**3. Tools to manipulate neural structures**

Many techniques have been developed to evaluate distinct brain structures. These methods allow for the direct manipulation of a defined brain area to manipulate their activity during behavior. Thus, they require intracranial access typically gained through stereotaxic surgery. Of these methods, the most widely used are lesions and microinjection. However, more modern tools have been developed that harness the capabilities of viral gene transfer to more precisely control cells and circuits. Each of these techniques, classic and contemporary, possesses inherent benefits and drawbacks that are discussed in detail below and summarized in **Table 1**.

#### **3.1. Classical tools**

#### *3.1.1. Lesions*

A classical method used to study brain function involves the removal or destruction of neural tissue. With this method, experimental lesions are made to defined brain structures through manual, chemical, or electrical means and can also be neurotransmitter specific. Popular neurotransmitter-specific lesioning agents include 6-hydroxydopamine (dopaminergic and noradrenergic neurons), 5,7-dihydroxytryptamine (serotonergic neurons), ibotenic acid [Nmethyl-D-aspartate (NMDA) receptor-containing neurons], kainic acid (kainate receptorcontaining neurons), and many others. Behavior is then examined in the absence of this tissue, thus providing insight into the involvement of the lesioned structure. Histology, such as simple cresyl violet stains or for markers of neuronal damage, is performed on neural tissue after behavior to confirm the location of damage. One issue that arises is the propensity for other brain structures to compensate for the damaged region. This may severely compromise the interpretation of results obtained from studies using a lesion procedure. Another issue is the difficulty encountered when using lesions to assess the effects at distinct phases of a behavior, as lesions cause irreversible damage to the region. Thus, with many animal models, it is difficult to determine whether ablation of a structure impacted the development (acquisition), performance (expression), extinction, or reinstatement of behavior. To affect a distinct phase of behavior, lesions can be made at specific times in the model. However, phase-specific lesions can unintentionally alter later behavior and can even impair the memory of earlier phases.


**Table 1.** Comparison of commonly used classical and contemporary tools in behavioral neuroscience.

#### *3.1.2. Intracranial microinjections*

interpretation of results obtained from studies using a lesion procedure. Another issue is the difficulty encountered when using lesions to assess the effects at distinct phases of a behavior, as lesions cause irreversible damage to the region. Thus, with many animal models, it is difficult to determine whether ablation of a structure impacted the development (acquisition), performance (expression), extinction, or reinstatement of behavior. To affect a distinct phase of behavior, lesions can be made at specific times in the model. However, phase-specific lesions can unintentionally alter later behavior and can even impair the memory of earlier phases.

**Lesion Microinjection Tracing Optogenetic Chemogenetic**

**2** Labeled spread; targeted to cells

**1** Single i.c. entry, but tracers may be toxic

**1** Increased with viral tracers and transgenic strains

**2** Requires micro injectors only

**1** Labeling of defined projections

**2** Labeled spread; targeted to cells

**0** Permanent **2** Milliseconds **1** Minutes to hours

**0** Permanent hardware, repeated i.c. access

**2** Using transgenic strains and viral promoters

**0** Cannula and fiber optics of custom length, multichannel light source

**2** With axonal light excitation

> opsins and wavelengths

n/a **2** Depends on

**2** Labeled spread; targeted to cells

> depending on actuator

**2** Single i.c. entry, activation by peripheral injection

**2** Using transgenic strains and viral promoters

**2** Requires microinjectors only

**2** With i.c.

**2** Depends on receptors and actuators

actuator infusion or anterograde/retro grade viruses

**Criteria Classical tools Contemporary tools**

**1** Spread difficult to determine

> hours depending on drug

**0** Permanent hardware, repeated i.c. access

**0** Receptors targeted across cell types

**0** Surgical stainless steel cannula, injectors custom gauge and length

**0** Indirect with disconnection procedure

**2** Inhibition and activation depend

**Table 1.** Comparison of commonly used classical and contemporary tools in behavioral neuroscience.

on drug

**0** Permanent **1** Minutes to

54 Recent Advances in Drug Addiction Research and Clinical Applications

Spatial resolution

Temporal resolution

Non invasive

Cell specificity

Minimal need for specialized equipment

Circuit specificity

Bidirectional Modulation Ability

**1** Depends on lesion type

**1** Single i.c. entry but involves intentional tissue damage

**0** Marginally possible with chemical lesions

**0** Currentgenerating device and electrodes for electrolytic

**0** Indirect with disconnection procedure

0 Inactivation only

0, low; 1, moderate; 2, high; i.c., intracranial.

The local administration of pharmacological agents into discrete brain targets is another strategy to control neural activity. This technique typically requires permanent surgical placement of guide cannula to allow for later access to otherwise inaccessible brain structures while animals are awake and behaving. Small volumes of drug solutions are then administered directly into the brain by threading a smaller gauge injector through the guide cannula. These solutions typically contain drugs that bind to distinct membrane proteins (receptors) expressed within the target brain region to enhance or inhibit local cellular activity during behavior. Similar to lesions, histology is performed afterwards to verify the site of microinjection. This procedure has several major advantages compared to lesions, most of which relate to its ability to produce more temporally specific effects. Unlike lesions, the effects of most pharmacological antagonists and agonists are temporary and can therefore be more precisely controlled and administered during distinct phases of behavioral procedures. This allows for more straight‐ forward interpretation of the effects of this manipulation on behavior. Additionally, this technique can provide insight into the neurochemical signals involved, as agents selective for distinct receptor types can be infused. To a lesser extent than lesions, microinjections also produce damage resulting in reactive gliosis from cannula installation and injector placement. Finally, it is difficult to ascertain the exact extent of diffusion of the administered solution. As diffusion may depend on a variety of factors, such as the volume injected and the nature of the solution (polarity, hydrophobicity), it is difficult to predict. Thus, it is not always clear that the site of infusion is the region directing the observed behavior. For this reason, it is often necessary to include additional groups that receive drug injections in locations proximal to the target structure.

#### **3.2. Contemporary tools**

In recent years, there has been a rapid emergence of novel tools engineered to control neuronal activity. Of benefit to these tools have been the advancements in recombinant viruses that are capable of gene transfer in the central nervous system (CNS). For example, viruses with low immunogenicity and cytotoxicity such as adeno-associated virus (AAV) can be delivered directly into the brain to safely and efficiently express recombinant genes [38]. This provides a means to site-specifically express proteins in the CNS that can be used to modulate the activity of cells in target brain tissue. Optogenetics and chemogenetics represent the two most widely used contemporary tools in behavioral neuroscience, as they can be applied in vivo to modulate neural activity in awake behaving mice and rats. As with the classical tools described above, these modern methods have inherent advantages and disadvantages (**Table 1**), which are detailed below.

#### *3.2.1. Optogenetics*

In this technique, neurons are genetically modified through intracranial injection of a viral vector to express photosensitive proteins. The most commonly used photosensitive receptors are channelrhodopsin (ChR; excitatory ion channel), halorhodopsin (NpHR; inhibitory ion pump), and archaerhodopsin (ArchT; inhibitory proton pump; reviewed in [39]). These lightgated proteins are activated by targeted illumination, causing rapid (millisecond timescale) depolarization or hyperpolarization of neurons (reviewed in [40]). By evoking or inhibiting spike activity with this light-protein interaction, the activity of distinct brain regions and cell types can be experimentally controlled, including during the performance of behavioral tasks [39]. Because these engineered opsins can be controlled by different light wavelengths, neural activity and behavior can be modulated bidirectionally (i.e., multiplexed), offering a major advantage to this technique. Moreover, the high temporal resolution afforded by this tool makes it ideal to examine the discrete phases of behavior. However, a major issue posed by optogenetics is the possibility of desensitization of the opsin, which can occur within seconds of photoactivation [41]. Thus, this is especially problematic for studies that require inhibition or activation of longer durations, such as is required in certain behavioral tasks that occur on the order of minutes. Repeated stimulation leading to the desensitization of the opsin may even produce opposing results. This is especially problematic in the case of the excitatory opsin ChR, as the desensitization of this receptor and repeated stimulation of the cell may result in a net inhibition, the opposite effect of what is initially intended. The extent of viral diffusion and resulting protein expression is easily measurable with this technique, as most viral constructs contain a fluorescent tag. However, similar to microinjections, implantable hardware is necessary to allow for intracranial insertion of fiber-optic probes. This technique also requires specialized equipment such as fiber-optic probes and programmable light sources, which can be costly. Tethering the animal to the external light source is also necessary, which may restrict the range of apparatuses that can be used and behaviors that can be assessed (although for recent developments in wireless technologies; see [42–46]). Recently, questions regarding the effect of illumination in brain structures have arisen, specifically in regards its thermal effects on neural tissue. It has been suggested that focal illumination, especially when intense and prolonged, can result in phototoxicity, heat-induced cell damage, and oxidative stress that independently alters cellular activity [47]. Even more problematic is evidence indicating that heat alone can increase neuronal firing rates [48]. In fact, even at commonly used intensities, the thermal effect of illumination is sufficient to increase cell firing rates [49]. Overall, optogenetics provides a unique tool to control neuronal activity with high spatiotemporal resolution. However, the required implantable hardware, specialized equipment, tethering, risk of desensitization, and light induction may render this tool less than ideal given the experimental question and design.

#### *3.2.2. Chemogenetics*

This relatively new technique involves the engineering of G protein-coupled receptors (GPCRs) to interact exclusively with small molecules that were otherwise unrecognized by the GPCR [50]. The most common of these mutated GPCRs are designer receptors exclusively activated by designer drugs (DREADDs) [51]. The engineered GPCRs possess no detectible constitutive activity and are robustly activated at nanomolar concentrations of otherwise pharmacologically inert compounds. The first established DREADDs were based on excitatory Gq-coupled and inhibitory Gi -coupled human muscarinic receptors M3 (hM3Dq) and M4 (hM4Di), respectively [51–53]. Receptors hM3Dq and hM4Di possess no affinity for the endogenous ligand acetylcholine and are robustly activated by the drug clozapine-N-oxide (CNO), a pharmacologically inert metabolite of clozapine, which is highly bioavailable and produces no pharmacological effect in rodents [51, 54]. Since their inception, other DREADDs have been engineered, which include a Gs-coupled muscarinic-based (rM3Ds) receptor and Gi -coupled KOR-based DREADD (KORD) [55–57]. Notably, the development of KORD with actuator salvinorin B allows for the bidirectional control of behavior when used in combination with hM3Dq receptors and CNO [57].

Comparable to optogenetics, DREADDs can be ectopically expressed in the CNS by focal infusion of a vector encoding for these receptors. A major advantage of chemogenetics over optogenetics, however, is the lack of required specialized equipment and need for permanently implanted hardware. In fact, DREADDs require just one initial intracranial entry to infuse the viral vector carrying the DREADD-encoding gene. The receptors can then be activated by a relatively noninvasive peripheral injection of an actuator. This is highly advantageous when performing sensitive behavioral procedures that are affected by excessive handling (e.g., CPP and self-administration) [58]. Similar to optogenetics, a more precise detection of viral spread and DREADD expression are possible with this technique, as they are designed to encode for DREADDs as well as a fluorescent marker.

Unlike optogenetics, DREADDs signal through canonical G-protein pathways. Once activated, the duration of the inhibition or activation produced by the DREADD can be long lasting. The duration of effect is also determined by the half-life of actuators, which may remain in central tissue and activate DREADDs for minutes to hours. In some cases, this low temporal resolution may serve as a major shortcoming of this technique. However, a protracted effect is often highly valued in studies where behavioral tasks are of longer duration and long-lasting effects of manipulations are desired. Another issue is that the presence of the receptor does not always indicate that it is a functionality. Additional measures may be necessary to demonstrate the function of these receptors in target tissue. Although it is theoretically possible, no studies have reported DREADD desensitization. However, this presents another reason for including some form of functional confirmation of DREADD effects.

In summary, DREADDs are a useful technique to control neuronal signaling in vivo. Consid‐ ering the sensitivity and duration of many behavioral tasks, the noninvasive nature of DREADD activation (i.e., peripheral drug injection) and longer time course of inhibition/ activation make chemogenetic strategies highly desirable in behavioral research.

#### **3.3. Summary**

gated proteins are activated by targeted illumination, causing rapid (millisecond timescale) depolarization or hyperpolarization of neurons (reviewed in [40]). By evoking or inhibiting spike activity with this light-protein interaction, the activity of distinct brain regions and cell types can be experimentally controlled, including during the performance of behavioral tasks [39]. Because these engineered opsins can be controlled by different light wavelengths, neural activity and behavior can be modulated bidirectionally (i.e., multiplexed), offering a major advantage to this technique. Moreover, the high temporal resolution afforded by this tool makes it ideal to examine the discrete phases of behavior. However, a major issue posed by optogenetics is the possibility of desensitization of the opsin, which can occur within seconds of photoactivation [41]. Thus, this is especially problematic for studies that require inhibition or activation of longer durations, such as is required in certain behavioral tasks that occur on the order of minutes. Repeated stimulation leading to the desensitization of the opsin may even produce opposing results. This is especially problematic in the case of the excitatory opsin ChR, as the desensitization of this receptor and repeated stimulation of the cell may result in a net inhibition, the opposite effect of what is initially intended. The extent of viral diffusion and resulting protein expression is easily measurable with this technique, as most viral constructs contain a fluorescent tag. However, similar to microinjections, implantable hardware is necessary to allow for intracranial insertion of fiber-optic probes. This technique also requires specialized equipment such as fiber-optic probes and programmable light sources, which can be costly. Tethering the animal to the external light source is also necessary, which may restrict the range of apparatuses that can be used and behaviors that can be assessed (although for recent developments in wireless technologies; see [42–46]). Recently, questions regarding the effect of illumination in brain structures have arisen, specifically in regards its thermal effects on neural tissue. It has been suggested that focal illumination, especially when intense and prolonged, can result in phototoxicity, heat-induced cell damage, and oxidative stress that independently alters cellular activity [47]. Even more problematic is evidence indicating that heat alone can increase neuronal firing rates [48]. In fact, even at commonly used intensities, the thermal effect of illumination is sufficient to increase cell firing rates [49]. Overall, optogenetics provides a unique tool to control neuronal activity with high spatiotemporal resolution. However, the required implantable hardware, specialized equipment, tethering, risk of desensitization, and light induction may render this tool less than

This relatively new technique involves the engineering of G protein-coupled receptors (GPCRs) to interact exclusively with small molecules that were otherwise unrecognized by the GPCR [50]. The most common of these mutated GPCRs are designer receptors exclusively activated by designer drugs (DREADDs) [51]. The engineered GPCRs possess no detectible constitutive activity and are robustly activated at nanomolar concentrations of otherwise pharmacologically inert compounds. The first established DREADDs were based on excitatory

(hM4Di), respectively [51–53]. Receptors hM3Dq and hM4Di possess no affinity for the endogenous ligand acetylcholine and are robustly activated by the drug clozapine-N-oxide


ideal given the experimental question and design.

56 Recent Advances in Drug Addiction Research and Clinical Applications

*3.2.2. Chemogenetics*

Gq-coupled and inhibitory Gi

The above-described tools provide a means through which to target and manipulate brain regions. These tools offer variable degrees of selectivity, with contemporary techniques typically being associated with higher precision in terms of spatial and neuronal targeting. The tools mentioned above do not encapsulate all available methods of discovering the neurobi‐ ology behind behaviors. Other commonly used methods not described in this chapter are intracranial electrical or self-stimulation, intracranial microdialysis, electrophysiology, immunohistochemistry (IHC), genetic knockout rodents, and many others that similarly assess the importance of a brain region and specific neurotransmitter systems to behavior.
