threshold of 0.4 volts above the
baseline activity during the CS period, but at least 80 msec after
CS onset, to avoid contamination of the CR measures by the
startle (alpha) response. The threshold level is typically less than
10% of the amplitude of the UR. Previous studies have shown
that the paired training protocol used in the present study estab-
lished associative eyeblink CRs in developing rats (Nicholson and
Freeman Jr. 2003a,b, 2004).
Unpaired training
Rat pups in both age groups were given six 200-trial sessions of
explicitly unpaired presentations of the CS and US. The same
time durations for the CS and US were used as in the paired
procedure. The intertrial interval (ITI) was set to average 15 sec to
match the total time spent in the conditioning chamber and the
temporal distribution of CS and US presentations with the paired
groups. The method for defining CRs was the same as that used
in the paired procedure.
Histology
After training was completed, rats were euthanized with a lethal
injection of sodium pentobarbital (90 mg/kg) and transcardially
perfused with 100 mL of physiological saline, followed by
300 mL of 3% formalin. The brains were post-fixed in formalin
for 2 d and then put in a solution of 10% sucrose in PBS before
sectioning. The brains were sectioned at 50 µm with a sliding
microtome. Sections were then stained with cresyl violet. The
cannula and stimulating electrode locations were determined by
examining serial sections.
Acknowledgments
Support for this research comes from National Institute for Neu-
rological Disorders and Stroke grant NS38890 to J.H.F.
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Received December 27, 2004; accepted in revised form March 22, 2005.
Development of eyeblink conditioning
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