4. DISCUSSION
In the current study, we compared in vitro hippocampal network
oscillations in three commonly used inbred mouse strains (B6J, B6N, 129)
with the aim of elucidating common oscillatory patterns in inbred mouse
strains that show aberrant emotional/cognitive behaviour (B6N and 129).
We further investigated the density of main GABAergic interneurons in
the hippocampal subregions and assessed hippocampal excitability via
measuring population spikes in the CA3 and CA1 subregions. We identified
three common oscillatory features in B6N and 129 strains that diverged
from the B6J strain: (1) Larger amplitude cholinergic gamma oscillations
in the CA3; (2) Higher incidence of CA1 SW with a specific increase of
SW propagation along the CA3-CA1 axis in the 129 strain; (3) Higher
correlation of CA3 and CA1 SW transients. We further detected
significant alterations in the number of specific inhibitory interneuron
populations in the hippocampal subregions and activation of CA3
associative network that are essential for generation and maintenance of
hippocampal network oscillations across three mouse strains. These
observations align well with the notion that in vitro network
oscillations provide useful reductionist models to investigate
neurobiological underpinnings of phenotypic differences and highlight
the importance of careful consideration of genetic background when
designing experiments.
We used in vitro network oscillations that are dependent on the
CA3 associative network activity as our experimental model. Hippocampal
CA3 subregion forms an extensive associative network with abundant local
recurrent excitatory connections that are under tight control of local
inhibitory interneurons (Li et al. , 1994; Le Duigou et
al. , 2014). These features make this circuit ideal for generation of
local field potential network oscillations such as SW-R and gamma
oscillations both in vivo and in vitro (Le Duigou et
al. , 2014). Both oscillation types show similar features when recordedin vitro or in vivo (Butler & Paulsen, 2015; Çalışkan &
Stork, 2018). Indeed, there is accumulating evidence that ex vivohippocampal network oscillations correlate with specific behaviours (Luet al. , 2011; Çalişkan et al. , 2016) or are modulated by
behavioural manipulations (Albrecht et al. , 2013; Mizunumaet al. , 2014; Çalışkan et al. , 2015) suggesting that our
findings might be relevant for behavioural phenotypes observed in
investigated inbred mouse strains.
In our study, B6J strain was used as control strain as it has been shown
to exhibit normal spatial learning, normal anxiety levels, normal
contextual fear memory and extinction (Gerlai, 1998a, 1998b, 2002;
Stiedl et al. , 1999; Rodgers et al. , 2002; Siegmundet al. , 2005; Camp et al. , 2012; Åhlgren & Voikar, 2019).
Of note, both B6N substrain and 129 strains have been consistently shown
to exhibit aberrant contextual fear memory / extinction and elevated
anxiety levels in comparison to B6J strain (Stiedl et al. , 1999;
Rodgers et al. , 2002; Siegmund et al. , 2005; Camp et
al. , 2012; Åhlgren & Voikar, 2019). We used hippocampal slices
obtained from the ventral-to-mid portion of the hippocampus.
Importantly, ventral hippocampus have been repeatedly shown to be
involved in stress adaptation, emotional memory formation and anxiety,
while the dorsal hippocampus is implicated in cognitive functions such
as spatial memory processing (Kjelstrup et al. , 2002; Fanselow &
Dong, 2010; Strange et al. , 2014). Therefore, our findings might
be more relevant to emotional phenotypes observed in these two mouse
strains.
Both B6N and 129 strains show significantly increased number of CA1 SW
events and correlation of SW events in the CA3 and CA1 (Fig. 1).
Additionally, 129 strain shows a facilitated propagation of SW events
from CA3 to CA1 region. These findings aligns well with our previous
work demonstrating that both increased propensity for SW-R generation in
the ventral CA1 and increased SW propagation along CA3-CA1 axis
correlate well with the aberrant fear extinction and persistent fear
memories (Çalişkan et al. , 2016). This is also supported byin vivo work showing causal involvement of SW-R in contextual
fear memory consolidation (Wang et al. , 2015; Girardeau et
al. , 2017; Ognjanovski et al. , 2017). Cholinergic hippocampal
gamma oscillations (Fig. 2) are significantly enhanced in mice with
anxious phenotype (B6N, 129) in comparison to a less anxious mouse
strain (B6J). This observation also supports our previous study
demonstrating reduced gamma oscillation in ex vivo slice
preparations of mice with lower anxiety (Albrecht et al. , 2013).
Indeed, heightened anxiety/emotionality appears to elicit increases ofin vivo gamma oscillations in the ventral hippocampus and
hypersynchronous gamma activity has been related to emotional
psychopathologies (Headley & Paré, 2013; Dunkley et al. , 2014,
2015; Stujenske et al. , 2014). Future studies testing whether
such oscillatory patterns are also observed in vivo are needed to
substantiate these in vitro findings. Furthermore, it would be
interesting to test whether cholinergic markers that are important for
gamma generation (e.g., M1 receptors) are altered in these mouse
strains.
Augmented network oscillations might be due to an altered CA3
associative collateral network activation in B6N substrain and 129
strain (Çalışkan et al. , 2015). To assess the activation of CA3
associative collateral network (Fig. 3), we analysed antidromic
populations spikes and synaptically-mediated orthodromic population
spikes triggered by antidromic stimulation of CA3 subregion (Behrenset al. , 2005; Fano et al. , 2012). We found no major effect
on the antidromic population spikes in the CA3 subregion except for a
minor decrease in the 129 strain at higher stimulation strengths.
However, the synaptically-mediated orthodromic population spikes were
significantly reduced primarily in 129 strain. Local CA3 GABAergic
interneurons control the excitation mediated by excitatory CA3-CA3
synapses (Li et al. , 1994; Le Duigou et al. , 2014). Thus,
a reduction in the excitatory interactions within the CA3 recurrent
collaterals might be due to an increased local inhibitory tonus in the
129 strain. Therefore, we analysed the expression of distinct markers
for inhibitory interneurons that are crucial for generation and
modulation hippocampal network oscillations (Fig. 4) (Mann et
al. , 2005; Oren et al. , 2006; Ellender et al. , 2010). We
used glutamate decarboxylase 67 (GAD67) as the pan-GABAergic marker.
Furthermore, somatostatin (SST) and parvalbumin (PV) were used to
quantify SST+ and PV+ interneuron populations. We observed a prominent
overall increase in the number of GAD67+ interneurons in the hippocampal
CA3-CA1 of 129 strain. This finding supports the notion that an
increased inhibition might be present in the 129 strain which, in turn,
can cause a reduction in the CA3 orthodromic population spikes for a
given antidromic stimulation.
Changes in the number of interneuron populations can either cause the
observed oscillatory features or reflect a compensatory mechanism.
During both CA3 SW events and carbachol-induced gamma oscillation
cycles, CA3 pyramidal cells receive a relatively stronger inhibitory
input in comparison to an excitatory input (Oren et al. , 2006;
Hajos et al. , 2013; Zemankovics et al. , 2013; Bazelotet al. , 2016). These compound intracellular excitatory/inhibitory
events with a relatively stronger inhibitory component can be recorded
as extracellular field potentials from the pyramidal cell layer as
positive going voltage deflections (Bazelot et al. , 2010, 2016;
Beyeler et al. , 2013). On the other hand, both perisomatic-region
targeting PV+ interneurons and distal dendrite-targeting SST+
interneurons appear to receive relatively stronger excitation in
comparison to inhibition during each gamma or SW event (Oren et
al. , 2006; Hajos et al. , 2013; Pangalos et al. , 2013).
Thus, a relatively increased inhibition during these oscillatory cycles
can cause such an increase in gamma oscillation power as observed in the
CA3 of both B6N and 129 strains.
Some evidence suggests that perisomatic inhibition onto pyramidal cells
provided by PV+ basket cells is the source of gamma-range field
potentials induced by carbachol (Gulyás et al. , 2010).
Inconsistently, recent evidence shows a paradoxical increase in
broadband gamma power in mice deficient for PV+ interneuron function,
however, with reduced cellular synchronization to gamma oscillations
(Guyon et al. , 2021). We observed a reduced PV+ interneuron
number in both CA3 and CA1 of B6N strain. It is tempting to speculate
that the reduction in PV+ interneuron number can cause the increased
gamma power observed in the CA3 and CA1 of B6N strain. However, we
detected an increased local gamma correlations in the CA3 and CA1
suggesting an increased gamma synchronization potentially mediated via
increased PV+ interneuron functionality. Thus, as an alternative
explanation, more efficient recruitment of PV+ interneurons by
excitatory pyramidal cells could compensate for reduced PV+ interneuron
abundance and increase perisomatic inhibition augmenting gamma field
potential oscillations. Such increase in perisomatic inhibition can also
lead to heightened SW amplitude as observed in the CA3 and CA1 of B6N
strain. Furthermore, the reduction in the number of PV+ interneuron
could alter the reciprocal interactions between PV+ interneurons during
SW events that mediate the generation of fast ripple oscillations
(Schlingloff et al. , 2014). In line, we observed a reduced CA3
ripple frequency in the B6N strain. Future studies assessing
excitation-inhibition balance using intracellular recordings during
field potential oscillations need to confirm whether such alterations
are present in the B6N strain.
On the other hand, the increased number of SST+ interneurons observed in
the hippocampus of 129 strain might underlie the increased gamma power.
Indeed, a recent study suggests that SST+ interneurons are necessary for
the maintenance of carbachol-induced gamma oscillations in hippocampal
slices prepared similarly to our study (Antonoudiou et al. ,
2020). Furthermore, the same study shows that optogenetic activation of
SST+ interneurons leads to an increase in the peak frequency of
cholinergic gamma oscillations in vitro . Indeed, in the 129
strain, the increased SST+ interneuron number was accompanied by faster
gamma oscillation cycles evident by an increased peak frequency of gamma
oscillations. However, it still remains to be elucidated how increased
number of SST+ interneurons could directly affect the entrainment of
pyramidal cells leading to augmented gamma oscillations as they mostly
target the distal dendrites of the pyramidal neurons in the CA3 (Müller
& Remy, 2014). Thus, it is likely that, during cholinergic gamma
oscillations, they modulate perisomatic inhibition via altering the
excitability of principal neurons and spiking precision of PV+
interneurons as previously described for the dentate gyrus circuit
(Savanthrapadian et al. , 2014). Alternatively, changes in the
GABA-A receptor subunit composition might also underlie the alterations
in the peak frequency of cholinergic gamma oscillations as previously
described (Heistek et al. , 2010).
Taken together, we identified several common oscillatory and cellular
features in the hippocampus of inbred mouse strains with aberrant
emotional/cognitive phenotype (B6N and 129). Noteworthy, while there is
substantial evidence for behavioural differences among C57/BL6
substrains (B6N vs. B6J), to the best of our knowledge, our study is the
first to show potential interneuronal and oscillatory correlates of
these differences in C57/BL6 substrains. Collectively, our study
provides further evidence for altered hippocampal physiology among
commonly used inbred mouse strains and cautions scientific community to
consider genetic background as an important variable when designing
experiments and interpreting their results.