Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Abstract

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs' coding properties and their modulation at the membrane level.

High Fructose Diet inducing diabetes rapidly impacts olfactory epithelium and behavior in mice

“…In order to monitor the effect of HFruD on the odorant elicited responses in individual OSNs, we performed patch-clamp recordings on MOR23 expressing neurons after 8 weeks of diet. These recordings were performed on the dendritic knob of GFP labeled neurons in an intact preparation as described previously 19 23 . In voltage clamp mode, all MOR23 neurons respond to increasing Lyral concentration stimuli with inward currents showing increasing maximum amplitude.…”

Daily oscillation of odorant detection in rat olfactory epithelium

“…To monitor whether OSNs exhibit daily variations in their physiological properties, as suggested by EOG, qPCR and western blot experiments, we performed patch‐clamp recordings on rats’ OSNs at ZT4 and ZT22. These recordings were carried out on the dendritic knobs of randomly chosen OSNs in an intact preparation as described earlier (Cadiou et al ., ; Jarriault & Grosmaitre, ). To characterise the total capacity of the transduction pathway, we stimulated OSNs at ZT4 ( n = 19 neurons) and ZT22 ( n = 21 neurons) with a mix of 100 μ m IBMX and 10 μ m Forskolin.…”

“…Patch‐clamp recordings of OSNs dendritic knobs were performed as described earlier (Grosmaitre et al ., ; Cadiou et al ., ; Jarriault & Grosmaitre, ). To assess the total capacity of the OSNs’ transduction pathway, we recorded the response to a mixture of IBMX and Forskolin diluted in Ringer or with Ringer alone.…”

The extremely broad odorant response profile of mouse olfactory sensory neurons expressing the odorant receptor MOR256‐17 includes trace amine‐associated receptor ligands

“…We thus investigated the activity of native OSNs expressing defined ORs from their endogenous locus in an environment that is relatively close to the in vivo situation. This well‐established method (Jarriault & Grosmaitre, ) has allowed us previously to characterize responses of OSNs expressing an OR with a narrow response profile such as MOR23 (Grosmaitre et al ., ) or S1 (Lam & Mombaerts, ), an OR with a broad response profile such as SR1 (Grosmaitre et al ., ), and the β2‐adrenergic receptor expressed in OSNs from an OR locus (Omura et al ., ). This preparation has been informative in a variety of experimental contexts such as ageing (Lee et al ., ), the effect of variations in OR sequence to odorant responses (Zhang et al ., ), prenatal development (Lam & Mombaerts, ), spontaneous activity (Connelly et al ., ), odorant‐induced plasticity (Cadiou et al ., ), and the correlation of OSN ciliary length with sensitivity in the septum and dorsal recess (Challis et al ., ).…”

“…Intact epithelial preparations were made as described previously (Ma et al ., ; Grosmaitre et al ., , ; Jarriault & Grosmaitre, ). Mice were deeply anaesthetized by intraperitoneal injection of ketamine/xylazine (150 and 10 mg/kg, respectively), and then decapitated.…”