SK3 K channel deficient mice have enhanced 5HT and DA neurotransmission and altered depression, anxi

1
SK3 K channel deficient mice have enhanced 5-HT-
and DA neurotransmission and altered depression-,
anxiety- and cognitive behaviors
J.P.R. Jacobsen1, P. Weikop1, H.H. Hansen1, J.D.
Mikkelsen1, J.P. Redrobe1, S. Pedersen1, D.
Holst1, C.T. Bond2, J.P. Adelman2 , P.
Christophersen1, N. R. Mirza1? 1NeuroSearch A/S,
Pederstrupvej 93, 2750 Ballerup, Denmark.
2Vollum Institute, Portland, Oregon, USA ?
max_at_neurosearch.dk
157.4
HYPOTHESIS Selective SK3 deficiency will result
in enhanced 5-HT and DA neurotransmission and an
antidepressant-like phenotype
RESULTS
Table 1. SK3 deficient T/T mice show no sensory
or motor impairments
Fig 3. SK3 deficient T/T mice show
antidepressant-like behaviors in the Forced
Swim and Tail Suspension tests
Fig 5. SK3 deficiency influence activity in a
gender- and context dependent fashion
Fig 6. SK3 deficient T/T have deficits in
working/short-term memory

• INTRODUCTION
• Small conductance calcium-activated K (SK1-3)
  channels regulate firing patterns, firing
  frequency and synaptic plasticity
• SK channels of the SK3 subtype is widely
  expressed in the brain and particularly enriched
  in 5-hydroxytryptophan (5-HT) and dopamine (DA)
  neurons in the pons and midbrain, respectively.
• Blocking SK (presumably SK3) channels on 5-HT
  and DA neurons increase firing activity
• Non SK-subtype selective channel blockers are
  reported to display antidepressant-like and
  pro-cognitive effects in rodents
• Decreased cerebellar SK2 channel function may
  cause severe ataxia, limiting the therapeutic
  potential of non-selective SK blockers/negative
  modulators
• In lieu of SK3 selective blocking/negatively
  modulating compounds, we here used conditional
  SK3 deficient (T/T) mice to assess the effect of
  SK3 loss-of-function on 5-HT- and DA
  neurotransmission as well as affective and
  cognitive behaviors.

Fig 1. SK3 deficient T/T mice have increased 5-HT
neurotransmission
Fig 3. Forced Swim and Tail Suspension tests.
Immobility was scored during the entire 360 sec
test. N 10-20. t- test. , p lt 0.05
Fig 4. SK3 deficient T/T female mice are more
anxious specifically in the Zero Maze
Fig 1. Increased extracellular levels of 5-HT (a)
after citalopram (20mg/kg) and the primary
metabolite, 5-HIAA (b), indicates enhanced 5-HT
function. N 10-11. Two-way RM-ANOVA
student-newman-keuls post-hoc test. , p lt 0.05
Fig 2. SK3 deficient T/T mice have increased DA
neurotransmission
Fig 5. Activity levels of SK3 deficient T/T mice
in different contexts In the open field
ambulatory distance in the males where higher or
lower under low and high illumination,
respectively. No differences in female mice were
seen (a, N 10-20). SK3 deficient T/T mice of
both sexes displayed increased exploratory
activity in a novel cage (b, N 12-18). Home
cage activity was normal (c, N 5-8). Two-way
RM-ANOVA student-newman-keuls post-hoc test , p
lt 0.05
Fig 6. Cognitive evaluation of SK3 deficient T/T
mice SK3 deficiency lead to decreased alternation
behavior, indicative of impaired working memory,
with no difference in arm entries (a, N 3-9).
SK3 deficient T/T mice displayed impaired
short-term memory in 5-trial inhibitory
avoidance, a putative model for ADHD behaviors
(b, N 10-12). In contrast, performance in
passive avoidance (c, N 8-15), object
recognition (d, N 9-16) and Morris water maze
(e, N 7-8) was normal. Two-way RM-ANOVA
student-newman-keuls post-hoc test , p lt 0.05
METHODS Mice In SK3 T/T mice the SK3 gene is
under control of a doxycycline-sensitive
gene-switch. Upon dietary treatment with
doxycycline SK3 gene transcription is suppressed,
resulting in near-abolishment of SK3 protein from
the brain (Bond et al, 2000, Science,
289(5486)1942-6). The colony was maintained on a
C57BL/6NTac background. WT littermates served as
controls. Mice were housed 8 per cage in mixed
genotype single-sex cages (21 x 37 x 25
cm). Sensory and motor assessment Presence of
basic reflexes were evaluated according Crawley
guidelines (Brain Res. 1999 835(1)18-26). The
rotorod test comprised two 8-80 RPM trials 60min
apart. The two recorded fall-latencies were
averaged. In beam walking the mice explored a
wooden beam (2.5 cm diameter, 50 cm length)
divided into 5 zones. Number of zone entries over
60 sec were counted. In the hot plate (55 oC)
test latency to first response, hind paw licking,
hind paw shake, jumping, whatever appeared first,
was recorded. Acoustic startle and pre-pulse
inhibition was assessed in startle chambers
according to standard protocols. Microdialysis
The measurements were performed in freely moving
animals. Guide cannulas were pre-implanted and
the probes (2 mm membrane-length) implanted 3 hrs
before sample-collection start. 5-HT, DA and
metabolites in the dialysates were quantified by
HPLC-EC. Tissue 5-HT, DA and metabolites Brains
were rapidly removed and frozen. Regions of
interest were dissected from still frozen tissue
slabs and stored at -80oC. Tissues were thawed,
homogenized in 0.1 M perchloric acid and the
filtered supernatant analyzed by
HPLC-EC Depression-like behaviors The tail
suspension test was done using an automated
pressure-gauge system from Med Associates. The
forced swim test was done in 4 L glass laboratory
beakers at 2325OC. Immobility was scored by a
digital videotracking system (Videotrack,
Viewpoint). Anxiety-like behaviors The zero
maze consisted of a circular runway (diameter of
53 cm) elevated 50 cm above the floor divided
into two open and two walled areas. Time in and
entries into the open areas were recorded. The
four plate test used the apparatus from Bioseb.
After a time-out of 15 sec the mouse was shocked
(punished) at each crossing between the four
squared zones of the apparatus floor. Number of
punished crossings were scored over 180 sec. The
open field was a rectangular, transparent perspex
box (60 x 60 cm floor area). The mouses center
of gravity was videotracked digitally
(Videotrack, Viewpoint). Time in center area (30
x 30 cm) was scored. Velocity of the center of
gravity distinguished ambulatory, non-ambulatory
(small) movements and immobility. Illumination
was either 20 or 100 lux. Novel and home cage
activity Activity was monitored using a
two-dimensional photobeam system (TSE Systems)
framing the cage. In the novel cage the mouse was
placed in a clean cage with bedding under very
low lighting (2 lux, to decrease anxiety) and the
activity monitored for 120 min. When
investigating home cage activity the mouse was
allowed to habituate to a test cage with chow and
water for 16 hrs where after activity was
recorded for 48 hrs. Cognitive assessment
Spontaneous alternations, object recognition and
passive avoidance were conducted according to
standard protocols. In 5-trial inhibitory
avoidance the mice were shocked upon entering the
dark compartment in a shuttle-box, immediately
returned to the home cage for 60 sec and the
trial then repeated for a total of 5 times.
Latencies to enter the dark compartment were
scored. The Morris water maze was executed over
two days, 6 trials per day, using a standard
protocol.

• CONCLUSION
• SK3 deficiency enhances 5-HT and DA
  neurotransmission
• SK3 deficiency leads to an antidepressant-like
  phenotype not confounded by general hyperactivity
• SK3 deficiency leads to selective cognitive
  deficit in working/short-term memory.
• Although it is tempting to ascribe the observed
  behavioral changes to the observed 5-HT and DA
  alterations, a definitive causal coupling is
  currently premature.
• Possibly, selective SK3 negative modulators
  could be antidepressants

Fig 4. Effect of SK3 deficiency on anxiety
behaviors .Female SK3 deficient T/T mice
displayed more anxiety-like behaviors in the zero
maze (a). No genotype effects we found In the
four plate test (b) or the open field (c). N
20-36 (zero maze), 14-16 (four plate test), 11-20
(open field). t- test., p lt 0.05
Fig 2. Increased extracellular basal levels of DA
(a) and the metabolites DOPAC (b) and HVA (c)
indicates enhanced DA function. Interestingly, no
DA difference was evident following cocaine
(30mg/kg). Two-way RM-ANOVA student-newman-keuls
post-hoc test. N 13. , p lt 0.05
Society for Neuroscience, Washington DC, 2008