1. Project Title: Rewarding effects of cholinergic antagonists in the rostromedial tegmental
nucleus of mice
Abstract: Drugs of abuse share the ability to increase forebrain dopamine (DA) levels and their
ability to do so critically contributes to their habit-forming effects. Most drugs activate the
dopamine system by acting through modulatory inputs to dopamine cell bodies in the ventral
tegmental area (VTA). A better understanding of the source and nature of modulatory inputs to
the VTA is needed. The laterodorsal tegmental nucleus (LDTg) and pedunculopontine tegmental
nucleus (PPTg) provide direct excitatory cholinergic input to VTA dopamine neurons that
contributes to drug reward. They also provide input to the rostromedial tegemental nucleus
(RMTg) which itself is a critical modulatory input important for drug reward. The experiments
outlined here will test the motivational effects of blocking cholinergic inputs to the RMTg,
which has previously be shown to activate brain dopamine signaling. The results will test
whether cholinergic control of RMTg inputs to the VTA contribute to appetitively motivated
behaviors. The results are expected to guide future development of treatment targets for drug
addiction.

2. Project Description: Drugs of abuse share in common the ability to increase levels of the
neurotransmitter dopamine (DA) in the forebrain. In particular drug-induced dopamine increases
in forebrain areas such as the nucleus accumbens are thought to be critically related to the habit-
forming effects of drugs and thus the development of addiction (Robinson,
2011). Pharmacotherapies that directly target the dopamine system have met with limited
clinical efficacy. The ventral tegmental area (VTA) in the midbrain where the cell bodies of
dopamine cells are found receives input from many different brain areas and many of these are
able to either increase or decrease forebrain DA levels (Watabe-Uchida et al., 2012). Several of
these modulatory sources of input contribute the rewarding effects of drugs of abuse and
targeting modulatory inputs to the dopamine system may provide an alternative strategy in the
development of treatment targets. Inputs to the VTA that utilize the neurotransmitters
acetylcholine (ACh) and gamma-aminobutyric acid (GABA), for example, each contribute to the
ability of opiates drugs (e.g. morphine) to activate brain dopamine systems (Johnson & North,
1992; Steidl & Yeomans, 2009; Steidl et al., 2011). The only known sources of acetylcholine
input to VTA dopamine neurons originate from the pedunculopontine tegmental nucleus (PPTg)
and laterodorsal tegmental nucleus (LDTg) (Oakman et al., 1995). These cholinergic inputs are
known to excite VTA dopamine neurons (Forster & Baha, 2000). A major source of GABA
input to the VTA comes from the rostromedial tegmental nucleus (RMTg), a recently identified
region very near to the VTA with distinct anatomical, physical, and behavioral properties (Jhou
et al., 2012). The RMTg provides inhibitory input to VTA DA neurons (Jhou et al. 2012) and
has been shown to have an important role in both positively and negatively motivated behaviors
(Hong et al. 2011). LDTg and PPTg send projections to the RMTg in parallel to those they send
to the VTA, suggesting the PPTg and/or LDTg may be in a position to indirectly control VTA
dopamine neuron activity (via a relay in the RMTg). In support of this Steidl’s lab has found
that blocking cholinergic input to the RMTg increases the ability of opiates to increase brain
dopamine activity in mice. Blocking cholinergic input to the RMTg is thus expected to be
rewarding and the experiments outlined here will test this hypothesis. We will use the
conditioned place preference (CPP) paradigm to test whether blockade of cholinergic input to the
RMTg is rewarding. CPP is a well established model that tests primary drug reward. In this
paradigm mice repeatedly experience the subjective effects of a drug treatment, e.g. RMTg
cholinergic receptor blockade, in one distinct environmental context. Mice are then given a
choice between this environment and an alternate (non-drug paired) environment. A greater
amount of time spent in the drug-paired environment indicates a conditioned place preference
and is operationally defined as an index of the rewarding efficacy of previous drug treatment.
Cholinergic receptors have two subcategories, nicotinic and muscarinic receptors. We will test
the effects of selectively blocking either muscarinic or nicotinic receptors in the RMTg.
Experiment:
Hypothesis: Blocking muscarinic cholinergic receptors in the RMTg will result in development
of a conditioned place preference.
Rationale: The RMTg is known to inhibit VTA dopamine neurons via its GABAergic
projections. The RMTg receives cholinergic input from the PPTg and LDTg. Blocking RMTg
muscarinic cholinergic receptors increases brain dopaminergic activity. The experiments
proposed here will determine whether the effects of muscarinic receptor blockade in RMTg are
motivationally significant (i.e. whether they are rewarding). The effects of RMTg nicotinic
cholinergic receptor blockade, the other main type of acetylcholine receptor, need to be tested in
parallel.

3. Methods: This experiments will use a conditioned place preference (CPP) paradigm. The CPP
apparatus consists of two distinct conditioning chambers that differ in several features. One is
black and has a wire grid floor, the other has a smooth floor and has black and white stripes. A
third neutral chamber connects the two conditioning chambers. In 8 mice we will test the
rewarding effects of RMTg atropine (a muscarinic antagonist) microinjection and in 8 mice we
will test the effects of RMTg mecamylamine (a nicotinic antagonist) microinjection. Each
mouse will be deeply anesthetized and implanted with guide cannulae (stainless steel tubes)
aimed at the RMTg. Cannulae are fixed to the mouse skull using stainless steel screws and
dental cement. A small volume of a high concentration drug solution can subsequently be locally
infused into the RMTg via these cannulae. Mice will be given seven to ten days for surgical
recovery before behavioral testing begins. CPP procedure: On day 1each mouse is allowed free
access to the entire CPP apparatus for 20 minutes. The amount of time spent in each chamber
will be measured to determine baseline chamber preferences. Days 2,4,6,8: The mouse is
confined to one of the two chambers for 20 minutes immediately after receiving an RMTg
injection of either atropine or mecamylamine. Days 3,5,7,9: The mouse will be confined to the
alternate chamber for 20 minutes immediately after receiving an RMTg injection of saline.
Chamber assignment and treatment order will be counterbalanced across mice. On day 10 the
mouse is again given free access to the entire apparatus and the amount of time spent in each
chamber is measured. An increase in the amount of time spent in the previously drug-paired
chamber relative to baseline indicates a conditioned place preference. Below is an expected
timeline for the experiment, taking place from May to August. This will be repeated for each
group: Surgery  Recovery (7-10 d)  Behavioral testing (10 d)  Histology and data analysis
If drug treatment is rewarding, the mouse should spend more time in the drug-paired chamber.
This will show that RMTg cholinergic receptor blockade is rewarding suggesting that cholinergic
inputs to the RMTg are part of a brain circuit that contributes to appetitively motivated
behaviors.
Histology: After the ten day behavioral training component mice will be transcardially perfused
using standard techniques. Mouse brains will be sectioned using a cryostat. The tissue will be
processed with cresyl violet dye to highlight cell bodies of interest and compare them to a brain
atlas. We will verify that injection sites were confined to the RMTg.
Student-Mentor Responsibilities:
The student will be responsible for training and handling the mice in the PPC chamber and
recording data for each session. The student will prepare the pharmacological injection solutions
and perform the injections for each group. The student will perform surgery to implant the
cannulae and transcardially perfuse the mice. The student will be responsible for harvesting
brain tissue, collecting samples from the tissue, and staining the tissue. The student will analyze
data after experiment completion.
The mentor will provide the student with necessary training pertaining to safety protocols and
surgery, data acquisition, pharmacological preparation, perfusions, brain harvesting, tissue
sampling, and tissue staining, along with overseeing the student’s progress and ensuring the
student’s safety. The mentor will be available at all times for discussing progress throughout the
project and aiding the student in any part of the experiment when necessary. Weekly meetings
will be held to ensure the research is moving in the right direction and to recognize and work
through any obstacles that may be encountered throughout the study. Meetings will continue
after the experiment to discuss the data, what was learned from the data, and how the data can be
used for the future (e.g. a poster presentation).

4. Budget:
Four microinjection syringes are needed to make intracranial injections (as four mice can be
tested simultaneously) and will cost $214.28. Guide cannulae to be implanted and dummy
cannulae to protect the guides, totaling $224.56. 20 injector cannulae (reusable) required to
perform injections, $151.20. The amounts of atropine and mecamylamine required will total to
$52.36. $357.60 will provide partial cost recovery for the mice needed. Grand total: $1000.00
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