“The impact on synaptic transmission by removal of

endocannabinoid signaling in specific neurons”. Mujica P.

Abstract

Endocannabinoids (eCBs) are powerful modulators of synaptic function throughout the CNS.
Alterations in the eCB system are observed in several neuropsychiatric disorders, suggesting that eCBs
modulate emotional and cognitive processes in associative areas such as the prefrontal cortex (PFC).
However, the underlying cellular mechanisms as well as the specific participating neural circuits are
unclear. Given the importance of synaptic inhibition in shaping neural activity and brain function, we study
eCB signaling at GABAergic synapses from a major population of inhibitory interneurons (INs) in the PFC.
By targeting dendritic regions of principal pyramidal cells where excitatory synapses act, somatostatin-
expressing (SOM-) INs can profoundly modulate synaptic gain and integration to influence pyramidal cell
output. The ability of eCBs to suppress GABAergic inhibition from SOM-INs would endow the eCB system
with the power to fine-tune information flow from multiple sources such as the thalamus, hippocampus
and other cortical areas to reshape associative cognitive processing. Preliminary findings suggest that
these GABAergic synapses are under eCB regulation. In this project, we will examine the physiological
impact of eCB modulation of SOM-IN mediated inhibition on synaptic function and cortical activity.
Specifically, we will conditionally delete the main cannabinoid receptor in the brain from these GABAergic
cells and determine whether this selective loss alters the balance of excitation and inhibition. In order to
develop this objective, we will record spontaneous excitatory and inhibitory postsynaptic responses, i.e.,
EPSCs and IPSCs, onto layer 2/3 pyramidal cells and compare the amplitude and frequency of this
events between conditional knockouts and littermate controls.

Introduction

In the cerebral cortex, information processing requires temporally and spatially coordinated synaptic
communication among excitatory and inhibitory cells. This balance is key to normal brain function and is
thought to be maintained through mechanisms that regulate synaptic strength and excitability 1. Several
findings have suggested that endocannabinoids (eCBs) by modulating synaptic strength are powerful
regulators of neural activity throughout the CNS 2. In the prefrontal cortex (PFC), eCBs have been
associated with neuropsychiatric disorders that reflect PFC dysfunction 3 and preclinical data has
demonstrated that eCB signaling is important in stress, emotional and cognitive functions 4. However, it
remains unclear exactly how eCBs regulate PFC function.
The eCB system comprises the endogenous compounds such as anandamide (AEA) and 2-
arachidonoylglycerol (2-AG) 5,6, the cannabinoid type 1 and type 2 receptors (CB1R and CB2R), the
enzymes responsible for eCB synthesis and metabolism and transporters that regulate eCB levels in the
synaptic cleft 7. CB1R is expressed predominantly in the CNS and is located in different neuronal
subpopulations 8,9, whereas the CB2R is mostly present peripherally in the immune system 10. Both are
seven transmembrane receptors that are coupled to G protein, G i/o 7. Activation of either cannabinoid
receptor reduces adenylyl cyclase (AC) activity, thus lowering cAMP levels and protein kinase A (PKA)
activity. Because CB1R is one of the most highly expressed G protein-coupled receptors in the brain 11,
and is widely distributed in many brain areas embodying the cortico-limbic system like prefrontal cortex,
hippocampus, cerebellum, striatum and amygdala 12, it is thought that this receptor subtype mediates
most of the effects of the eCB system on emotional, cognitive and memory processes.
The principal mechanism by which eCBs regulate synaptic function is through retrograde
signaling 13. This retrograde signaling is involved in short-term and long-term forms of synaptic plasticity 14
and contributes to certain forms of learning and memory 15,16. eCBs are released from neurons in activity-
dependent manners, act retrogradely on presynaptic CB1 cannabinoid receptors and modulate
transmitter release 17. eCB mobilization can be triggered either by strong neuronal depolarization 18 or by
activation of Gq-coupled receptors such as group I metabotropic glutamate receptors (mGluRI) and
M1/M3 muscarinic acetylcholine receptors 19,20 through activation of phospholipase Cβ (PLCβ) 21. More
recently, it has been revealed that eCBs can act in non-retrograde ways, where the postsynaptically
produced eCBs activate postsynaptic CB1Rs or transient receptor potential vanilloid type 1 (TRPV1)
channels. Moreover, eCBs can signal via astrocytes to indirectly modulate presynaptic or postsynaptic
function, thereby triggering gliotransmission 2.
Although CB1Rs are abundantly expressed in the PFC, little is known about the specific neural
circuits through which eCBs signal and exactly how eCBs regulate PFC function. Deletion or
pharmacological blockade of CB1Rs on GABA interneurons disrupt eCB-mediated plasticity in many brain
regions 30. Additionally, genetic deletion of CB1R from GABAergic neurons (GABA/CB1 -/-) leads to
behavioral changes such as enhanced interest to social stimuli 31 and enhanced neuroinflammation in
ageing 32 as well as hyperphagia 33 and developmental deficits 34. Exogenous activation of CB1Rs on
GABAergic interneurons disrupts hippocampal-dependent learning in vivo 38 and inhibits LTD of excitatory
synapses in the amygdala in vitro 27. Taken together, these findings suggest that CB1Rs at GABAergic
synapses play a role in modulating synaptic plasticity and may underlie certain learning and memory
deficits. It is currently unclear whether cortical circuits are involved.
Previous studies have shown that activation of CB1Rs in neocortex 22,23 can suppress inhibitory
transmission by reducing GABA release. In the PFC, eCBs trigger I-LTD, but only in conjunction with the
dopaminergic system 25. It remains unclear whether the eCB system alone can persistently suppress
GABAergic synapses. Furthermore, studies of eCB-mediated synaptic plasticity at GABAergic synapses
have largely focused on the cholecystokinin-expressing (CCK-) interneurons in the hippocampus. These
interneurons constitute a small percentage of GABAergic cells in the cortex 24, calling into question the
significance of eCB signaling at inhibitory synapses in the PFC. Recent work suggests three principal
groups of interneurons in the cortex: cells co-expressing either the calcium (Ca +2) binding protein
parvalbumin (PV), the peptide transmitter somatostatin (SOM), and the serotonin 5HT3a receptor 24. The
PV group accounts for ~40% of GABAergic neurons and includes fast spiking basket cells and chandelier
cells. The SOM group represents ~30% of GABAergic neurons and includes the Martinotti cells. The
5HT3aR group, accounts for ~30% of the total interneuron population and includes the small group of
CCK-positive cells 24. It is possible that inhibition from GABAergic cells other than CCK-interneurons are
under eCB regulation in the PFC.
The rich diversity of GABA-producing cell types mediates widely distributed and precisely
positioned inhibition. The specific subdomain of GABAergic contact (e.g. dendrite, soma or axon) critically
determines the impact of synaptic inhibition in shaping neuronal activity in the neocortex. SOM-INs
primarily target distal dendrites of excitatory neurons 26, where they regulate Ca +2 signaling, synaptic
integration, and dendritic spiking 27 and mediate feedback inhibition, being most excited by local cortical
pyramidal cell. Evidence suggests that SOM-INs may contribute to emotional and cognitive brain
processing. Cortical tissue from schizophrenic patients shows decreased expression of markers for
multiple distinct interneuron populations, including those that co-express SOM 28,29. We hypothesized that
CB1Rs present in SOM-INs may be a target by which eCBs exert control over synaptic function.
Preliminary results in the lab show that synaptic inhibition from SOM-INs in the PFC are modified by eCB
signaling (unpublished). In this project, we will investigate the role of eCB signaling at inhibitory synapses
from this major population of GABAergic interneurons.

Hypothesis

If the eCB system normally regulates efficacy of these inhibitory synapses, removal of the CB1R from
SOM-INs should augment dendritic inhibition, thereby disrupting the fine balance between
excitation/inhibition and modify cortical excitability and plasticity.
General Aim

Determine the role of eCB modulation at specific GABAergic synapses in shaping inhibitory and excitatory
synaptic transmission

Specific Aims

 Determine whether the lack of eCB modulation of dendritic inhibition changes the balance of excitation
and inhibition in the PFC circuit by comparing basal inhibitory and excitatory synaptic transmission
between control and conditional KO littermates. Record spontaneous EPSCs/IPSCs and determine the
amplitude and frequency of this events.

Methods
Slice Preparation
Experiments are performed in acute prefrontal cortical slices (300 μm thick) taken from male and female
mice (P26-P40). For these experiments, we crossed conditional CB1R mice with SOM-Cre mice to
generate transgenic mice lacking CB1R expression in all SOM-interneurons. Controls are littermates in
which Cre is not expressed but both cnr1 alleles that code for C1Rs are floxed. For slice preparation, the
animals are anesthetized with isoflurane, decapitated and their brains rapidly removed and placed in ice-
cold artificial cerebrospinal fluid (ACSF) containing (in mM): 127 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5
KCl, 1 MgCl2, 2 CaCl2 and 20 glucose (pH=7,4) bubbled with 95% O2 and 5% CO2 . Coronal slices are
cut in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 7
MgCl2, 0.5 CaCl2, 20 glucose, 11.6 sodium ascorbate and 3.1 sodium pyruvate, bubbled with 95% O2
and 5% CO2 and transferred to ACSF. After an incubation period of 10 min at 34°C, the slices are
maintained at 20–22 °C for at least 30 min before use .

Electrophysiology and analysis

Experiments are conducted at room temperature (20–22°C), in a submersion-type recording chamber.
Whole-cell patch-clamp recordings are obtained from layer 2/3 pyramidal cells (200-300 μm from the pial
surface) identified with video-infrared/differential interference contrast. For voltage-clamp recordings, cells
are held at -65 mV to isolate EPSCs or +10 mV to isolate IPSCs, and glass electrodes (5.5–6.5 MΩ) are
filled with internal solution containing (in mM): 130 CsGluconate, 10 HEPES, 4 MgCl2, 4 Na2ATP, 0.4
NaGTP and 10 sodium creatine phosphate, adjusted to pH 7.3 with CsOH. Recording are made from
visually identified pyramidal-shaped somata of principal neurons in the PFC. Electrophysiological
recordings are acquired with a Multiclamp 700B amplifier, filtered at 4 kHz, and digitized at 10 kHz. Data
are analyzed in custom-written IgorPro software. Statistical tests will be performed using p <0.05 in
GraphPad Prism.

Results
To determine whether loss of eCB modulation of dendritic inhibition changes the balance of excitation and
inhibition in the PFC circuit, we aimed to compare basal inhibitory and excitatory synaptic transmission
between control and conditional KO littermates by recording spontaneous inhibitory and excitatory
postsynaptic currents in layer 2/3 pyramidal cells of acute brain slices from mice (P25-P30). Preliminary
results suggest a tendency for both the frequency and amplitude of sIPSCs to be decreased in KO mice
for CB1 receptor (fig.1). Unfortunately, we could not detect any sEPSCs in either KO or wt controls. Thus,
we are unable to determine whether excitatory/inhibitory balance is altered (data not shown).
Discussion

Here, in this project, by comparing spontaneous inhibitory postsynaptic responses, we show a trend
towards decreased basal inhibition in the PFC of mice in which CB1Rs have been selectively removed
from dendrite-targeting SOM-INs. This result, at first, seems contrary to our prediction of an enhanced
dendritic inhibition in conditional KOs. However, the sIPSCs we record most likely arise from multiple IN
sources. In fact, the spontaneous events are probably predominated by perisomatic PV-INs which
synapse closer to the soma and are more active. Maybe in this mouse model would be compensatory
changes through development because neuronal expression of CB1Rs at embryonic and early postnatal
stages as well as in adult progenitor cells, implicate a role of these receptors in cell migration and
differentiation. Also, developmental regulation of GABA synapse is affected due to CB1R deletion 35,36.
Another reason could be that homeostatic decrease PV-IN mediated inhibition if SOM-IN inhibition
increases to maintain E/I. To address developmental compensation, we can use viral transfection instead
of crossing mices, using DREADs to stimulate SOM-IN or inhibit PV-IN. Finally, to look at SOM-
INmediated inhibition, we can use optogenetic stimulation of ChR2 previously injected in the mice. For
E/I, we will need to raise temperature and/or lower Mg to get more sEPSC events. Also we will examine
evoked responses.

eCBs have a critical role in regulating neural activity in the brain through the control of both
inhibitory and excitatory neurotransmission. Alterations in the eCB system are observed in several
psychiatric disorders, including emotional processing, locomotion and feeding behaviors. Although CB1R
is expressed widely in the nervous system, the pattern is not uniform. Activation of CB1Rs in interneurons
reduces GABA release and thereby disinhibits pyramidal neurons. SOM-INs that make up ~30% of the
GABAergic interneurons in the cortex through their inhibitory contact on dendritic arbors regulate Ca +2
signaling, synaptic integration, and dendritic excitability. If these cortical GABAergic synapses are under
the control of eCBs, deregulation of this system will have profound consequences for information
processing and cognitive functions.

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