US2005015820A1PendingUtilityA1

Assessment of neurons in the arcuate nucleus to screen for agents that modify feeding behavior

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Priority: Sep 24, 2001Filed: Sep 24, 2002Published: Jan 20, 2005
Est. expirySep 24, 2021(expired)· nominal 20-yr term from priority
Inventors:Michael CowleyRoger D. ConeMalcolm LowAndrew Butler
A61P 7/00A61P 43/00A61P 3/04G01N 2333/665G01N 33/5088G01N 33/5058A61P 1/14G01N 2333/70571C07K 14/665A01K 2267/03C12Q 1/6897C07K 2319/60A01K 2227/105A01K 67/0275C12N 2830/008G01N 33/5082A01K 2217/05
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Claims

Abstract

Screening methods of use in identifying agents that affect caloric intake, food intake, appetite, and energy expenditure are disclosed herein. These methods are used to identify agents of use in treating obesity, or that can be used to decrease the weight of a subject. These methods can also be used to identify agents of use in treating anorexia or cachexia and can be used to increase appetite and to increase the weight and lean body mass of a subject.

Claims

exact text as granted — not AI-modified
1 . A method for screening for an agent that affects caloric intake, comprising 
 contacting a histological section of an arcuate nucleus from a mouse expressing a marker in proopiomelanocortin neurons with an agent to be tested, wherein the mouse comprises a transgene comprising a nucleic acid encoding the marker operably linked to a proopiomelanocortin nucleic acid sequence, wherein the proopiomelanocortin nucleic acid sequence directs expression of the marker in proopiomelanocortin neurons in the arcuate nucleus of the mouse; and    assaying for an electrophysiological response of a proopiomelanocortin neuron in the histological section, thereby determining if the agent affects caloric intake.    
     
     
         2 . The method of  claim 1 , wherein the agent specifically binds a receptor on the proopiomelanocortin neuron.  
     
     
         3 . The method of  claim 1 , wherein the agent specifically binds a melanocortin receptor or a μ-opioid receptor.  
     
     
         4 . The method of  claim 3 , wherein the agent specifically binds the melanocortin 3 receptor.  
     
     
         5 . The method of  claim 1 , wherein the agent specifically binds a receptor on a neuropeptide Y neuron.  
     
     
         6 . The method of  claim 5 , wherein the agent specifically binds the Y2 receptor.  
     
     
         7 . The method of  claim 1 , wherein the electrophysiological response comprises induction of current or a change in the membrane potential of the proopiomelanocortin neuron.  
     
     
         8 . The method of  claim 1 , wherein the electrophysiological response comprises depolarization of the proopiomelanocortin neuron.  
     
     
         9 . The method of  claim 1 , wherein the marker is a fluorescent protein.  
     
     
         10 . The method of  claim 9 , wherein the fluorescent protein is green fluorescent protein.  
     
     
         11 . The method of  claim 1 , wherein assaying an electrophysiological response comprises a whole cell recording.  
     
     
         12 . The method of  claim 1 , wherein assaying an electrophysiological response comprises determining a resting membrane potential of a proopiomelanocortin neuron.  
     
     
         13 . The method of  claim 1 , wherein assaying an electrophysiological response comprises determining a GABA-mediated inhibitory postsynaptic current or determining the action potential frequency of a proopiomelanocortin neuron.  
     
     
         14 . The method of  claim 1 , wherein the proopiomelanocortin nucleic acid sequences comprises a proopiomelanocortin regulatory nucleic acid sequence and a nucleic acid sequence encoding a marker.  
     
     
         15 . The method of  claim 1 , wherein the agent is a polypeptide, a chemical compound, a salt, or a neurotransmitter.  
     
     
         16 . The method of  claim 1 , wherein the agent is not PYY and is not a polypeptide fragment of PYY that is more than 10 amino acids in length.  
     
     
         17 . The method of  claim 1 , wherein the agent is not a polypeptide.  
     
     
         18 . A compound identified by the method of  claim 16 .  
     
     
         19 . A compound identified by the method of  claim 17 .  
     
     
         20 . A method for screening for an agent that affects appetite, comprising 
 contacting a histological section of an arcuate nucleus from a mouse expressing a marker in proopiomelanocortin neurons with an agent to be tested, wherein the mouse comprises a transgene comprising a nucleic acid encoding the marker operably linked to a proopiomelanocortin nucleic acid sequence, wherein the proopiomelanocortin nucleic acid sequence directs expression of the marker in proopiomelanocortin neurons in the arcuate nucleus of the mouse; and    assaying for an electrophysiological response of a proopiomelanocortin neuron in the histological section, thereby determining if the agent affects appetite.    
     
     
         21 . The method of  claim 20 , wherein the agent specifically binds a receptor on the proopiomelanocortin neuron.  
     
     
         22 . The method of  claim 20 , wherein the agent specifically binds a melanocortin receptor or a μ-opioid receptor.  
     
     
         23 . The method of  claim 22 , wherein the agent specifically binds the melanocortin 3 receptor.  
     
     
         24 . The method of  claim 20 , wherein the agent specifically binds a receptor on a neuropeptide Y neuron.  
     
     
         25 . The method of  claim 24 , wherein the agent specifically binds the Y2 receptor.  
     
     
         26 . The method of  claim 20 , wherein the electrophysiological response comprises induction of current or a change in the membrane potential of the proopiomelanocortin neuron.  
     
     
         27 . The method of  claim 20 , wherein the electrophysiological response comprises depolarization of the proopiomelanocortin neuron.  
     
     
         28 . The method of  claim 20 , wherein the marker is a fluorescent protein.  
     
     
         29 . The method of  claim 28 , wherein the fluorescent protein is green fluorescent protein.  
     
     
         30 . The method of  claim 20 , wherein assaying an electrophysiological response comprises a whole cell recording.  
     
     
         31 . The method of  claim 20 , wherein assaying an electrophysiological response comprises determining a resting membrane potential of a proopiomelanocortin neuron.  
     
     
         32 . The method of  claim 20 , wherein assaying an electrophysiological response comprises determining a GABA-mediated inhibitory postsynaptic current or determining the action potential frequency of a proopiomelanocortin neuron.  
     
     
         33 . The method of  claim 20 , wherein the proopiomelanocortin nucleic acid sequences comprises a proopiomelanocortin regulatory nucleic acid sequence and a nucleic acid sequence encoding a marker.  
     
     
         34 . The method of  claim 20 , wherein the agent is a polypeptide, a chemical compound, a salt, or a neurotransmitter.  
     
     
         35 . The method of  claim 20 , wherein the agent is not PYY and is not a polypeptide fragment of PYY that is more than 10 amino acids in length.  
     
     
         36 . The method of  claim 20 , wherein the agent is not a polypeptide.  
     
     
         37 . A compound identified by the method of  claim 35 .  
     
     
         38 . A compound identified by the method of  claim 36 .  
     
     
         39 . A method for screening for an agent that affects food intake, comprising 
 contacting a histological section of an arcuate nucleus from a mouse expressing a marker in proopiomelanocortin neurons with an agent to be tested, wherein the mouse comprises a transgene comprising a nucleic acid encoding the marker operably linked to a proopiomelanocortin nucleic acid sequence, wherein the proopiomelanocortin nucleic acid sequence directs expression of the marker in proopiomelanocortin neurons in the arcuate nucleus of the mouse; and    assaying for an electrophysiological response of a proopiomelanocortin neuron in the histological section, thereby determining if the agent affects food intake.    
     
     
         40 . The method of  claim 39 , wherein the agent specifically binds a receptor on the proopiomelanocortin neuron.  
     
     
         41 . The method of  claim 39 , wherein the agent specifically binds a melanocortin receptor or a μ-opioid receptor.  
     
     
         42 . The method of  claim 41 , wherein the agent specifically binds the melanocortin 3 receptor.  
     
     
         43 . The method of  claim 39 , wherein the agent specifically binds a receptor on a neuropeptide Y neuron.  
     
     
         44 . The method of  claim 43 , wherein the agent specifically binds the Y2 receptor.  
     
     
         45 . The method of  claim 39 , wherein the electrophysiological response comprises induction of current or a change in the membrane potential of the proopiomelanocortin neuron.  
     
     
         46 . The method of  claim 39 , wherein the electrophysiological response comprises depolarization of the proopiomelanocortin neuron.  
     
     
         47 . The method of  claim 39 , wherein the marker is a fluorescent protein.  
     
     
         48 . The method of  claim 47 , wherein the fluorescent protein is green fluorescent protein.  
     
     
         49 . The method of  claim 39 , wherein assaying an electrophysiological response comprises a whole cell recording.  
     
     
         50 . The method of  claim 39 , wherein assaying an electrophysiological response comprises determining a resting membrane potential of a proopiomelanocortin neuron.  
     
     
         51 . The method of  claim 39 , wherein assaying an electrophysiological response comprises determining a GABA-mediated inhibitory postsynaptic current or determining the action potential frequency of a proopiomelanocortin neuron.  
     
     
         52 . The method of  claim 39 , wherein the proopiomelanocortin nucleic acid sequences comprises a proopiomelanocortin regulatory nucleic acid sequence and a nucleic acid sequence encoding a marker.  
     
     
         53 . The method of  claim 39 , wherein the agent is a polypeptide, a chemical compound, a salt, or a neurotransmitter.  
     
     
         54 . The method of  claim 39 , wherein the agent is not PYY and is not a polypeptide fragment of PYY that is more than 10 amino acids in length.  
     
     
         55 . The method of  claim 39 , wherein the agent is not a polypeptide.  
     
     
         56 . A compound identified by the method of  claim 53 .  
     
     
         57 . A compound identified by the method of  claim 54 .  
     
     
         58 . A method for screening for an agent that affects caloric intake, energy expenditure, appetite, or food intake, comprising 
 contacting a histological section of an arcuate nucleus, with an agent to be tested, wherein proopiomelanocortin neurons in the histological section express a heterologous marker that distinguishes the proopiomelanocortin neurons from other cells in the histological section; and    assaying for an electrophysiological response of a proopiomelanocortin neuron in the histological section, thereby determining if the agent affects caloric intake, appetite, energy expenditure, or food intake.    
     
     
         59 . The method of  claim 58 , wherein the heterologous marker is expressed from a transgene.  
     
     
         60 . The method of  claim 58 , wherein the agent specifically binds a receptor on the proopiomelanocortin neuron.  
     
     
         61 . The method of  claim 58 , wherein the agent specifically binds a melanocortin receptor or a μ-opioid receptor.  
     
     
         62 . (canceled).  
     
     
         62 . The method of  claim 58 , wherein the agent specifically binds a receptor on a neuropeptide Y neuron.  
     
     
         63 . The method of  claim 62 , wherein the agent specifically binds the Y2 receptor.  
     
     
         64 . The method of  claim 58 , wherein the electrophysiological response comprises induction of current or a change in the membrane potential of the proopiomelanocortin neuron.  
     
     
         65 . The method of  claim 58 , wherein the electrophysiological response comprises depolarization of the proopiomelanocortin neuron.  
     
     
         66 . The method of  claim 58 , wherein the heterologous marker is a fluorescent protein.  
     
     
         67 . The method of  claim 66 , wherein the fluorescent protein is green fluorescent protein.  
     
     
         68 . The method of  claim 58 , wherein assaying an electrophysiological response comprises a whole cell recording.  
     
     
         69 . The method of  claim 58 , wherein assaying an electrophysiological response comprises determining a resting membrane potential of a proopiomelanocortin neuron.  
     
     
         70 . The method of  claim 58 , wherein assaying an electrophysiological response comprises determining a GABA-mediated inhibitory postsynaptic current or determining the action potential frequency of a proopiomelanocortin neuron.  
     
     
         71 . The method of  claim 58 , wherein the proopiomelanocortin nucleic acid sequences comprises a proopiomelanocortin regulatory nucleic acid sequence and a nucleic acid sequence encoding proopiomelanocortin.  
     
     
         72 . The method of  claim 58 , wherein the agent is not PYY and is not a polypeptide fragment of PYY that is more than 10 amino acids in length.  
     
     
         73 . The method of  claim 58 , wherein the agent is not a polypeptide.  
     
     
         74 . A compound identified by the method of  claim 72 .  
     
     
         75 . A compound identified by the method of  claim 73 .  
     
     
         76 . The method of  claim 1 , wherein assaying for an electrophysiological response of a proopiomelanocortin neuron comprises a loose cell attached patch recording.  
     
     
         77 . The method of  claim 20 , wherein assaying for an electrophysiological response of a proopiomelanocortin neuron comprises a loose cell attached patch recording.  
     
     
         78 . The method of  claim 39 , wherein assaying for an electrophysiological response of a proopiomelanocortin neuron comprises a loose cell attached patch recording.  
     
     
         79 . The method of  claim 58 , wherein assaying for an electrophysiological response of a proopiomelanocortin neuron comprises a loose cell attached patch recording.  
     
     
         80 . The method of  claim 61 , wherein the agent specifically binds the melanocortin 3 receptor.

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