Culturing primary rat inner medullary collecting duct cells.

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons.

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophila's advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.

Hydroxyurea-induced partial mushroom body ablation does not affect acquisition and retention of olfactory differential conditioning in honeybees.

The mushroom bodies (MBs), a paired structure in the insect brain, play a major role in storing and retrieving olfactory memories. We tested whether olfactory learning and odor processing is impaired in honeybees in which MB subunits were partially ablated. Using hydroxyurea (HU) to selectively kill proliferating cells, we created honeybees with varying degrees of MB lesions. Three-dimensional reconstructions of brains were generated to analyze the drug-induced morphological changes. These reconstructions show that, with few exceptions, only the MBs were affected by the drug, while other brain areas remained morphometrically intact. Typically, lesions affected only the MB in one hemisphere of the brain. To preclude HU-induced physiologic deficits in the antennal lobe (AL) affecting olfactory learning, we measured the responses to odors in the AL using an in vivo calcium imaging approach. The response patterns did not differ between the AL of intact versus ablated brain sides within respective specimens. We, therefore, carried out side-specific classical discriminative olfactory conditioning of the proboscis extension reflex (PER) with control bees and with HU-treated bees with or without MB ablations. All experimental groups learned equally to discriminate and respond to a rewarded (CS+) but not to an unrewarded (CS-) conditioned stimulus during acquisition and retention tests. Thus, our results indicate that partial MB lesions do not affect this form of elemental olfactory learning.