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Contents by Research Area: • Transmission • Transmission • Transmission • Transmission • Chemogenetics Tocris Product Guide Series Neurotransmission Research

Contents Page Principles of Neurotransmission 3 Dopaminergic Transmission 5 Glutamatergic Transmission 6 Opioid Peptide Transmission 8 Serotonergic Transmission 10 Chemogenetics in Neurotransmission Research 12 14 Addiction 18 20 List of Acronyms 22 Neurotransmission Research Products 23 Featured Publications and Further Reading 34

Introduction Neurotransmission, or synaptic transmission, refers to the passage of signals from one to another, allowing the spread of information via the propagation of action potentials. This process is the basis of communication between within, and between, the peripheral and central nervous systems, and is vital for memory and cognition, muscle contraction and co-ordination of organ function. The following guide outlines the principles of dopaminergic, opioid, glutamatergic and serotonergic transmission, as well as providing a brief outline of how neurotransmission can be investigated in a range of neurological disorders. Included in this guide are key products for the study of neurotransmission, targeting different systems. The use of small molecules to interrogate neuronal circuits has led to a better understanding of the under- lying mechanisms of disease states associated with neurotransmission, and has highlighted new avenues for treat- ment. Tocris provides an innovative range of high performance life science reagents for use in neurotransmission research, equipping researchers with the latest tools to investigate neuronal network signaling in health and disease. A selection of relevant products can be found on pages 23-33.

Key Neurotransmission Research Products

Box Number Title Page Box Number Title Page Box 1 Dopaminergic Transmission 5 Box 6 15 Box 2 Glutamatergic Transmission 7 Box 7 and its Metabolites 17 Box 3 Opioid Transmission 9 Box 8 Addiction 19 Box 4 Serotonergic Transmission 11 Box 9 Epilepsy 20 Box 5 Chemogenetic Compounds: DREADD ligands 13 and PSEMs

2 | NEUROTRANSMISSION RESEARCH Principles of Neurotransmission

The majority of neurotransmission occurs across chemical syn- cross the synaptic cleft and bind to their spe- apses, where an endogenous neurotransmitter is released by cific receptors. These maybe -gated ion channels (LGICs) the presynaptic neuron and detected by receptors on the post- or G protein-coupled receptors (GPCRs), with some neuro- synaptic neuron (Figure 1). Neurotransmitters can be broadly transmitters having receptors in both categories. Binding of split into three categories; amino acids including glutamate and a neurotransmitter to a LGIC causes a , amines including (DA), (5-HT) in the structure of the protein, allowing the passage of ions and (NE), and such as dynorphin, the through the channel. Passage of ions through channels that are enkephalins and Y. selective for positively-charged cations results in depolariza- tion of the postsynaptic membrane and initiation of an action While the amino acids glutamate and glycine are found in all potential in the postsynaptic neuron. In contrast, passage of cells of the body, other neurotransmitters are only synthe- ions through negatively-charged, anion selective channels sized by neurons. Following synthesis, neurotransmitters are results in hyperpolarization of the postsynaptic membrane, taken up and stored in synaptic vesicles, ready for release. The so inhibiting action potential initiation. Binding of a neuro- release of a neurotransmitter is triggered by the arrival of action transmitter to a GPCR results in the activation of G proteins, potentials in the axon terminal of the presynaptic neuron, open- which are then able to act on to modulate intracellular ing voltage-gated Ca2+ channels and allowing influx of ions. signaling pathways. The end result of this is the modulation of The resulting elevation in intracellular Ca2+ concentration activity of other proteins, including ion channels and enzymes. causes synaptic vesicles to merge with the presynaptic mem- brane, releasing the neurotransmitter into the synaptic cleft by Once a neurotransmitter has bound to its , it is cleared exocytosis. from the synaptic cleft to allow another wave of synaptic

Figure 1 | Principles of Neurotransmission

Presynaptic neuron 5-HT1,5 Postsynaptic neuron DAT D2,3,4 Opioid receptors G Group II and III mGluRs i/0 SERT (–)

AC ATP 5-HT Glu 4,6,7 D Opioid 1.5 G (–) proteins s cAMP PDE Precursor proteins PKA Ca2+ DA 5-HT2 Group I mGluRs IP3 CaMK Gq/11 5-HT PLC DAG PKC (–) Mg2+ CaMK Glu NMDARs Ca2+ Increased 5-HT neuronal 3 excitability

Na+ AMPARs and Kainate receptors

This simplified schematic shows the main events during dopaminergic, glutamatergic, opioid peptide and serotonergic neurotransmission. DA and 5-HT are both biogenic amines that are derived from amino acids, while glutamate itself is an and opioid peptides are cleaved from precursor proteins. All neurotransmitters undergo exocytosis from the presynaptic membrane and cross the synaptic cleft where they bind to their specific receptors. These receptors may be ligand gated ion channels, such as ionotropic glutamate receptors, or G protein-coupled receptors, such as all subtypes of opioid receptor. Passage of ions through a ligand gated alters the excitability of a neuron. The action of neurotransmitters at GPCRs alters intracellular signaling pathways, with the specific pathway being dependent on the G protein-coupled to the receptor.

www.tocris.com | 3 Tocris Product Guide Series transmission. Neurotransmitter molecules are taken up by the concentration gradient. This depolarizes the membrane presynaptic neuron, or by other cell types such as astrocytes, past the threshold for action potential initiation. As Nav chan- via specific transporters. Neurotransmitters may then nels become inactivated, preventing the flow of Na+, voltage- be metabolized to be reused for further production, or they can gated potassium channels (Kv channels) open allowing the be recycled into synaptic vesicles. efflux of+ K . This causes repolarization of the , as the balance of ion movement across the membrane leads to The strength of the synaptic connection between two neurons the cytosol being more negatively charged than extracellular depends on a range of factors. These include the number of fluid. When K channels are open the cell membrane is highly individual between two neurons, the probability of v permeable to K+, but permeability to Na+ is low as Na channels neurotransmitter release at the presynaptic membrane and the v are still inactivated. This leads to a period of hyperpolarization size of the post-synaptic potential induced by binding of the until K channel close, and the resting membrane potential is neurotransmitter to its receptor. The presence of neurotrans- v re-established. mitter receptors on the pre-synaptic membrane also regulates the release of neurotransmitters, through both positive and Propagation of an action potential occurs as a wave of depolar­ negative feedback loops. Synaptic connection strength is a key ization that spreads along an axon. When an area of the mem- factor in cognitive processes including memory formation. brane becomes depolarized, it opens neighboring Nav channels, which then allow depolarization of that section of the mem- Action Potentials brane. The inactivation of Nav channels in the preceding sec- An action potential is the signal that conveys information along tion of membrane ensures that an action potential travels in a neuron and is also the trigger for release of a neurotrans- only one direction alone an axon. Some axons in the central mitter at a . Physically, an action potential is the rapid nervous system have a sheath around them, composed of mye- reversal of the resting membrane potential, caused by opening lin, which acts as an electrical insulator. Nav and Kv channels and closing of voltage-gated ion channels. At rest, the cytosol are localized on gaps in the myelin sheath, known as nodes of of a neuron is negatively charged (polarized) with respect to Ranvier. Myelination increases that speed of action potential extracellular fluid, due to the distribution of ions across the propagation as the action potential effectively ‘hops’ along an cell membrane. axon, occurring only at nodes of Ranvier in a process known as saltatory conduction. An action potential is initiated by the opening of voltage-gated + + Na channels (Nav channels) allowing influx of Na down its

Figure 2 | DDC in Human Brain Figure 3 | D1R in Human Brain Figure 4 | DAT1 in Human Brain

Detected in immersion-fixed paraffin- D1R detected in immersion-fixed paraffin- DAT1 detected in immersion-fixed sections embedded sections of human brain embedded sections of human brain (caudate of human brain () using (substantia nigra) using a Goat Anti-Human/ nucleus) using a Mouse Anti-Human a Mouse Anti-Human/Mouse/Rat DAT1

Mouse/Rat DDC Antigen Affinity-Purified Dopamine D1R Monoclonal Antibody (R&D Monoclonal Antibody (Novus Biologicals, Polyclonal Antibody (R&D Systems, Cat. No. Systems, Cat. No. MAB8276). The tissue Cat. No. NBP2-46649). The tissue was AF3564). The tissue was stained using the was stained using the Anti-Mouse HRP-DAB stained using HRP and DAB (brown) and Anti-Goat HRP-DAB Cell & Tissue Staining Cell & Tissue Staining Kit (R&D Systems, counterstained with hematoxylin (blue. Kit (R&D Systems, Cat. No. CTS008; brown) Cat. No. CTS002; brown) and counterstained Tocris, Cat. No. 5222). Specific staining and counterstained with hematoxylin (blue. with hematoxylin (blue. Tocris, Cat. No. was localized to the dopamine neuron Tocris, Cat. No. 5222). Specific staining was 5222). Specific staining was localized to the nuclei and fibres. localized to the neuronal . neuronal cytoplasm.

4 | NEUROTRANSMISSION RESEARCH Dopaminergic Transmission

neurotransmitters, including norepinephrine and epinephrine, Products by Category Page and its activity is a rate-limiting step in catecholamine synthe- sis. Following synthesis, DA is released at the synaptic cleft and ...... 25 binds to its receptors on the postsynaptic membrane. cAMP...... 26 Catechol O-Methyltransferase...... 27 All DA receptors are GPCRs and they can be split into two Decarboxylases...... 27 families based on their structure, function and pharmaco-

Dopamine D1-like Receptors...... 27 logical properties: D1-like receptors and D2-like receptors. The Dopamine D2-like Receptors...... 28 D1-like receptors, D1R (Figure 3) and D5R, are coupled to the Dopamine Receptors: Non-selective Compounds...... 28 G protein Gs and activate adenylyl cyclase to increase the intra- Dopamine Transporters...... 28 cellular concentration of the second messenger cyclic

Monoamine Oxidase...... 31 monophosphate (cAMP). The 2D -like receptors, D2R, D3R and Vesicular Monoamine Transporters...... 33 D4R, are coupled to the Gi/o pathway, which directly inhibits the formation of cAMP by inhibiting adenylyl cyclase. DA is the major catecholamine neurotransmitter in the mam- The actions of DA and other catecholamines in the synaptic malian brain and is involved in a range of functions includ- cleft are terminated by their reuptake into the presynaptic neu- ing locomotion, neuroendocrine secretion, cognition and rons, via selective Na+-dependent transporters; DA is taken up emotion. It is metabolized from the amino acid in a by the (DAT; Figure 4). Once returned two-step process. First the conversion of L-tyrosine to levo- to synaptic vesicles by the vesicular monoamine transporters dopa (L-DOPA) is catalyzed by (TH), then (VMATs), DA may be recycled for release or broken down by L-DOPA is converted to DA by DOPA decarboxylase (DDC; (MAO), located on the outer mitochon- Figure 2). TH is a marker for neurons expressing catecholamine drial membrane.

Box 1: Dopaminergic Transmission See pages 23-33 for a full list of targets and related products OH N OH PMe H N 3 N O Ru2+ N N H HO CO H 2 2 N N Me NHNH2 HO NC N

(S)-(-)- (0455) SB 277011A (4207) RuBi-Dopa (4932)

Aromatic L-amino acid decarboxylase inhibitor Selective D3 antagonist Caged dopamine; exhibits two-photon sensitivity

Me N OH O2N OH N N H N OH O N OH

NO2 Cl OR-486 (0483) GBR 12783 (0513) SKF 82958 (5719) LE 300 (1674)

COMT inhibitor Potent and selective DA uptake inhibitor D1 Potent and selective D1 antagonist

Box 1: Dopaminergic Transmission

www.tocris.com | 5 Tocris Product Guide Series Glutamatergic Transmission

GluN1, GluN2A-D and GluN3A or B. Alternative splicing of the Products by Category Page GluN1, GluN2 and GluN3A subunits add to the heterogeneity of NMDA receptors. Adenylyl Cyclase...... 25 AMPA Receptors...... 25 AMPA and Kainate receptors have a simple ligand-gated cAMP...... 26 mechanism of action, however the gating of NMDARs is more Glutamate Transporters...... 29 complex. The opening and closing of NMDARs requires the Kainate Receptors...... 30 binding of D- (Tocris, Cat. No. 0226) and glycine (Tocris, Metabotropic Glutamate Receptors...... 30 Cat. No. 0219) as well as glutamate. Additionally, the flow of NMDA Receptors...... 32 ions through NMDARs is mediated by the binding of Mg2+ or Phospholipase C...... 32 Zn2+ to specific sites in the receptor. Only upon depolarization of the cell membrane, displacing Mg2+/Zn2+, does the influx of Na+ and Ca2+ and the efflux of+ K occur. Glutamate is an amino acid and is the most abundant excita- tory neurotransmitter in the nervous system. Under healthy Following receptor activation, glutamate is released into the conditions, enough glutamate is obtained from the diet and synaptic cleft from where it is taken up into neurons and glia it does not need to be synthesized, however it can be synthe- by glutamate transporters, also known as excitatory amino sized from α-ketoglutarate, a product of the citric acid cycle. acid transporters (EAATs). The EAAT family has five mem- Two types of exist: metabotropic glutamate bers, although EAAT2 expressed on glia is responsible for receptors (mGluRs), which are GPCRs, and ionotropic gluta- most glutamate reuptake in the central nervous system. Once mate receptors, which are LGICs. mGluRs are encoded by a taken up into glia, glutamate is converted into glutamine, single gene and are composed of an extracellular region con- which is transported back into presynaptic neurons where it taining the glutamate , a transmembrane region, is converted into glutamate once more for further synaptic and an intra­cellular region that is coupled to a G protein. There transmission. Glutamate is packaged into synaptic vesicles by are three subtypes of mGluRs based on receptor structure and vesicular glutamate transporters (VGLUTs; Figure 5). physiological activity. Group I mGluRs are primarily located on Figure 5 | VGLUT2 in Human Brain postsynaptic membranes and are coupled to the Gq/11 pathway and activation of phospholipase C (PLC). In contrast, group II and group III mGluRs are mainly located on the presynaptic membrane, are coupled to the Gi/o pathway and prevent the formation of cAMP, leading to presynaptic inhibition. Ionotropic GluRs (iGluRs, Figure 6) are LGICs that are tetra­ mers formed by homo- or hetero-oligomeric assembly of sub­ units, all of which are encoded by individual genes. The specific combination of subunits determines the activity of specific ago- nists and antagonists at iGluRs. They are divided into three subgroups based on and sequence similarity, and are named for the that selectively bind to them. AMPA receptors are the most commonly found receptor in the nervous system where they mediate fast synaptic transmis- sion. They are composed of subunits designated GluA1, GluA2, GluA3 and GluA4, and may also be associated with transmem- brane AMPA receptor regulatory proteins (TARPs) as auxiliary VGLUT2 detected in immersion-fixed paraffin-embedded sections subunits. Kainate receptors modulate neurotransmission and of using a Mouse Anti-Human/Mouse/Rat VGLUT2 Monoclonal Antibody (Novus Biologicals, Cat. No. NBP2-46641). can be located pre- or postsynaptically. Like AMPA recep- The tissue was stained using HRP and DAB (brown) and tors, they are composed of subunits designated GluK1, GluK2, counterstained with hematoxylin (blue. Tocris, Cat. No. 5222). GluK3, GluK4 and GluK5. NMDA receptors have a critical role Specific staining was localized to glutamatergic synapses in the in synaptic plasticity and are composed of subunits designated molecular and granular layers of the cerebellum.

6 | NEUROTRANSMISSION RESEARCH

Figure 6 | Glutamate Receptor Expression A B

A) GluR1 (AMPA receptor subunit) was detected in immersion-fixed SK-N-BE human neuroblastoma cells using a Mouse Anti-Rat GluR1 Monoclonal Antibody (Novus Biologicals, Cat. No. NBP2‑22399). The cells were stained using a ATTO 488-conjugated goat anti-mouse secondary antibody (green). The cells were also stained for F-Actin (Texas Red®‑X phalloidin; red) and counterstained with DAPI (blue. Tocris, Cat. No 5748). Specific staining was localized to the cell membrane and cell junctions. B) KA2 (Kainate receptor subunit) was detected in immersion-fixed, paraffin-embedded sections of human brain cortex using a Goat Anti-Human KA2/GRIK5/ Glutamate Receptor KA2 Antigen Affinity-Purified Polyclonal Antibody (Novus Biologicals, Cat. No. NBP1‑36959). The tissue was stained using alkaline phosphatase-streptavidin and chromogen (red) and counterstained with hematoxylin (blue. Tocris, Cat. No. 5222).

Box 2: Glutamatergic Transmission H N O N See pages 23-33 for a full list of targets and related products N N O N

OMe N

O2N - N N O CO2H H NO S N 2 2 2Na+ NO - 2 O2N N O O NH2

MDNI-caged-L-glutamate (5785) NBQX disodium salt (1044) GT 949 (6578) Stable photoreleaser of L-glutamate Potent AMPA antagonist; more water soluble Potent and selective positive form of NBQX (Cat. No. 0373) of EAAT2

H2N CO2Na

NaO2C Me CO H HN 2 H

N CO2H Me H O

(+)-MK 801 (0924) (0222) LY 341495 disodium salt (4062) Non-competitive NMDA antagonist; Kainate agonist; excitant and neurotoxin Potent and selective group II mGlu antagonist; acts at ion channel site disodium salt of LY 341495 (Cat. No. 1209)

NH CO2H 2 H N HO2C 2 PO(OH)2 H H PO(OH)2

D-AP5 (0106) L-AP4 (0103) Potent and selective NMDA antagonist; Selective group III mGlu agonist more active form of DL-AP5 (Cat. No. 0105)

www.tocris.com | 7 Box 2: Glutamatergic Transmission Tocris Product Guide Series Opioid Peptide Transmission

pathways. The highest expression of the δ opioid receptor Products by Category Page (DOR) is found in the basal ganglia and neocortical brain regions, while the κ opioid receptor (KOR) is widely distributed Adenylyl Cyclase...... 25 throughout the brain, including the , substan- cAMP...... 26 tia nigra, and . The µ opioid receptor δ Opioid Receptors...... 28 (MOR) exists on presynaptic membranes in the periaqueductal κ Opioid Receptors...... 30 gray and the dorsal horn of the spinal cord. The NOP receptor µ Opioid Receptors...... 31 (nociceptin/orphanin FQ receptor) is also widely expressed in NOP Receptors...... 32 the brain and specifically binds nociceptin (Figure 7). Receptors: Non-selective Compounds...... 32 In general, endogenous opioids are not highly selective for one specific type of opioid receptor, due to the extensive Endogenous opioid peptides are released from precursor similarities in receptor structure, function and intracellular proteins by proteolytic cleavage, and act at opioid receptors signaling pathways. Also opioid receptors can form homo- and with differing selectivity. Proenkephalin is cleaved to release heteromeric complexes with each other, and with non-opioid Met-enkephalin, and to a lesser extent Leu-enkephalin. Leu- receptors, which can change their response to a specific opi- enkephalin is predominantly cleaved from prodynorphin, along oid peptide. These physical interactions between receptors are with dynorphin A and dynorphin B. Pro-opiomelanocortin key to their pharmacological and physiological properties. All is cleaved to release β-endorphin, the largest endogenous opioid peptides are broken down following receptor binding opioid peptide, while the more recently discovered nociceptin by peptidases. For example, enkephalins are broken down into is cleaved from pronociceptin. inactive metabolites by two metalloproteases; neprilysin

All opioid receptors are GPCRs coupled to the Gi/o pathway and and aminopeptidase N. so inhibit the formation of cAMP and its downstream signaling

Figure 7 | Opioid Receptors A B C

A) NOP (OPRL1) was detected in fixed sections of rat using using a Rabbit Anti-Human/Mouse/Rat ORL1/OPRL1 Antigen Affinity‑Purified Polyclonal Antibody (Novus Biologicals, Cat. No. NBP2‑21065). The tissue was stained (green) and counterstained with DAPI (blue. Tocris, Cat. No 5748). B) MOR was detected in perfusion-fixed frozen sections of rat spinal cord using a Rabbit Anti-Rat μ Opioid R/OPRM1 Monoclonal Antibody (R&D Systems, Cat. No. MAB8629). The tissue was stained using the NorthernLights™ 557-Conjugated Anti‑Rabbit IgG Secondary Antibody (R&D Systems, Cat. No. NL004; red) and counterstained with DAPI (blue. Tocris, Cat. No 5748). Specific staining was localized to the dorsal horn.C ) KOR detected in immersion-fixed paraffin-embedded sections of human medulla using a Mouse Anti‑Human KOR Monoclonal Antibody (R&D Systems, Cat. No. MAB38951). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (R&D Systems, Cat. No. CTS013). The tissue was stained with the Anti‑Mouse HRP‑DAB Cell & Tissue Staining Kit (R&D Systems, Cat. No. CTS002; brown) and counterstained with hematoxylin (blue. Tocris, Cat. No. 5222).

8 | NEUROTRANSMISSION RESEARCH

Box 3: Opioid Transmission See pages 23-33 for a full list of targets and related products

O

Et2N H N N OMe OH HO N Me N N N

Me N O O N O (and ) HO H OH OH

nor-Binaltorphimine (0347) SNC 80 (0764) (±)-J 113397 (2598) Selective κ antagonist Highly selective non-peptide δ agonist Potent and selective NOP antagonist

N N OH OH Cl N O O N Cl Tyr-D-Ala-Gly-NMe-Phe-Gly-ol Me N O O CO2Me HO O (and enantiomer) HO H H

Naloxone (0599) (±)-U-50488 (0495) β-Funaltrexamine (0926) DAMGO (1171) Non-selective Selective κ agonist Irreversible and selective μ antagonist Selective μ agonist

Box 3: Opioid Transmission

Life Science Literature from Tocris Tocris provides a wide range of scientific literature, including the following titles:

Product Guides & Listings Life Science Posters Scientific Reviews

• G-Protein Coupled Receptors • Learning and Memory • Nicotinic ACh Receptors • Ion Channels • Pain • 5-HT Receptors • Neurodegeneration • Epilepsy • GABA Receptors • Pain Research • Depression • Dopamine Receptors

For a complete selection of Tocris literature please visit www.tocris.com/literature.php

www.tocris.com | 9 Tocris Product Guide Series Serotonergic Transmission

Figure 8 | TPH-1 in Rat Brain Products by Category Page

5-HT Transporters...... 23

5-HT1 Receptors...... 23 5-HT2 Receptors...... 23 5-HT3-7 Receptors...... 23 Adenylyl Cyclase...... 25 cAMP...... 26 Hydroxylases...... 30

IP3 Receptors...... 30 Monoamine Oxidase...... 31 Phospholipase C...... 32 Vesicular Monoamine Transporters...... 33

5-HT is an amine neurotransmitter derived from the amino acid , by tryptophan hydroxylase (TPH; Figure 8) TPH-1 detected in perfusion-fixed frozen sections using and 5-HTP decarboxylase. a Goat Anti-Human TPH-1 Antigen Affinity-Purified Polyclonal Antibody (R&D Systems, Cat. No. AF5276). Once released into the synaptic cleft, 5-HT binds to its recep- The tissue was stained using the NorthernLights™ tors. There are seven families of 5-HT receptors (Figure 9). 557-Conjugated Donkey Anti-Goat IgG Secondary 5-HT1, 5 receptors are GPCRs, coupled to the Gi/o pathway and Antibody (R&D Systems, Cat. No. NL001; red) and counterstained with DAPI (blue. Tocris, Cat. No 5748). so inhibit the formation of cAMP. In contrast, 5-HT4, 6, 7 recep- Specific staining was localized to neurons. tors are coupled to the Gs pathway and so increase the produc- tion of cAMP. 5-HT2 receptors are coupled to the Gq/11 pathway and activate PLC, which hydrolyzes phosphatidylinositol (PIP2) Like DA, following binding to its receptor 5-HT is taken up into to diacylglycerol (DAG) and triphosphate (IP3). The neurons via a selective transporter, SERT. It can then be broken only 5-HT receptor that is not a GPCR is the 5-HT3 receptor, down by MAO or recycled for further synaptic transmission via which is a cation selective LGIC. repackaging into synaptic vesicles by VMATs.

Figure 9 | 5-HT Receptors in the Human Brain Cortex A B

A) 5-HT1F was detected in immersion-fixed paraffin-embedded sections using a Rabbit Anti‑Human Antigen Affinity-Purified 5-HT1F Antibody (Novus Biologicals, Cat. No. NBP1‑02371). The tissue was stained using alkaline phosphatase-streptavidin and chromogen (red) and

counterstained with hematoxylin (blue. Tocris, Cat. No. 5222). B) 5‑HT2C was detected in immersion-fixed paraffin-embedded sections using a Mouse Anti-Human 5‑HT2C Monoclonal Antibody (R&D Systems, Cat. No. MAB5764). Before incubation with the primary antibody, the tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (R&D Systems, Cat. No. CTS013). The tissue was stained using the Anti-Mouse HRP‑DAB Cell & Tissue Staining Kit (R&D Systems, Cat. No. CTS002; brown) and counterstained with hematoxylin (blue. Tocris, Cat. No. 5222). Specific staining was localized to neuronal cell bodies and processes.

10 | NEUROTRANSMISSION RESEARCH

Box 4: Serotonergic Transmission See pages 23-33 for a full list of targets and related products

F3C

NH O O N O F O NHMe N N N OMe NHSO2Me H H

CP 94253 (1317) (0927) GR 125487 (1658)

Potent and selective 5-HT1B agonist 5-HT Potent and selective 5-HT4 antagonist; active in vivo

2+

H N N 2 N NH PMe N N 3 O Ru - .2PF6 N N H N 2 OMe N N N OH N

F

RS 127445 (2993) RuBi-5-HT (3856) WAY 100635 (4380)

High affinity and selective 5-HT2B antagonist RuBi-caged serotonin Potent 5-HT1A antagonist; also D4 agonist

Cl O O HN O O N S MeN N Cl H N OMe H MeO

Tropisetron (2459) SB 399885 (3189)

Potent 5-HT3 antagonist; also α7 nAChR Potent and selective 5-HT6 antagonist

Detection of Neurotransmitters in vivo with RNAscope® Identification, visualization and characterizationBox of 4: specific Serotonergic cell types Transmission within the nervous system is difficult. RNAscope® in situ hybridization assay (ISH) (from ACD) can be used for the detection of RNA in the central and peripheral nervous system. The probes used are highly specific and sensitive with advanced signal amplification allowing visualization of single RNA transcripts. This enables researchers to detect RNA expression associated with proteins for which there are no reliable antibodies. RNAscope® ISH can be used for characterization of cell types, validation of genetic modifications, validation of mRNA expression and for determining subcellular localization of mRNA expression.

Figure 10 | DA Receptors in the Mouse DA receptors were detected using RNAscope multiplexing fluorescent ISH in fresh frozen mouse striatum, DAPI counterstaining. Two distinct populations

of neurons expressing either D1R (Drd1, red) or D2R (Drd2, green) were identified. Areas indicated by green and red arrows are shown at higher magnification in the right hand panels.

www.tocris.com | 11 Tocris Product Guide Series Chemogenetics in Neurotransmission Research

Major advances in neuroscience methods have allowed Chemogenetic manipulation of neuronal activity can be researchers to selectively manipulate neural systems in awake achieved through genetically modified GPCRs known as animals, with two key techniques emerging; optogenetics and DREADDs (Designer Receptors Activated by Designer chemogenetics. Chemogenetic experiments require the intro- ; Figure 11) or though chimeric ion channels known duction of genetically engineered receptors or ion channels as PSAMs (Pharmacologically Selective Actuator Modules). into specific brain areas, via viral vector expression systems. The binding of DREADD ligands to Gαq-DREADDs, such as Ligands, that are inert except for their specific action at those hM3Dq, provokes neuronal firing, whereas binding to Gαi- receptors/ion channels, are then administered. Binding of the DREADDs, such as hM4Di results in inhibition of neuronal ligand to its target initiates changes in downstream intracel- activity. PSAMs containing the ion pore domain of cation lular signaling pathways or opening of an ion channel pore, selective channels, such as 5-HT3, result in neuronal activa- enabling controlled activation or inhibition of neuronal activ- tion upon binding of a PSEM (Pharmacologically Selective ity, depending on the specific receptor/ion channel and ligand Effector Molecule). In contrast, PSAMs containing the ion used. Similarly, optogenetics allows the modulation of neuronal pore domain of an anion selective channel, such as the glycine activity via expression of light-sensitive ion channels. However, receptor, are inhibitory. activation or inhibition of neuronal activity is initiated by implanted fibre optics, rather than small molecules.

Figure 11 | Mechanism of action of DREADD ligands

DREADD agonist 21 (#5548) N-oxide 2HCl (#6329) Salvinorin B (#5611)

hM1Dq, hM5Dq hM4Di hM3Dq KORD

G Gαq αi Gαi Gαq

PLC AC

IP3 DAG cAMP ATP

Ca2+ PKC PKA EPAC action Activation Activate neuronal activity Inhibit neuronal activity Inhibition

Binding of DREADD ligands to Gαq-DREADDs provokes neuronal firing, whereas binding to Gαi-DREADDs results in inhibition of neuronal activity. Clozapine N-oxide dihydrochloride and DREADD agonist 21 are non-selective muscarinic DREADD agonists and so can activate or inhibit neuronal activity, depending on the specific receptor being expressed. Salvinorin B is selective for the KORD receptor, which is coupled to GαI signaling; consequently binding results in inhibition of neuronal activity.

GPCR Chemogenetics: DREADDs

12 | NEUROTRANSMISSION RESEARCH

Box 5: Chemogenetics Compounds: DREADD Ligands and PSEMs See pages 23-33 for a full list of targets and related products

O- CH3 + N NH N

N N 2HCl N N N 2HCl N Cl

N N H H

Clozapine N-oxide dihydrochloride (6329) DREADD agonist 21 dihydrochloride (6422) (5549) Activator of muscarinic DREADDs; water soluble Potent muscarinic DREADD agonist; water soluble Potent muscarinic DREADD agonist version of Clozapine N-oxide (Cat. No. 4936) version of DREADD agonist 21 (Cat. No. 5548)

O (Z)

(E)

O O H H N HO N N O MeO Me H N Me OMe MeO O O N

PSEM 308 (6425) Salvinorin B (5611) PSEM 89S (6426) L141F L141F,Y115F L141F L141F,Y115F PSAM -GlyR and PSAM -5-HT3 Activates the κ-opioid DREADD (KORD) PSAM -GlyR and PSAM -5-HT3 chimeric ion channel agonist chimeric ion channel agonist

Box 5: Chemogenetic compounds

Chemogenetics Research Bulletin Produced by Tocris, the chemogenetics research bulletin provides an introduction to chemogenetic methods to manipulate neuronal activity. It outlines the development of RASSLs, DREADDs and PSAMs, and the use of chemogenetic compounds. DREADD ligands and PSEMs available from Tocris are highlighted.

Text excerpt: “All DREADDs have some common features that make them ideal for use in neuroscience experiments. Firstly, DREADDs exhibit no response to endogenous ligands due to genetic mutations within their ligand binding sites that abolish binding. This means that any activity of the DREADD will be solely due to the specific DREADD ligand applied. Secondly, expression of DREADDs in vitro or in vivo, has no effect on cellular activity, neuronal function or baseline behaviors, prior to the addition of the DREADD ligand (Sternson & Roth, 2014).”

www.tocris.com | 13 Tocris Product Guide Series Depression Research

Major depressive disorder (MDD) is an affective disorder char- Products by Category Page acterized by the core symptoms of depressed mood and loss of interest and/or pleasure, often accompanied by sleep distur- 5-HT Transporters...... 23 bances, fatigue and altered ability to concentrate. The pervad- 5-HT Receptors...... 23 1 ing biochemical theory to explain the neurobiological causes 5-HT Receptors...... 23 2 of MDD is the monoamine hypothesis, which suggests that 5-HT Receptors...... 23 3-7 an imbalance in monoamine (5-HT, DA and NE) signaling is Transporters...... 25 to blame. This theory grew from the observation that various α-receptors...... 25 drugs known to alter monoamine neurotransmission mimic or Adrenergic β-receptors...... 25 alleviate the symptoms of depression. For example, the VMAT2 Adrenergic Transporters...... 25 inhibitor (Tocris, Cat. No. 2742), originally intro- AMPA Receptors...... 25 duced as an , has an inhibitory effect on Dopamine D -like Receptors...... 27 1 monoamine transmission and is associated with a lowering Dopamine D -like Receptors...... 28 2 of mood. Also, MAO inhibitors that block the breakdown of Dopamine Transporters...... 28 monoamines and were originally introduced to treat tubercu- Glutamate Transporters...... 29 losis, are associated with a marked elevation in mood. Selective Kainate Receptors...... 30 5-HT reuptake inhibitors (SSRIs) are currently the most pre- κ Opioid Receptors...... 30 scribed , although compounds including non- Metabotropic Glutamate Receptors...... 30 competitive NMDAR antagonists, agents and Monoamine Oxidases...... 31 opioid signaling modulators are under investigation as potential NMDA Receptors...... 32 therapies. Vesicular Monoamine Transporters...... 33 Currently available animal models and tests to investigate MDD neurobiology and identify new treatments are limited, as subjective measures used to study mood disorders in humans

Figure 12 | Positive Affective Bias Induced by Antidepressants

A 15 B 15 C 15

*** 10 ** 10 10 ** *** * 5 5 ** 5

% Choice bias 0 % Choice bias 0 % Choice bias 0 fluoxetine-paired substrate -paired substrate -5 -paired substrate -5 -5 0.0 0.3 1.0 3.0 0.0 0.1 0.3 1.0 3.0 0.0 0.1 0.3 1.0 Dose (mg/kg) Dose (mg/kg) Dose (mg/kg)

D E F 15 15 15 *** *** 10 10 ** 10 ** 5 5 5

% Choice bias 0 % Choice bias 0 % Choice bias 0 -paired substrate -paired substrate -5 -paired substrate -5 -5 0.0 1.0 3.0 10.0 0.0 0.1 0.3 1.0 0.0 0.3 1.0 3.0 10.0 0.0 10.0 30.0 Dose (mg/kg) Dose (mg/kg) Dose (mg/kg)

Fluoxetine (A), citalopram (B), reboxetine (C), venlafaxine (D) and agomelatine (E) induce a significant positive affective bias in the ABT in rats. The neurokinin 1 aprepitant (F) failed to cause a significant change in affective bias. * p<0.05, ** p<0.01, *** p<0.005. Adapted from Stuart et al, 2013.

14 | NEUROTRANSMISSION RESEARCH

Box 6: Antidepressants See pages 23-33 for a full list of targets and related products NHMe

Me2N F Me2N OH

O Cl NC Cl MeO

Citalopram (1427) (2395) Venlafaxine (2917) Highly potent and selective 5-HT uptake inhibitor 5-HT re-uptake inhibitor Dual 5-HT/noradrenalin re-uptake inhibitor

Me N

H N O NH N N Cl O O

NMe2

Mirtazapine (2018) (0457) Reboxetine (1982)

Potent 5-HT2 antagonist; also 5-HT3, 5-HT re-uptake inhibitor Potent and selective noradrenalin

H1 and α2 antagonist uptake inhibitor; orally active

cannot be replicated in animals, i.e. you can’t ask a rodent how Using ABT, they showed the SSRIs fluoxetine (Tocris, Cat. No it ‘feels’. Recent advances in cognitive neuropsychologicalBox testing 6: Antidepressants 0927), citalo­pram (Tocris, Cat. No. 1427) and clomipramine in humans have provided an opportunity to translate these tests (Tocris, Cat. No. 0457); the selective NE reuptake inhibi- to develop methods of the assessment of depressive behaviors tor reboxetine (Tocris, Cat. No. 1982); the mixed 5-HT/NE in rodents. reuptake inhibitor venlafaxine (Tocris, Cat. No. 2917); and the 5-HT antagonist (Tocris, Cat. No. 2018) induced Acute and long-term antidepressant treatments induce a posi- 2 a significant positive affective bias, replicating results seen in tive shift in emotional processing, also known as cognitive humans (Figure 12). They also showed induction of a negative affective bias, in humans (Pringle et al, 2011). In their paper, affective bias by compounds associated with negative bias in Stuart et al describe the evaluation of an affective bias test humans, including the CB receptor inverse ago- (ABT) for rats, using a range of compounds with acute effects 1 nist (Tocris, SR 141716A, Cat. No. 0923) and the on affective bias in humans (Stuart et al, 2013). In the ABT, FG 7142 (Tocris, Cat. No. rats encounter two independent positive experiences (the 0554). Acute psychosocial stress and environmental enrich- association of food reward with a specific digging material) in ment also induced significant negative and positive affective learning sessions. Affective bias is then quantified using a pref- bias, respectively (Stuart et al, 2013). erence test, in which both previously rewarded digging mat­ erials are presented together and the rat’s choice in recorded.

www.tocris.com | 15 Tocris Product Guide Series

Depression – continued

Figure 13 | Antidepressant-like Effects of KOR Antagonists in the Forced Swim Test

A Sprague Dawley Wistar Kyoto Saline 50 # nor-BNI (1 mg/kg) -BNI (5 mg/kg) 45 nor nor-BNI (10 mg/kg) 40 * * 35

30

25 ** Count 20 *

15

10 # 5

0 Immobility Swimming Climbing Immobility Swimming Climbing

B Sprague Dawley C Wistar Kyoto 50 50 Saline Saline 45 45 DIPPA (5 mg/kg) DIPPA (1 mg/kg) 40 DIPPA (10 mg/kg) 40 DIPPA (2.5 mg/kg) * * 35 35 DIPPA (5 mg/kg) DIPPA (10 mg/kg) 30 30 ** 25 25 Count Count 20 20

15 15 * * 10 10

5 5

0 0 Immobility Swimming Climbing Immobility Swimming Climbing

Nor-BNI (A) and DIPPA (B and C) significantly reduced immobility and increased swimming behavior in WKY rats, but not SD rats. # indicates a significant difference between saline-treated groups across the two strains, # p<0.001. Asterisks indicate a significant difference within each strain, compared to the saline-treated group, * p<0.05, ** p<0.01. Adapted from Carr et al, 2010.

Modulators of opioid signaling are currently under investiga- (nor-BNI, Tocris, Cat. No. 0347) and DIPPA (Tocris, Cat. No. tion for their efficacy as antidepressants. The expression of KOR 0794), in WKY rats when compared to the Sprague-Dawley is increased in Wistar-Kyoto (WKY) rats (Pearson et al, 2006), (SD) rat strain, using the forced swim test (Figure 13). C-fos a strain originally developed as a normotensive control for the expression, a marker of neuronal activity, was altered in the spontaneously hypertensive rat strain, which is known to have piriform cortex and following KOR antago- increased sensitivity towards stress (Pare, 1992; 1994). KOR nist treatment. Also, direct administration of nor-BNI into the agonists cause depressive behaviors in commonly used rodent piriform cortex induced antidepressant-like effects implicating models (McLaughlin et al, 2006a), while KOR antagonists pro- this area in the antidepressant-like activity of KOR antagonists duce an antidepressant-like effect (McLaughlinet al, 2006b). (Carr et al, 2010). In their paper, Carr et al confirmed the antidepressant-like effects of the selective KOR antagonistsNor -Binaltorphimine

16 | NEUROTRANSMISSION RESEARCH

Ketamine Metabolites for the Treatment of Depression neuropathic pain. Also, 2R,6R- (Tocris, Ketamine (Tocris, Cat. No. 3131) is a non-competitive NMDA Cat. No. 6094) and 2S,6S-Hydroxynorketamine (Tocris, Cat. receptor antagonist that demonstrates a rapid and robust anti- No. 6095) both reduce intracellular D-serine concentrations depressant effect in patients, occurring within a few hours of required for NMDA receptor opening, displaying more potent administration and with long-lasting effects. This gives keta- inhibition than other ketamine metabolites. mine advantages over current antidepressants, which generally 2R,6R-Hydroxynorketamine displays rapid and persistent have a long lead time before showing their beneficial effects and antidepressant effects in various animal models of depression. have a high non-response rate. However, the use of ketamine is Unlike other metabolites, it does not displace the noncompeti- hampered by its liability for abuse and side effects tive NMDA antagonist MK-801 (Tocris, Cat. No. 0924) from even at low doses. its binding site in the NMDA ion channel suggesting another Recent investigations have indicated that metabolites of keta- specific site of action. It causes increases in AMPA receptor mine may be responsible for its antidepressant effects. In vivo, mediated excitatory postsynaptic potentials (EPSPs), causing ketamine is converted to metabolites including an increase in glutamatergic signaling with subsequent upregu- (Tocris, Cat. No. 1970), hydroxyketamines, dehydroxynorketa- lation of synaptic AMPA receptors. Additionally, even at high mines and hydroxynorketamines. (R)- and (S)-Norketamine doses 2R,6R-Hydroxynorketamine shows none of the addictive (Tocris, Cat. Nos. 5996 and 6112, respectively) both modulate and dissociative side effects of ketamine. NMDA receptor activity and act as analgesics in rat models of

Box 7: Ketamine and its Metabolites See pages 23-33 for a full list of targets and related products

O Cl O O NH2 NH2 HO

NHMe (and enantiomer) Cl Cl

Ketamine (3131) Norketamine (1970) cis-6-Hydroxynorketamine (5982) Non-competitive NMDA antagonist Potent non-competitive NMDA antagonist; Enhances AMPA currents; antidepressant antinociceptive

O NH2 HO

Cl

2R,6R-Hydroxynorketamine (6094) Enhances AMPA currents; decreases D-serine (NMDA co-agonist); lacks ketamine-related side effects

Box 7: Ketamine and its Metabolites

www.tocris.com | 17 Tocris Product Guide Series Addiction Research

Figure 14 | Brain Circuits Associated with Addiction Products by Category Page

5-HT Transporters...... 23 PFC 5-HT1 Receptors...... 23 5-HT Receptors...... 23 2 ACG 5-HT3-7 Receptors...... 23 Acetylcholine Nicotinic Receptors...... 24 OFC AMPA Receptors...... 25 HIP NAcc Cannabinoid CB1 Receptors...... 26 VP Cannabinoid CB2 Receptors...... 26 Amyg Dopamine D1-like Receptors...... 27 Dopamine D2-like Receptors...... 28 Dopamine Transporters...... 28 δ Opioid Receptors...... 28

GABAA Receptors...... 28 GABAA-ρ Receptors...... 29 Glutamate Transporters...... 29 Four interacting circuits are invovled in addiction, modulating Kainate Receptors...... 30 reward prediction and pleasure (green), memory (red), motivation κ Opioid Receptors...... 30 (blue) and cognition (purple) to varying extents depending on the individual and the addictive substance in question. Use of addictive µ Opioid Receptors...... 31 substances leads to adaptive changes, for example in the ‘top-down’ NMDA Receptors...... 32 control over urges and impulses provided by the function of the PFC/OFC connection. AGC – Anterior cingulate gyrus; Amyg – Amygdala, HIP – Hippocampus, NAcc – Nucleus accumbers, OFC – orbitao-frontal cortex, PFC – Prefrontal cortex, VP – Ventral Drug addiction refers to the processes by which people become palladium. Adapted from Baler & Volkow, 2006. dependent on the use of drugs, or even gambling, to the point where it interferes with and eventually takes over their life. Not all those who take drugs become addicted; there This underlies the reward/reinforcement aspect of are several factors that predispose to addiction, including class use, leading to drug abuse and addiction (Rice & Cragg, 2008). of drug, family history of addiction and propensity for with- In vitro investigations have suggested that cocaine may have drawal reaction and cravings. There are three defining features allosteric actions at D R, known to play a role in the abuse of addiction: compulsion to take the drug, loss of control of 2 related side effects of cocaine. At low concentrations, known limiting intake and withdrawal symptoms when the drug is not to inhibit DAT, cocaine enhances the ability of , withheld. a selective D2-like receptor agonist (Tocris, Cat. No. 1061), to Using preclinical and human imaging, the brain circuits reduce labeled DA efflux from synaptosomes, evoked by+ K involved in addiction have been characterized. There are (Ferraro et al, 2010b). This was further investigated in vivo, four interacting circuits that are involved to varying extents, demonstrating that when given in combination with quin- depending on the addictive substance. The reward circuit is a pirole, cocaine significantly increased quinpirole-induced dopaminergic pathway from the ventral tegmental area (VTA) hyperlocomotion in rats (Figure 15). Cocaine also enhanced to the nucleus accumbens (NAcc). The hippocampus and amyg­ the Gi/o coupling of D2Rs, possibly by either an allosteric direct dala regulate the encoding and reactivation of memories asso- or indirect enhancing effect (Ferraroet al, 2010a). These results ciated with drug use, with the hippocampus being responsible suggest a novel mechanism for cocaine, with relevance for for place memories and the amygdala being responsible for the understanding the rewarding aspect of cocaine addiction. emotional aspect of memories. A separate pathway, involving Extinction of drug addiction promotes abstinence from drug areas of the prefrontal cortex and anterior cingulate cortex, is seeking behaviors. This is an active learning process involving responsible for cognitive control and relies on the balance of inhibition of learned motivations and behaviors, and is depend- glutamatergic and GABAergic signaling to exert ‘top-down’ ent on the infralimbic prefrontal cortex (ilPFC). As well as its control of the motivation and pleasure areas (Figure 14). established role in feeding as a motivated behavior, the hypo­ As outlined above, the rewarding aspects of drug addiction is also thought to play a role in reinstatement of drug originate in a dopaminergic circuit. Cocaine (Tocris, Cat. addiction and associated reward seeking behaviors. Various No. 2833) is a competitive inhibitor of DAT and so blocks the lines of evidence suggest a commonality between the circuits reuptake of DA and increases dopaminergic transmission. responsible for reinstatement of drug seeking behaviors and

18 | NEUROTRANSMISSION RESEARCH

Figure 15 | Effect of Cocaine on Drug-Induced Locomotion those controlling feeding. Using complementary functional and neuroanatomical techniques, Marchant et al investigated the 8000 role of the medial dorsal (MDH) in the extinc- < ** tion of alcohol seeking behavior in rats. Neurons projecting 6000 from ilPCF to MDH are known to be necessary for extinction * in vivo, and MDH neurons projecting to the paraventricular 4000 nucleus of the thalamus (PVN) are also thought to be involved. MDH to PVN neurons showed expression of dynorphins, and 2000 ** * infusion of the KOR agonist (±)-U-50488 (Tocris, Cat. No. 0495) into the PVN prevented reinstatement of alcohol-seeking

Distance traveled (cm γ 120 min) 0 behavior. This indicates that PVN KOR activation can inhibit Veh Coc 0.625 Veh Coc 0.625 Veh Coc 0.625 Veh Veh Quin 0.5 Quin 0.5 Quin 1 Quin 1 alcohol-seeking behaviors in rats, affecting a neuronal circuit involving MDH and ilPCF (Marchant et al, 2010). Cocaine (0.625 mg/kg) significantly enhances quinpirole (0.5 or 1 mg/kg) -induced hyperlocomotion, in a concentration dependent manner, but has no effect on locomotion when given alone. Coc – cocaine, Quin – quinpirole, Veh – vehicle. * p<0.05, ** p<0.01 vs Veh+Veh group. ^ p<0.05 vs Veh+Quin 1 group. Adapted from Ferraro et al, 2010.

Box 8: Addiction See pages 23-33 for a full list of targets and related products

N N H H CO2Me

NH2 O H O NHMe HO O OH O O Cocaine (2833) D- (2813) (5158) (±)-MDMA (3027) Inhibitor of monoamine transporters Induces dopamine, 5-HT Narcotic opioid analgesic Inhibitor of 5-HT and dopamine uptake; and noradrenalin release hallucinogenic

Me

OH

N N Me Me OH N

Phencyclidine (2557) (-)- (1570) (-)- (3546)

Non-competitive NMDA antagonist Natural cannabinoid; GPR55 antagonist, weak CB1 Prototypical nAChR agonist

antagonist, CB2 inverse agonist and AMT inhibitor

Box 8: Addiction

www.tocris.com | 19 Tocris Product Guide Series Epilepsy Research

receptors have been identified in TLE, while at a cellular level Products by Category Page inflammation, neuronal cell death and reactive astrocytosis are seen. AMPA Receptors...... 25

GABAA Receptors...... 28 NMDARs mediate synaptic plasticity, a process essential GABAA-ρ Receptors...... 29 to most forms of learning and memory. Full activation of Kainate Receptors...... 30 NMDARs requires binding of D-serine or glycine as an allos- Metabotropic Glutamate Receptors...... 30 teric co-agonist. D-serine, which binds at a separate binding site NMDA Receptors...... 32 to glutamate, is released from astrocytes in a Ca2+-dependent manner, regulating activation of NMDARs and therefore synaptic plasticity. Epilepsy is a common neurological disorder, frequently result- ing from traumatic brain injury and acquired pathology In their investigation, Klatte et al showed that D-serine levels including tumors or infection. Epileptic seizures arise from are reduced in a rat model of TLE, induced by treatment with abnormal, excessive or synchronous neuronal activity, resulting the pilocarpine (Tocris, Cat. No. 0694). in sensory disturbances, loss of consciousness and/or convul- This resulted in desaturation of synaptic and extrasynaptic sions. Epilepsy can also be caused by mutations in a single gene, NMDARs and deficits in hippocampal long-term potentiation however in most cases the exact cause is unknown. (LTP), the strengthening of the connection between neurons that is thought to underlie learning and memory. Exogenous Seizures arise when there is a disruption in the balance of exci- application of D-serine (Tocris, Cat. No. 0226) rescues hippo­ tation and inhibition within neuronal circuits. The activity of campal LTP in the pilocarpine TLE model, which is reversed a network of neurons, rather than of a single neuron, must be by the potent D-serine/glycine site antagonist CGP 78606 altered to cause a seizure. Many mechanisms that occur during (Tocris, Cat. No. 1493). TLE induction impairs working mem- epileptogenesis promote cell activity synchronization, through ory in rats, as shown by an increase in distance travelled in changes to neurotransmission in glutamatergic interneurons the Morris water maze. D-serine treatment ameliorated these and GABAergic connections. behavioral deficits (Klatteet al, 2013) (Figure 16). These results The most common form of epilepsy is temporal lobe epilepsy suggest that D-serine has a beneficial effect on cognitive (TLE), which is associated with cognitive changes. Changes in changes seen in TLE. the expression of various ion channels and neurotransmitter

Box 9: Epilepsy A full list of targets and related products are listed on pages 23-33

H2N N NH2

CO2H NH2 N N N Cl O NH CO Na 2 2 Cl (4098) Valproic acid (2815) (1611) (0806)

Inhibitor of neuronal NaV channels; Increases GABA levels; Inhibits glutamate release; Increases brain GABA; anticonvulsant anticonvulsant anticonvulsant anticonvulsant

H O - Me N NC N O .2Na+ HO2C CO2H H H N - 2 O2N N O

LY 379268 (2453) MPEP (1212) CNQX disodium salt (1045)

Highly selective group II mGlu agonist Potent mGlu5 antagonist; positive Potent non-NMDA iGluR antagonist; water soluble

allosteric modulator at mGlu4 version of CNQX (Cat. No. 0190)

Box 9: Epilepsy

20 | NEUROTRANSMISSION RESEARCH

Figure 16 | Assessment of D-serine and LTP in a Pilocarpine Induced TLE Model

A 400 B ** n. s. ** 350 (10) (10) (13) (14) 200 HPLC 300 (14) (12) ** ** 200 150 Sham Post-SE 150 50 fEPSP slope (% baseline)

50 % D-serine content

0 0 Native D-ser D-ser Cortex Hippocampus + CGP Sham C D Sham, D-serine 1800 1800 Post-SE 1600 1600 Post-SE, D-serine 1400 1400 1200 1200 1000 1000 800 ** 800 Distance (cm) Distance (cm) 600 *** 600 400 400 200 200 0 0 1 2 3 4 5 6 7 1 2 3 4 5 6 Day Trial (first day)

A) Following TLE induction, D-serine levels are significantly decreased in the cortex and hippocampus, determined by high performance liquid chromatography (HPLC). B) Induction of TLE significantly impairs LTP in the hippocampus, which is reversed by exogenous application of D-serine. The action of D-serine is blocked by addition of CGP 78608. C) TLE induction impairs working memory, shown by increased distance travelled in the Morris water maze on consecutive trials on the first day of training. D-serine had no significant effect on working memory. D) On the first trial of consecutive days of testing, TLE induction increased the distanced travelled to reach the platform, showing a cognitive deficit that was significantly improved by D-serine. n.s. – not significant, * p<0.05, ** p<0.01, *** p<0.005. Adapted from Klatte et al, 2013.

www.tocris.com | 21 Tocris Product Guide Series

List of Acronyms

Acronym Definition Acronym Definition

5-HT Serotonin MDH Medial dorsal hypothalamus

cAMP Cyclic mGluR Metabotropic glutamate receptors

COMT Catechol O-Methyltransferase MOR µ-opioid receptor

DA Dopamine NAcc Nucleus accumbens

DAG Diacylglycerol NMDARs NMDA receptors

DAT Dopamine transporter NE Norepinephrine

DDC Dopa decarboxylase NOP Nociceptin/orphanin FQ receptor

DOR δ-opioid receptor PLC Phospholipase C

DREADDs Designer Receptors Activated by Designer Drugs PSAM Pharmacologically Selective Actuator Module

EAAT Excitatory amino acid transporters PSEM Pharmacologically Selective Effector Molecule

EPSP Excitatory postsynaptic potential PVN Paraventricular nucleus of the hypothalamus

GPCRs G protein-coupled receptors SERT

ilPFC Infralimbic prefrontal cortex SSRI Selective serotonin reuptake inhibitor

IP3 Inositol 1,4,5-trisphosphate TH Tyrosine hydroxylase

KOR κ-opioid receptor TLE Temporal lobe epilepsy

LGIC Ligand gated ion channel TPH Tryptophan hydroxylase

LTP Long term potentiation VGLUT Vesicular glutamate transporter

MAO Monoamine oxidase VMATs Vesicular

MDD Major depressive disorder VTA Ventral tegmental area

22 | NEUROTRANSMISSION RESEARCH Neurotransmission Research Products

Class Cat. No. Product Name Primary Action Unit Size 5-HT Transporters Inhibitors 1427 Citalopram Highly potent and selective 5-HT uptake inhibitor 10 mg 50 mg 4798 (S)- Potent 5-HT and NA reuptake inhibitor; also blocks dopamine reuptake 10 mg 50 mg 4796 Selective serotonin reuptake inhibitor (SSRI) 10 mg 50 mg 0927 Fluoxetine 5-HT reuptake inhibitor 10 mg 50 mg

5-HT1 Receptors

Agonists 1080 (R)-(+)-8-Hydroxy-DPAT Selective 5-HT1A agonist; enantiomer of 8-Hydroxy-DPAT hydrobromide 10 mg (Cat. No. 0529) 50 mg

0529 8-Hydroxy-DPAT Selective 5-HT1A agonist; also has moderate affinity for 5-HT7 10 mg 50 mg

1317 CP 94253 Potent and selective 5-HT1B agonist 10 mg 50 mg

2451 LY 344864 Potent and selective 5-HT1F agonist 10 mg 50 mg

2854 Selective 5-HT1A partial agonist 10 mg 50 mg

Antagonists 1477 GR 127935 Potent and selective 5-HT1B/1D antagonist 10 mg 50 mg

1221 SB 224289 Selective 5-HT1B antagonist 10 mg 50 mg

1253 (S)-WAY 100135 Potent and selective 5-HT1A antagonist 10 mg 50 mg

4380 WAY 100635 Potent 5-HT1A antagonist; also D4 agonist 10 mg 50 mg

5-HT2 Receptors

Agonists 5171 NBOH-2C-CN High affinity and selective 5-HT2A agonist 10 mg 50 mg

2592 TCB-2 High affinity and potent 5-HT2A agonist 10 mg 50 mg

1801 WAY 161503 Potent and selective 5-HT2C agonist 10 mg 50 mg

Antagonists 4081 LY 266097 Selective 5-HT2B antagonist 10 mg 50 mg

4173 MDL 100907 Potent and selective 5-HT2A antagonist 10 mg 50 mg

1955 Potent 5-HT2 antagonist 10 mg 50 mg

2993 RS 127445 High affinity and selective 5-HT2B antagonist 10 mg 50 mg

2901 SB 242084 Selective 5-HT2C antagonist; brain penetrant 10 mg 50 mg

5-HT3-7 Receptors

Agonists 1968 AS 19 Potent 5-HT7 agonist 10 mg 50 mg

4374 BIMU 8 Potent 5-HT4 agonist 5 mg 25 mg

1205 SR 57227 Potent and selective 5-HT3 agonist 10 mg 50 mg

5589 WAY 181187 High affinity and selective 5-HT6 agonist 10 mg 50 mg

www.tocris.com | 23 Tocris Product Guide Series

Class Cat. No. Product Name Primary Action Unit Size

Antagonists 1658 GR 125487 Potent and selective 5-HT4 antagonist; active in vivo 10 mg 50 mg

2891 Ondansetron Selective 5-HT3 antagonist 10 mg 50 mg

1612 SB 269970 Potent and selective 5-HT7 antagonist; brain penetrant 10 mg 50 mg

3189 SB 399885 Potent and selective 5-HT6 antagonist 10 mg 50 mg Acetylcholine Muscarinic Receptors

Agonist 3569 Xanomeline Functionally selective M1 agonist 10 mg 50 mg

Antagonists 2292 AQ-RA 741 High affinity and selective M2 antagonist 10 mg 50 mg

2507 J 104129 Potent and selective M3 antagonist 10 mg

1071 Pirenzepine Selective M1 antagonist 100 mg

0909 Tropicamide Selective M4 antagonist 100 mg

3727 VU 0255035 Highly selective M1 antagonist 5 mg 25 mg

Modulators 3634 VU 0238429 Selective positive allosteric modulator of M5 receptors 10 mg 50 mg

4295 VU 0357017 Positive allosteric modulator of M1 receptors 10 mg 50 mg

3383 VU 152100 Positive allosteric modulator of M4 receptors 10 mg 50 mg Acetylcholine Nicotinic Receptors Agonists 0684 (±)- High affinity nAChR agonist 10 mg 3546 (-)-Nicotine Prototypical nAChR agonist 50 mg 6140 NS 6784 α7 nAChR agonist 10 mg 50 mg 3092 PHA 543613 Potent and selective α7 nAChR agonist 10 mg 5559 SSR 180711 Selective α7 nAChR partial agonist 10 mg 50 mg 3754 Selective α4β2 nAChR partial agonist; orally active 10 mg 50 mg Antagonists 2133 α- Selective α7 nAChR antagonist 1 mg 1340 α- MII Potent and selective α3β2 and β3 nAChR antagonist 500 µg 3123 α-Conotoxin PnIA Selective α3β2 nAChR antagonist 500 µg 2349 Dihydro-β-erythroidine α4β2, muscle type and Torpedo nAChR antagonist 10 mg 50 mg 2843 Non-competitive nAChR antagonist 10 mg 50 mg 1029 Methyllycaconitine Selective α7 neuronal nAChR antagonist 5 mg 25 mg Modulators 5963 CMPI Potent positive allosteric modulator of α4β2 nAChRs; also inhibitor of 10 mg (α4)2(β2)3, muscle-type and Torpedo nAChRs 5112 NS 9283 Positive allosteric modulator of α4β2 nAChRs 10 mg 50 mg 2498 PNU 120596 Positive allosteric modulator of α7 nAChRs; active in vivo 5 mg 25 mg Other 2736 Sazetidine A α4β2 nAChR ligand; may act as an agonist or a desensitizer 10 mg Acetylcholine Receptors: Non-selective Compounds Agonists 2810 Carbamoylcholine Non-selective agonist 100 mg

24 | NEUROTRANSMISSION RESEARCH

Neurotransmission Research Products – continued

Class Cat. No. Product Name Primary Action Unit Size Acetylcholine Transporters Inhibitors 0653 (±)- Inhibits ACh transport 250 mg Others 5725 ML 352 High affinity and selective presynaptic transporter (CHT) inhibitor 10 mg 50 mg Adenylyl Cyclase Activators 1099 Forskolin Adenylyl cyclase activator 10 mg 50 mg Inhibitors 3834 KH 7 Selective soluble adenylyl cyclase inhibitor 10 mg 50 mg 1435 SQ 22536 Adenylyl cyclase inhibitor 10 mg 50 mg Adrenergic α Receptors

Agonists 1052 A 61603 α1A agonist 10 mg 50 mg

5160 Potent and highly selective α2 agonist 10 mg 50 mg

Antagonists 2937 Selective α2 antagonist 10 mg 50 mg

2964 α1 antagonist 50 mg

0623 α1 and α2B antagonist; also MT3 antagonist 100 mg

0987 RS 79948 Potent and selective α2 antagonist 10 mg 50 mg Adrenergic β Receptors

Agonists 1499 CL 316243 Highly selective β3 agonist 10 mg 50 mg

1448 Potent and selective β2 agonist 10 mg 50 mg 1747 Isoproterenol Standard selective β agonist 100 mg

0950 Selective β1 partial agonist 10 mg 50 mg

Antagonists 2685 Potent and non-selective β antagonist; also α1 antagonist 50 mg

1024 CGP 20712 Highly potent and selective β1 antagonist 10 mg 50 mg

1511 SR 59230A Potent and selective β3 antagonist 10 mg 50 mg

Inverse Agonists 0821 ICI 118,551 Highly selective β2 inverse agonist 10 mg 50 mg Adrenergic Transporters Inhibitors 3067 Selective inhibitor of noradrenalin transporters 50 mg 1982 Reboxetine Potent and selective noradrenalin uptake inhibitor; orally active 10 mg 50 mg 2011 Tomoxetine Potent and selective noradrenalin re-uptake inhibitor 10 mg 50 mg AMPA Receptors Agonists 0254 (S)-AMPA Selective AMPA agonist; active isomer of (RS)-AMPA (Cat. No. 0169) 1 mg 10 mg 50 mg Antagonists 2312 DNQX disodium salt Selective non-NMDA iGluR antagonist; water-soluble salt of DNQX 10 mg (Cat. No. 0189) 50 mg 2555 GYKI 53655 Non-competitive AMPA antagonist; also kainate antagonist 10 mg 50 mg 2766 Naspm Ca2+-permeable AMPA antagonist 10 mg 1044 NBQX disodium salt Potent AMPA antagonist; more water-soluble form of NBQX (Cat. No. 0373) 1 mg 10 mg 50 mg

www.tocris.com | 25 Tocris Product Guide Series

Class Cat. No. Product Name Primary Action Unit Size

Modulators 2980 CX 546 AMPA potentiator 10 mg 50 mg 0713 Positive allosteric modulator of AMPA receptors; inhibits AMPA 10 mg desensitization 50 mg 6278 JNJ 55511118 High affinity and selective negative modulator of AMPA receptors 5 mg containing TARP-γ8 25 mg Caged Compounds

5785 MDNI-caged-L-glutamate Stable photoreleaser of l-glutamate 10 mg

1490 MNI-caged-L-glutamate Stable photoreleaser of l-glutamate 10 mg 50 mg 2224 MNI-caged-NMDA Caged NMDA 10 mg 3991 NPEC-caged-serotonin Caged serotonin 10 mg 50 mg 4932 RuBi-Dopa Caged dopamine; exhibits two-photon sensitivity 10 mg 3400 RuBi-GABA Caged GABA; excitable at visible wavelengths 10 mg cAMP Antagonists 1337 cAMPS-Rp cAMP antagonist 1 mg Others 5255 6-Bnz-cAMP Cell-permeable cAMP analog 1 mg 1140 8-Bromo-cAMP Cell-permeable cAMP analog 10 mg 50 mg 1141 Dibutyryl-cAMP Cell-permeable cAMP analog 10 mg 50 mg

Cannabinoid CB1 Receptors

Agonists 1319 ACEA Potent and highly selective CB1 agonist 5 mg 25 mg

1782 (R)-(+)-Methanandamide (in Potent and selective CB1 agonist; supplied in water-soluble emulsion 5 mg Tocrisolve™ 100) 25 mg

Antagonists 1117 AM 251 Potent CB1 antagonist; also GPR55 agonist 1 mg 10 mg 50 mg

5443 AM 6545 High affinity and selective CB1 antagonist 10 mg 50 mg

Inverse Agonists 1115 AM 281 Potent and selective CB1 inverse agonist 10 mg 50 mg

0923 SR 141716A Selective CB1 inverse agonist 10 mg 50 mg

Modulators 2957 Org 27569 Potent allosteric modulator of CB1 receptors 10 mg 50 mg

5321 PSNCBAM-1 Negative allosteric modulator of CB1 receptors 10 mg 50 mg

Cannabinoid CB2 Receptors

Agonists 3088 HU 308 Potent and selective CB2 agonist 10 mg 50 mg

1343 JWH 133 Potent and selective CB2 agonist 10 mg

Inverse Agonists 1120 AM 630 Selective CB2 inverse agonist 10 mg 50 mg

5039 SR 144528 High affinity and selective CB2 inverse agonist 10 mg 50 mg

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Neurotransmission Research Products – continued

Class Cat. No. Product Name Primary Action Unit Size Cannabinoid Receptors: Non-selective Compounds Agonists 1298 2-Arachidonylglycerol Endogenous and non-selective CB agonist; potent GPR55 agonist 10 mg 1339 Endogenous and non-selective CB agonist 5 mg 25 mg 0949 CP 55,940 Potent and non-selective CB agonist 10 mg 50 mg 1038 WIN 55,212-2 Highly potent and non-selective CB agonist 10 mg 50 mg Cannabinoid Transporters Inhibitors 1116 AM 404 Anandamide transport inhibitor 10 mg 50 mg

1570 (-)-Cannabidiol AMT inhibitor; also GPR55 antagonist, weak CB1 antagonist, CB2 inverse 10 mg agonist; natural cannabinoid 50 mg Catechol O-Methyltransferase Inhibitors 0483 OR-486 COMT inhibitor 50 mg 5864 COMT inhibitor; also inhibits aggregation 10 mg 50 mg Chemogenetics DREADD Ligands 4936 Clozapine N-oxide Activator of muscarinic DREADDs 10 mg 50 mg 6329 Clozapine N-oxide Activator of muscarinic DREADDs; water-soluble version of Clozapine 10 mg dihydrochloride N-oxide (Cat. No. 4936) 50 mg 5548 DREADD agonist 21 Potent muscarinic DREADD agonist 10 mg 50 mg 6422 DREADD agonist 21 Potent muscarinic DREADD agonist; water-soluble version of DREADD 10 mg dihydrochloride agonist 21 (Cat. No. 5548) 50 mg 5549 Perlapine Potent muscarinc DREADD agonist 10 mg 50 mg 5611 Salvinorin B Activates the κ-opioid DREADD (KORD) 1 mg L141F L141F,Y115F PSEMs 6425 PSEM 308 PSAM -GlyR and PSAM -5-HT3 chimeric ion channel agonist 5 mg 25 mg L141F L141F,Y115F 6426 PSEM 89S PSAM -GlyR and PSAM -5-HT3 chimeric ion channel agonist 5 mg 25 mg Inhibitors 4385 Donepezil Potent AChE inhibitor 10 mg 50 mg 0622 Physostigmine inhibitor 10 mg Decarboxylases Inhibitors 0455 (S)-(-)-Carbidopa Aromatic L-amino acid decarboxylase inhibitor 25 mg 0584 L-(-)-α- Aromatic L-amino acid decarboxylase inhibitor 1 g

Dopamine D1-like Receptors

Agonists 1701 A 77636 Potent and selective D1-like agonist; orally active 10 mg 50 mg

0884 Selective D1-like agonist 5 mg 25 mg

1447 SKF 81297 D1 agonist 10 mg 50 mg

5719 SKF 82958 D1 agonist 10 mg 50 mg

Antagonists 1674 LE 300 Potent and selective D1 antagonist 10 mg

2299 SCH 39166 High affinity D1-like antagonist 10 mg 50 mg

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Class Cat. No. Product Name Primary Action Unit Size

Dopamine D2-like Receptors

Agonists 4552 A 412997 Selective D4 agonist 5 mg 25 mg

1243 (+)-PD 128907 High affinity D3 agonist (D3 ≥ D2 > D4) 10 mg 50 mg

1061 (-)-Quinpirole Selective D2-like agonist 10 mg 50 mg

2773 Selective D2 agonist 10 mg 50 mg

Antagonists 1003 L-741,626 High affinity D2 antagonist 10 mg 50 mg

1002 L-745,870 Highly selective D4 antagonist 10 mg 50 mg

4207 SB 277011A Selective D3 antagonist 10 mg 50 mg Dopamine Receptors: Non-selective Compounds

Antagonists 0444 Clozapine with some D4 selectivity; also 5-HT2A/2C antagonist 50 mg 500 mg Other 3788 L-DOPA Dopamine precursor 50 mg Dopamine Transporters Inhibitors 0513 GBR 12783 Potent and selective DA uptake inhibitor 10 mg 50 mg 0421 GBR 12909 Selective DA uptake inhibitor; also σ ligand 10 mg 50 mg δ Opioid Receptors Agonists 1431 DPDPE Selective δ agonist 1 mg 0764 SNC 80 Highly selective non-peptide δ agonist 10 mg 50 mg Antagonists 0740 Naltrindole Selective non-peptide δ antagonist 10 mg 50 mg Modulators 5983 BMS 986187 Potent positive allosteric modulator of δ receptors 10 mg 50 mg Fatty Acid Amide Hydrolase (FAAH) Inhibitors 4355 TC-F 2 Potent, reversible and selective FAAH inhibitor 10 mg 50 mg 4612 URB 597 Potent and selective FAAH inhibitor 10 mg 50 mg

GABAA Receptors

Agonists 3250 L-838,417 GABAA partial agonist; displays subtype selectivity 10 mg 50 mg

0289 Muscimol Potent GABAA agonist; also GABAA-ρ partial agonist 1 mg 10 mg 50 mg

Antagonists 2503 (-)- methiodide Water-soluble GABAA antagonist 10 mg 50 mg

1128 GABAA antagonist 1 g

1262 SR 95531 Competitive and selective GABAA antagonist 10 mg 50 mg 1328 Flumazenil Benzodiazepine antagonist 10 mg 50 mg

1327 L-655,708 Benzodiazepine inverse agonist; selective for α5-containing GABAA 10 mg receptors 50 mg 0655 Zolpidem Benzodiazepine agonist 10 mg 50 mg

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Neurotransmission Research Products – continued

Class Cat. No. Product Name Primary Action Unit Size

Modulators 3653 Positive allosteric modulator of GABAA receptors 10 mg

2867 GABAA modulator; also indirect NMDA antagonist and Kv7 channel activator 10 mg 50 mg

2531 Ganaxolone Potent positive allosteric modulator of GABAA receptors 10 mg 50 mg Others 1471 GABA-mimetic; selectively interacts with β2- and β3-subunit containing 10 mg GABAA receptors 50 mg

GABAA-ρ Receptors

Antagonists 0379 SKF 97541 GABAA-ρ antagonist; also highly potent GABAB agonist 10 mg 50 mg

0807 THIP GABAA-ρ antagonist; also GABAA agonist 50 mg

1040 TPMPA Selective GABAA-ρ antagonist 10 mg 50 mg

GABAB Receptors

Agonists 0796 (R)- Selective GABAB agonist; active enantiomer of (RS)-Baclofen 10 mg (Cat. No. 0417) 50 mg

Antagonists 1246 CGP 52432 Potent and selective GABAB antagonist 10 mg 50 mg

1088 CGP 54626 Potent and selective GABAB antagonist 10 mg 50 mg

1248 CGP 55845 Potent and selective GABAB antagonist 10 mg 50 mg

Modulators 1513 CGP 7930 Positive allosteric modulator of GABAB receptors 10 mg 50 mg GABA Miscellaneous Compounds Other 0344 GABA Endogenous agonist 1 g 0806 Gabapentin Increases brain GABA; anticonvulsant 10 mg 50 mg GABA Transporters Inhibitors 1779 NNC 711 Selective GAT-1 inhibitor 10 mg 50 mg 0768 GABA uptake inhibitor; also inhibits glutamate release and blocks 25 mg

NaV channels 1561 (S)-SNAP 5114 GABA uptake inhibitor 10 mg 50 mg Glutamate Transporters Inhibitors 0111 Dihydrokainic acid EAAT2 (GLT-1)-selective non-transportable inhibitor of L-glutamate and 1 mg L-aspartate uptake 10 mg 50 mg 1223 DL-TBOA Selective non-transportable inhibitor of EAATs 10 mg 50 mg 2532 TFB-TBOA High affinity EAAT1 and EAAT2 blocker 1 mg 10 mg 2652 WAY 213613 Potent, non-substrate EAAT2 inhibitor 10 mg 50 mg GPR55 Agonists 1297 Abn-CBD Selective GPR55 agonist; neurobehaviorally inactive 10 mg 2797 O-1602 Potent GPR55 agonist 10 mg Antagonists 4959 CID 16020046 Selective GPR55 antagonist 5 mg 25 mg G Proteins (Heterotrimeric) Inhibitors 5642 CMPD101 Potent and selective GRK2/3 inhibitor 10 mg 3090 Gallein Inhibitor of βγ signaling 50 mg

Other 3097 Pertussis Toxin Catalyzes ADP-ribosylation of Gi, Go and Gt 50 µg

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Class Cat. No. Product Name Primary Action Unit Size Hydroxylases Inhibitors 0938 p-Chlorophenylalanine Tryptophan hydroxylase inhibitor 100 mg

IP3 Receptors

Antagonists 1224 2-APB IP3 receptor antagonist; also TRP 10 mg 50 mg 2+ Others 1280 (-)-Xestospongin C Reported inhibitor of IP3-dependent Ca release 10 µg Kainate Receptors Agonists 0269 Potent and selective kainate agonist 1 mg 0222 Kainic acid Kainate agonist; excitant and neurotoxin 1 mg 10 mg 50 mg Antagonists 2728 ACET Potent kainate antagonist; displays selectivity for GluK1-containing 10 mg receptors 1045 CNQX disodium salt Potent non-NMDA iGluR antagonist; more water soluble form of CNQX 1 mg (Cat. No. 0190) 10 mg 50 mg 3621 UBP 310 GluK1-selective kainate antagonist 10 mg 50 mg κ Opioid Receptors Agonists 3195 Dynorphin A Endogenous κ agonist 1 mg 5519 (-)-Pentazocine κ agonist; antinociceptive 10 mg 0495 (±)-U-50488 Selective κ agonist 25 mg Antagonists 0794 DIPPA Selective and irreversible κ antagonist 10 mg 50 mg 0347 nor-Binaltorphimine Selective κ antagonist 10 mg 50 mg Ketamine and Metabolites 5982 cis-6-Hydroxynorketamine Enhances AMPA currents: antidepressant 10 mg 50 mg 6094 2R,6R-Hydroxynorketamine Enhances AMPA currents; decreases D-serine (a NMDA co-agonist); lacks 10 mg ketamine-related side effects 6095 2S,6S-Hydroxynorketamine Decreases D-serine (a NMDA co-agonist); antidepressant 10 mg 3131 Ketamine Non-competitive NMDA antagonist 50 mg 4379 (S)-(+)-Ketamine NMDA antagonist; enantiomer of ketamine (Cat. No. 3131); 50 mg neuroprotective. 1970 Norketamine Potent non-competitive NMDA antagonist; antinociceptive 10 mg 50 mg 5996 (R)-Norketamine NMDA ; metabolite of ketamine (Cat. No. 3131) 10 mg 6112 (S)-Norketamine NMDA receptor modulator; metabolite of ketamine (Cat. No. 3131) 10 mg MAGL Inhibitors 3836 JZL 184 Potent MAGL inhibitor 10 mg 50 mg 4872 KML 29 Highly potent and selective MAGL inhibitor 10 mg 50 mg Metabotropic Glutamate Receptors

Agonists 2385 AMN 082 Selective mGlu7 agonist 10 mg 50 mg

3695 CHPG Sodium salt Selective mGlu5 agonist; sodium salt of CHPG (Cat. No. 1049) 10 mg 50 mg

1302 (S)-3,4-DCPG Potent and selective mGlu8a agonist 10 mg 50 mg 0975 DCG IV Highly potent group II mGlu agonist; also NMDA agonist 1 mg 10 mg 50 mg

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Class Cat. No. Product Name Primary Action Unit Size

0805 (S)-3,5-DHPG Selective group I mGlu agonist; active enantiomer of 3,5-DHPG 5 mg (Cat. No. 0342) 10 mg 0103 L-AP4 Selective group III mGlu agonist 1 mg 10 mg 50 mg 0188 L- Group I mGlu agonist; also AMPA agonist 1 mg 10 mg 50 mg 5064 LY 379268 disodium salt Selective group II mGlu agonist; sodium salt of LY 379268 (Cat. No. 2453) 10 mg 50 mg Antagonists 0972 CPPG Potent group III mGlu antagonist 5 mg 25 mg

2333 JNJ 16259685 Highly potent, mGlu1-selective non-competitive antagonist 10 mg 50 mg 4062 LY 341495 disodium salt Potent and selective group II mGlu antagonist; disodium salt of LY 341495 1 mg (Cat. No. 1209) 10 mg 50 mg

1237 LY 367385 Selective mGlu1a antagonist 10 mg 50 mg

2963 MMPIP Potent and selective allosteric mGlu7 antagonist 1 mg 10 mg 50 mg

1212 MPEP Potent mGlu5 antagonist; positive allosteric modulator of mGlu4 receptors 10 mg 50 mg

2921 MTEP Potent and selective mGlu5 antagonist 10 mg 50 mg

Modulators 4416 ADX 10059 Negative allosteric modulator of mGlu5 receptors 10 mg 50 mg

4048 BINA Selective positive allosteric modulator of mGlu2 receptors 10 mg 50 mg

3283 LY 487379 Selective positive allosteric modulator of mGlu2 receptors 10 mg 50 mg

4982 ML 337 Selective negative allosteric modulator of mGlu3 receptors 10 mg 50 mg

4323 VU 0360172 Positive allosteric modulator of mGlu5 receptors 10 mg 50 mg

5377 VU 0483605 Positive allosteric modulator of mGlu1 receptors 10 mg 50 mg Monoamine Oxidase Inhibitors 1095 (R)-(-)-Deprenyl MAO-B inhibitor 1 g 4395 Reversible MAO-A inhibitor 10 mg 50 mg 4308 Selective and irreversible MAO-B inhibitor 50 mg µ Opioid Receptors Agonists 1171 DAMGO Selective μ agonist 1 mg 1055 Endomorphin-1 Potent and selective μ agonist 5 mg Antagonists 1560 CTAP Potent and selective μ antagonist 1 mg 1578 CTOP Potent and selective μ antagonist 1 mg 0926 β-Funaltrexamine Irreversible and selective μ antagonist 10 mg 50 mg

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Class Cat. No. Product Name Primary Action Unit Size NMDA Receptors Agonists 3406 GLYX 13 NMDA partial agonist; acts at the glycine site 1 mg 0114 NMDA Selective NMDA agonist 50 mg 500 mg Antagonists 0247 (R)-CPP Potent NMDA antagonist; more active enantiomer of (RS)-CPP 10 mg (Cat. No. 0173) 50 mg 0106 D-AP5 Potent and selective NMDA antagonist; more active form of DL-AP5 1 mg (Cat. No. 0105) 10 mg 50 mg 100 mg 0773 NMDA antagonist; acts at ion channel site 50 mg 0924 (+)-MK 801 Non-competitive NMDA antagonist; acts at ion channel site 10 mg 50 mg 5018 PEAQX Potent and GluN2A-selective NMDA antagonist 10 mg 4801 QNZ 46 GluN2C/GluN2D-selective NMDA non-competitive antagonist 10 mg 50 mg 1594 Ro 25-6981 GluN2B-selective NMDA antagonist 1 mg 10 mg 50 mg 4154 TCN 201 GluN1/GluN2A-selective NMDA antagonist 10 mg 50 mg NOP Receptors Agonists 0910 Nociceptin Endogenous NOP agonist 1 mg 3240 SCH 221510 Potent and selective NOP agonist 10 mg 50 mg Antagonists 2598 (±)-J 113397 Potent and selective NOP antagonist 10 mg 50 mg 3573 SB 612111 Selective NOP antagonist 10 mg 50 mg Opioid Receptors: Non-selective Compounds Agonists 1889 [Leu5]-Enkephalin Endogenous opioid agonist 25 mg Antagonists 0599 Naloxone Non-selective opioid antagonist 100 mg 0677 Naltrexone Non-selective opioid antagonist 100 mg Other 3137 Neuropeptide FF Antiopioid neuropeptide; endogenous NPFF1 and NPFF2 agonist 1 mg Phospholipase C Activators 1941 m-3M3FBS Phospholipase C activator 10 mg Inhibitors 1437 D609 Selective PC-PLC inhibitor 10 mg 50 mg 1842 RHC 80267 Diacylglycerol lipase inhibitor 10 mg 50 mg 1268 U 73122 Phospholipase C inhibitor 10 mg 50 mg Purinergic (P2X) Receptors

Agonists 3312 BzATP P2X7 agonist; also P2X1 and P2Y1 partial agonist 1 mg

Antagonists 3579 5-BDBD Potent P2X4 antagonist 10 mg 50 mg

2972 A 438079 Competitive P2X7 antagonist 10 mg 50 mg

5545 BX 430 Selective P2X4 allosteric antagonist 10 mg 50 mg

5299 JNJ 47965567 Potent and selective P2X7 antagonist; brain penetrant 10 mg 50 mg

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Neurotransmission Research Products – continued

Class Cat. No. Product Name Primary Action Unit Size

2548 NF 110 Potent P2X3 antagonist 10 mg 50 mg

1391 NF 449 Highly selective P2X1 antagonist 10 mg 50 mg Purinergic (P2Y) Receptors

Agonists 1624 2-Methylthioadenosine Potent agonist at P2Y1, and P2Y13 10 mg diphosphate

2157 MRS 2365 Highly potent and selective P2Y1 agonist 1 mg

2502 MRS 2693 trisodium salt Selective P2Y6 agonist 1 mg

3892 NF 546 Selective P2Y11 agonist 10 mg

3280 2-ThioUTP tetrasodium salt Potent and selective P2Y2 agonist 1 mg

Antagonists 4890 AR-C 118925XX Selective and competitive P2Y2 antagonist 5 mg

0900 MRS 2179 tetrasodium salt Selective P2Y1 antagonist 10 mg 50 mg

2159 MRS 2500 Highly potent and selective P2Y1 antagonist 1 mg

2146 MRS 2578 Selective P2Y6 antagonist 10 mg 50 mg

3983 PSB 0739 Highly potent P2Y12 antagonist 10 mg 50 mg Purinergic Receptors Non-selective compounds Agonists 4080 ATPγS tetralithium salt Non-selective P2 agonist; analog of ATP (Cat. No. 3245) 10 mg 1062 2-Methylthioadenosine Non-selective P2 agonist 10 mg triphosphate 3209 α,β-Methyleneadenosine Non-selective P2 agonist 10 mg 5ʹ-triphosphate Antagonist 0625 PPADS tetrasodium salt Non-selective P2 antagonist 10 mg 50 mg Vesicular Monoamine Transporters Inhibitors 2742 Reserpine Inhibitor of vesicular monoamine transport 1 g 2175 Potent inhibitor of vesicular monoamine transport 10 mg 50 mg Others 5911 FFN 200 Selective fluorescent VMAT2 substrate 10 mg 5043 FFN 206 Fluorescent VMAT2 substrate 10 mg

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Further Reading and Featured Publications

Dopaminergic Transmission Beaulieu et al. (2015) Dopamine receptors – IUPHAR Review 13. Br J Pharmacol. 172, 1. Bertler & Rosengren (1959) Occurrence and distribution of dopamine in brain and other tissues. Experientia. 15, 10. Eisenhofer et al. (2004) Catecholamine : a contemporary view with implications for physiology and medicine. Pharmacol Rev. 56, 331. Kebabian & Calne (1979) Multiple receptors for dopamine. . 277, 93. Laverty & Taylor (1970) Effects of intraventricular 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine) on rat behavior and brain catecholamine metabolism. Br J Pharmacol. 40, 836. Marsden (2006) Dopamine: the rewarding years. Br J Pharmacol. 147, S136.

Glutamatergic Transmission Anderson & Swanson (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia. 32, 1. Curtis & Watkins (1965) The pharmacology of amino acids related to gamma-aminobutyric acid.Pharmacol Rev. 17, 347. MacDermott et al. (1986) NMDA-receptor activation increases cytoplasmic concentration in cultured spinal cord neurones. Nature. 321, 519. McCulloch et al. (1974) The differential sensitivity of spinal interneurones and Renshaw cells to Kainate and N-methyl-D-aspartate. Exp Brain Res. 21, 515. Nakanishi (1992) Molecular diversity of glutamate receptors and implications for brain function. Science. 258, 597. Nicoletti et al. (2011) Metabotropic glutamate receptors: from the workbench to the bedside. Neuropharmacology. 60, 1017. Traynelis et al. (2010) Glutamate receptor ion channels: structure, regulation and function. Pharmacol Rev. 62, 405. Watkins & Jane (2006) The glutamate story.Br J Pharmacol. 147, S100.

Opioid Peptide Transmission Feng et al. (2012) Current research on opioid receptor function. Curr Drug Targets. 13, 230. Kelly (2013) Efficacy and ligand bias at the µ-opioid receptor.Br J Pharmacol. 169, 1430. Noble et al. (2015) The opioid receptors as targets for drug abuse .Br J Pharmacol. 172, 3964. Schwarzer (2009) 30 years of dynorphins – new insights on their functions in neuropsychiatric diseases. Pharmacol Ther. 123, 353. Toll et al. (2016) Nociceptin/Orphanin FQ receptor structure, signaling, ligands, functions, and interactions with opioid systems. Pharmacol Rev. 68, 419.

Serotonergic Transmission Bradley et al. (1986) Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology. 25, 563. Blakely et al. (1994) Molecular physiology of norepinephrine and serotonin transporters. J Exp Biol. 196, 263. Hannon & Hoyer (2008) Molecular biology of 5-HT receptors. Behav Brain Res. 195, 198. Twarog & Page (1953) Serotonin content of some mammalian tissues and urine and a method for its determination. Am J Physiol. 175, 157. Yohn et al. (2017) The role of 5-HT receptors in depression.Mol Brain. 10, 28.

Chemogenetics Campbell & Marchant (2018) The use of chemogenetics in behavioural neuroscience: receptor variants, targeting approaches and caveats. Br J Pharmacol. 175, 994. Sternson & Roth (2014) Chemogenetic tools to interrogate brain functions. Annu Rev Neurol. 37, 387. Vardy et al. (2016) A new DREADD facilitates the multiplex chemogenetic interrogation of behavior. Neuron. 86, 936.

34 | NEUROTRANSMISSION RESEARCH

Depression Carr et al. (2010) Antidepressant-like effects of kappa-opioid receptor antagonists in Wistar Kyoto rats. Neuropsychopharmacology. 35, 752. McLaughlin et al. (2006a) Prior activation of kappa opioid receptors by U50,488 mimics repeated forced swim stress to potentiate cocaine place preference conditioning. Neuropsychopharmacology. 31, 787. McLaughlin et al. (2006b) Social defeat stress-induced behavioral responses are mediated by the endogenous kappa opioid system. Neuropsychopharmacology. 31, 1241. Pare (1992) The performance of WKY rats on three tests of emotional behavior.Physiol Behav. 51, 1051. Pare (1994) Openfield, learned helplessness, conditioned defensive burying, and forced-swim tests in WKY rats. Physiol Behav. 55, 433. Pearson et al. (2006) Identifying genes in monoamine nuclei that may determine stress vulnerability and depressive behavior in Wister-Kyoto rats. Neuropsychopharmacology. 31, 2449. Pringle et al. (2011) A cognitive neuropsychological model of antidepressant drug action. Prog Neuropyschopharmacol Biol Psychiatry. 35, 1586. Stuart et al. (2013) A translational rodent assay of affective biases in depression and antidepressant therapy. Neuropsychopharmacology. 38, 1625.

Ketamine Metabolites in Depression Berman et al. (2000) Antidepressant effects of ketamine in depressed patients.Biol Psychiatry. 47, 351. Holtman et al. (2008) Effects of norketamine in rodent models of persistent pain.Pharmacol Biochem Behav. 90, 676. Zanos et al. (2016) NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature. 533, 481. Zarate et al. (2006) A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression. Arch Gen Pyschiatry. 62, 856.

Addiction Baler & Volkow (2006) Drug addiction: the neurobiology of disrupted self-control. Trends Mol Med. 12, 559. Ferraro et al. (2010a) A novel mechanism of cocaine to enhance dopamine D2-like receptor mediated neurochemical and behavioral effects. Anin vivo and in vitro study. Neuropsychopharmacology. 37, 1856. Ferraro et al. (2010b) Nanomolar concentrations of cocaine enhance D2-like agonist-induced inhibition of K+-evoked [3H]-dopamine efflux from rat striatal synaptosomes: a novel action of cocaine.J Neural Transm. 117, 593. Marchant et al. (2010) Medial dorsal hypothalamus mediated the inhibition of reward seeking after extinction.J Neurosci. 30, 14102. Rice & Cragg (2008) Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway. Brain Res Rev. 58, 303.

Epilepsy Goldman & Coulter (2013) Mechanisms of epileptogenesis: a convergence on neuronal circuit dysfunction. Nat Rev Neurosci. 14, 337. Klatte et al. (2013) Impaired D-serine-mediated cotransmission mediates cognitive dysfunction in epilepsy. J Neurosci. 33, 13066.

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