**4. Tools to manipulate specific neural pathways**

Historically, the direct manipulation of neural circuits has been a challenging task, with much of the difficulty due to limited methodologies. In the past, many of the tools used possessed relatively low selectivity and provided indirect manipulation. Several strategies, however, have been designed using both classical and contemporary tools to probe the neural circuitry underlying behavior. This section describes several commonly used strategies and includes a discussion of their merits and weaknesses.

#### **4.1. Classical tools**

Historically, disconnection procedures involving lesions and pharmacological microinjections have been used to evaluate neural circuitry. This strategy involves the disruption of two directly connected brain regions to assess whether their interaction is involved in behavior (e.g., [59–62]). Typically, a unilateral lesion or inactivating microinjection [e.g., γ-aminobutyric acid (GABA) agonists or channel blocker] is made in source regions and another lesion or microinjection is made in the contralateral hemisphere of its terminal target. Thus, if a behavior is dependent on a source-target interchange, then their contralateral disconnection should be more disruptive to behavior than ipsilateral disconnection or unilateral manipulation of each region individually. However, a major weakness of this strategy is the indirect nature of the manipulation on the circuit. Indeed, the imprecision of this method has at times been proven problematic resulting in significant reductions in behavior with ipsilateral and unilateral manipulations alone (e.g., [60]). This is likely due to the inability of this technique to directly target distinct yet intermixed populations of target-projecting neurons within source regions. Instead, each region is broadly manipulated leading to the inhibition of their overall activity and output throughout the brain.

To help visualize and identify the circuit, neuronal tracing has sometimes been used in conjunction with these classical tools. In these studies, tracing agents are injected into the brain to label neurons in a manner that is retrograde (axon terminal back to the soma), anterograde (soma to axon terminal), or transsynaptic (to adjacent neurons retrogradely or anterogradely). In studies of behavior, circuit involvement is inferred by colabeling of neuronal activity markers such as c-Fos with the tracer through IHC (e.g., [63]). Cells that are immunopositive for both the tracer and activity marker are then used to identify afferent or efferent projection neurons that were activated during behavior. When used in tandem with disconnection procedures, this provides a means to visually assess the impact of disconnection on circuit activity (e.g., [62]). Although these procedures help to label neurons within the circuit and gauge their activity, they still fall short of allowing isolated modulation of the circuit. Overall, this remains a major weakness of classical techniques, as results only offer an indirect measure of neural circuit involvement in behavior.

#### **4.2. Contemporary tools**

Optogenetic and chemogenetic strategies have provided a refined and more selective means to directly manipulate neural circuits. This is principally due to viral transduction, as proteins (opsins and DREADDs) are trafficked downstream from soma to axon terminals (anterograde) and therefore expressed on presynaptic boutons as well as to cell bodies [64]. In the case of optogenetics, illumination can then be targeted to axon terminals, which results in the depolarization or hyperpolarization of the neuron. This strategy has been successfully implemented in many behavior studies (reviewed in [64]). However, a concern that arises when using this method is the possibility of antidromic stimulation of the cell. The stimulation of terminals may result in the back-propagation of an action potential that activates the neuron and its collateral inputs to other regions outside the circuit of interest. Therefore, with this strategy, there is a potential for the activation of multiple circuits, which diminishes the selectivity of the manipulation.

**4. Tools to manipulate specific neural pathways**

58 Recent Advances in Drug Addiction Research and Clinical Applications

discussion of their merits and weaknesses.

and output throughout the brain.

of neural circuit involvement in behavior.

**4.2. Contemporary tools**

**4.1. Classical tools**

Historically, the direct manipulation of neural circuits has been a challenging task, with much of the difficulty due to limited methodologies. In the past, many of the tools used possessed relatively low selectivity and provided indirect manipulation. Several strategies, however, have been designed using both classical and contemporary tools to probe the neural circuitry underlying behavior. This section describes several commonly used strategies and includes a

Historically, disconnection procedures involving lesions and pharmacological microinjections have been used to evaluate neural circuitry. This strategy involves the disruption of two directly connected brain regions to assess whether their interaction is involved in behavior (e.g., [59–62]). Typically, a unilateral lesion or inactivating microinjection [e.g., γ-aminobutyric acid (GABA) agonists or channel blocker] is made in source regions and another lesion or microinjection is made in the contralateral hemisphere of its terminal target. Thus, if a behavior is dependent on a source-target interchange, then their contralateral disconnection should be more disruptive to behavior than ipsilateral disconnection or unilateral manipulation of each region individually. However, a major weakness of this strategy is the indirect nature of the manipulation on the circuit. Indeed, the imprecision of this method has at times been proven problematic resulting in significant reductions in behavior with ipsilateral and unilateral manipulations alone (e.g., [60]). This is likely due to the inability of this technique to directly target distinct yet intermixed populations of target-projecting neurons within source regions. Instead, each region is broadly manipulated leading to the inhibition of their overall activity

To help visualize and identify the circuit, neuronal tracing has sometimes been used in conjunction with these classical tools. In these studies, tracing agents are injected into the brain to label neurons in a manner that is retrograde (axon terminal back to the soma), anterograde (soma to axon terminal), or transsynaptic (to adjacent neurons retrogradely or anterogradely). In studies of behavior, circuit involvement is inferred by colabeling of neuronal activity markers such as c-Fos with the tracer through IHC (e.g., [63]). Cells that are immunopositive for both the tracer and activity marker are then used to identify afferent or efferent projection neurons that were activated during behavior. When used in tandem with disconnection procedures, this provides a means to visually assess the impact of disconnection on circuit activity (e.g., [62]). Although these procedures help to label neurons within the circuit and gauge their activity, they still fall short of allowing isolated modulation of the circuit. Overall, this remains a major weakness of classical techniques, as results only offer an indirect measure

Optogenetic and chemogenetic strategies have provided a refined and more selective means to directly manipulate neural circuits. This is principally due to viral transduction, as proteins

Several chemogenetic-based strategies have been used to modulate neural circuit activity. First, a functional disconnection procedure methodologically similar to that used with lesions or microinjections has been reported by Mahler et al. [65]. In this study, hM4Di receptors were unilaterally expressed in ventral tegmental area (VTA) dopamine (DA) cells and contralater‐ ally in rostral ventral pallidum (RVP) cells. The contralateral disconnection of RVP-VTA DA reduced cued reinstatement of cocaine seeking relative to both unilateral RVP inhibition and unilateral VTA DA inhibition. Despite these positive results, it is unclear whether the simul‐ taneous inhibition of RVP and VTA DA, regardless of hemisphere, would have been sufficient to produce a similar effect. Thus, with this technique, it is important to include ipsilateral as well as unilateral controls. In summary, the lack of precision and necessary inclusion of numerous controls renders this a less desirable strategy for the targeted modulation of neural circuits.

Another DREADD-based method that has been used involves the intracranial injection of CNO. The principle behind this strategy is similar to that of the optogenetic circuit-selective method outlined above. Here, like illumination, CNO is targeted to the terminal region of DREADD-expressing cells [65, 66]. The focal infusion of CNO therefore serves to activate/ inhibit DREADD-expressing cells and/or presynaptic neurotransmitter release from DREADD-expressing nerve terminals [66]. In this manner, the activity of defined neural circuits can be more precisely controlled. However, given that this strategy requires intracra‐ nial microinjections, it also carries with it the disadvantage of requiring permanently indwel‐ ling hardware (i.e., guide cannula and obturators), repeated intracranial entries, and a resulting increased risk of tissue damage.

An alternative approach to this involves the intersection of multiple viruses that are injected into serially connected nuclei. Typically, a retrograde virus encoding for cre recombinase is injected in a target region and a cre-inducible virus encoding for DREADD is injected into the source region. In this manner, the activity of a specific source's inputs to the target region (i.e., projection neurons) can be controlled by the systemic injection of CNO. This approach has been successfully implemented using canine adenovirus (CAV-2) to retrogradely infect source region cells and selectively express DREADDs in a specific neural projection [67–69]. Notably, this strategy provides an ideal way to control circuits, especially during behavior. Not only does this method provide a high degree of selectivity, it does not require implantable hardware and can be robustly activated by a simple peripheral injection of an actuator. Theoretically, it is possible for axon collaterals from source region cells to express DREADDs, which given the systemic nature of DREADD activation may lead to activity in sites outside the circuit. Hence, studies using this method should be careful to address this potential caveat.

#### **4.3. Summary**

Overall, several techniques involving classical and contemporary tools have been used to manipulate defined neural circuits. On one hand, classical tools provide indirect manipulation and generally require the use of multiple controls to carefully and appropriately interpret results derived using these strategies. Conversely, contemporary tools involving viralmediated gene transfer confer greater circuit selectivity and have been successfully used to manipulate serial projections form source to target region.
