No evidence tube entrapment distresses rodents in typical empathy tests.

In the first two experiments an empty tube open at one end was placed in different locations. Male hamsters, tested one at a time, tended to stay close to the tube or in it. During the first minute of the first 4 sessions of Experiment 3, the hamster was unrestrained. If it entered the tube, it was locked within the tube. If it did not enter the tube during the first min, it was placed in it, and the tube was locked. Fifteen min later, the tube was opened, and the hamster was unrestrained for a further 20 min. The tube remained open during Session 5. Hamsters spent more time near the tube than predicted by chance and continued to enter the tube although tube-occupancy duration did not differ from chance levels. In Experiment 4, male rats were tested in two groups: rats in one group had been previously trapped in a tube and rats in the other group allowed to freely explore the test space. For the first two min of each of four 20-min sessions, trapped-group subjects were permitted to move about the chamber unless they entered the tube. In that case, they were locked in for the remainder of the session. If, after two min, they did not enter the tube, they were locked in it for the remaining 18 min. Free rats were unrestricted in all sessions. In Session 5, when both groups were permitted to move freely in the chamber, trapped and free rats spent more time in and near the tube than predicted by chance. These data show tube restraint does not seem to distress either hamsters or rats.

Rats (Rattus norvegicus) find occupancy of a restraint tube rewarding.

Two experiments evaluated whether rats' occupancy of a restraint tube is reinforcing. In Experiment 1, each rat in the 0-min group moved freely in a chamber where a wall blocked access to a restraint tube. After 10 min the wall was removed, permitting 15 min of chamber access and tube entry. The other 2 groups were locked in the tube for 10 and 20 min respectively before release into the chamber for 15 min. Across sessions, rats locked up for 10 and 20 min entered the tube more frequently than rats in the 0-min group, and during the first 2 sessions rats in the 20-min group stayed in the tube longer than the other groups. Over sessions this difference disappeared. However, for all groups and sessions the mean percentage of session time in the tube exceeded chance expectations. This result suggests tube occupation was reinforcing. In Experiment 2's Phase 1, rats could enter an open tube. On exiting, the tube door closed. A lever press opened the door for the rest of the 1-hr session. In Phase 2, these rats were locked in the tube for 10 min before the door opened. Upon exiting, the door closed. As in Phase 1, a lever press opened the door for the rest of the session. The latency between pressing and tube entry decreased over sessions, indicating that tube entry reinforced lever pressing. These results are difficult to reconcile with accounts of rat empathy based on the thesis that tube restraint distresses occupants.

Does a rat free a trapped rat due to empathy or for sociality?

This report evaluates whether a rat releasing a trapped rat from a restraint tube is better explained as due to its empathic motivation or to the pursuit of social contact. In the first condition, each of six rats chose in an E maze between entering an empty goal box versus entering a goal box where its entrance caused a rat trapped in a restraint tube to be released. Rats preferred the goal box with the trapped rat over the empty goal box. In the second condition, these rats chose between releasing a restraint-tube-trapped rat in one goal box and another rat in the second goal box that was not locked into its restraint tube. Rats showed no preference between alternatives. In the third condition, rats chose between a goal box containing a rat with an open restraint tube and an empty goal box. Rats preferred the rat with the open restraint tube over the empty goal box. These results support attributing the response of releasing a rat from a restraint tube to the reinforcing power of social contact rather than interpreting this response as empathically motivated.

Odor-Cued Bitter Taste Avoidance.

In the course of our ongoing studies of odor-cued taste avoidance (OCTA) to measure olfactory capabilities in animals, we observed that mice could rapidly learn to use the vapor of the classical bitterant quinine hydrochloride to avoid contact with the tastant. Here we expand on this observation to determine which among several compounds generally classed as bitter could be detected at a distance. Since mice were initially naïve we were able to assess whether the vapors of the bitter compounds tested were innately aversive as are their tastes. CD-1 mice could readily use vapor cues from quinine hydrochloride, denatonium benzoate (DB), and 6-propyl-2-thiouracil to avoid their taste. Although mice did not hesitate to make contact with these solutions on their first exposure, they did learn to do so typically after only 1 or 2 exposures. Bilaterally bulbectomized mice did not learn or retain the ability to avoid quinine and DB solutions by vapor alone, implicating olfaction as the mode of detection. Saturated aqueous solutions of sucrose octaacetate and caffeine which are bitter to humans and some strains of mice were not aversive in our studies. The very low vapor concentrations of the 3 bitterant solutions that mice detected at a distance, suggest that impurities in the reagent grade solutions, rather than the bitter molecules themselves were the basis of detection. Implications of these findings for taste testing and the role of odor in food acceptance/rejections decisions are discussed.

Does a rat release a soaked conspecific due to empathy?

In Experiment 1, rats choosing in an E maze preferred to release a rat standing in a pool of water to dry ground over a rat already standing on dry ground. Five additional experiments showed that the choosing rat's preference for releasing the wet rat was maintained by two separable outcomes: (1) the social contact offered by the released rat and (2) the reinforcing value of proximity to a pool of water. These results call into question Sato et al.'s (Anim Cogn 18:1039-1047, 2015) claim to have demonstrated that a rat's releasing of a wet rat to dry ground is empathically motivated.

Odor-cued taste avoidance: a simple and robust test of mouse olfaction.

In odor-cued taste avoidance (OCTA), thirsty mice, offered either an odorized nonaversive fluid (S+) or an odorized aversive fluid (S-), quickly learn to use odor to avoid drinking the S-. Acquisition of both odor detection and odor discrimination tasks is very rapid with learning evidenced in most cases by either long response times or total avoidance on the second presentation of the S- stimulus. OCTA is perhaps one of the simplest conditioning procedures for assessing olfaction in mice; it requires only a test box, drinkometer circuit, and thirsty mice accustomed to drinking in the apparatus. Its advantages over the most commonly used alternatives, habituation-dishabituation, and the mouse dig test, are discussed.

Roles of olfactory system dysfunction in depression.

The olfactory system is involved in sensory functions, emotional regulation and memory formation. Olfactory bulbectomy in rat has been employed as an animal model of depression for antidepressant discovery studies for many years. Olfaction is impaired in animals suffering from chronic stress, and patients with clinical depression were reported to have decreased olfactory function. It is believed that the neurobiological bases of depression might include dysfunction in the olfactory system. Further, brain stimulation, including nasal based drug delivery could provide novel therapies for management of depression.

Desire for social contact, not empathy, may explain "rescue" behavior in rats.

Ben-Ami Bartal et al. (Science 334:1427-1430, 2011) showed that a rat in an open space (free rat) would touch the front door of a restraining tube to open its rear door, thereby enabling a rat trapped within (trapped rat) to enter a larger space that was farther away from the free rat. Since opening the rear door distanced the trapped rat from the free rat, Ben-Ami Bartal et al. argued free-rat behavior could not be motivated by the pursuit of social contact. Instead, this rat was empathically motivated, its goal being to reduce the presumed distress of the rat trapped in the restraining tube. In two experiments, we show that (a) a free rat will not learn to touch the front door to open the rear door when it is the first condition of the experiment; (b) over time, a trapped rat will often return to a restraining tube despite its presumed aversiveness; and (c) a free rat experienced in touching the front door will continue to touch it even if touching does not free the trapped rat. We explain these results and Ben-Ami Bartal et al.'s in terms of two processes, neophobia and the pursuit of social contact. When first placed in a restraining tube, neophobia causes the trapped rat to escape the tube when the rear door is opened. Across sessions, neophobia diminishes, permitting the rats' pursuit of social contact to emerge and dominate free- and trapped-rat behavior.

Accessory olfactory bulb function is modulated by input from the main olfactory epithelium.

Although it is now established that sensory neurons in both the main olfactory epithelium and the vomeronasal organ may be activated by both general and pheromonal odorants, it remains unclear what initiates sampling by the vomeronasal organ. Anterograde transport of wheat germ agglutinin-horseradish peroxidase was used to determine that adequate intranasal syringing with zinc sulfate interrupted all inputs to the main olfactory bulb but left intact those to the accessory olfactory bulb. Adult male treated mice were frankly anosmic when tested with pheromonal and non-pheromonal odors and failed to engage in aggressive behavior. Treated juvenile females failed to show puberty acceleration subsequent to exposure to bedding from adult males. Activation of the immediate early gene c-Fos and electrovomeronasogram recording confirmed the integrity of the vomeronasal system in zinc sulfate-treated mice. These results support the hypothesis that odor detection by the main olfactory epithelium is required to initiate sampling by the vomeronasal system.

A simple 2-transistor touch or lick detector circuit.

An easily constructed and inexpensive battery operated circuit is described for use as a lickometer or contact detector in behavioral studies with rodents.

Response accuracy and odor sampling time in mice trained to discriminate between enantiomers of carvone and those of terpinen-4-ol.

Response accuracy and odor-sampling times were used to compare the ability of mice to detect (+)-carvone and (+)-terpinen-4-ol and to discriminate between enantiomers of carvone and of terpinen-4-ol. Except for increased odor sampling when mice were first exposed to the (+)-carvone odor, there was no difference in odor-sampling time or response accuracy in tests of odor detection or in discriminating between enantiomers of these odorants. These results fail to support the suggestion that odorants that produce different patterns of olfactory bulb activation should be easier to discriminate than those that produce much more similar patterns of bulbar activation.

Olfaction and olfactory epithelium in mice treated with zinc gluconate.

OBJECTIVE: We assessed whether a nasal spray containing zinc gluconate (ZG) compromises the integrity of olfactory epithelium and olfactory function. METHODS: Axonal transport of horseradish peroxidase from olfactory epithelium to the olfactory bulb was studied in 2- to 21-day survival mice given intranasal injections of 2, 8, or 50 microL of ZG (approximately 4, 15, and 94 times the equivalent recommended human dose). Other similarly treated mice were tested using precision olfactometry to detect and discriminate odors. RESULTS: Anatomic changes were graded as a function of dose and survival time. Two microliter injections had no discernable effect. while the 50 microL volume produced substantial deafferentation of input to the olfactory bulb in short-survival cases. Nearly complete restitution of input occurred within 3 weeks. At each volume and survival time, zinc sulfate (ZS) had a greater effect. Behaviorally, 2 microL and 8 microL ZG-treated mice and those given multiple injections of 2 microL ZG performed as well as controls, whereas those given 50 microL were hyposmic but not anosmic. ZS-treated mice performed more poorly, and those injected with 50 microL were anosmic for the first 8 to 10 test days. CONCLUSIONS: A massive dose of a ZG nasal spray did cause a transient disruption of the olfactory epithelium and compromised olfaction. More moderate volumes, even those far in excess of a recommended dose, were largely without effect on odor detection and discrimination tasks. These outcomes fail to support the claims from recent clinical case reports that use of a ZG-containing nasal spray can produce anosmia.

Odor-sampling time of mice under different conditions.

Response accuracy and odor sample times on positive (S+) and negative (S-) trials were recorded for mice trained on a variety of go, no-go odor detection and discrimination tasks. Odor sample time was relatively stable over extended training on the same task, increased during acquisition of difficult tasks, relatively insensitive to reinforcement magnitude, and, in some cases, provided more information regarding task difficulty and discrimination than did response accuracy. Mice generally sampled longer on S- trials in simple odor detection tasks but longer on S+ trials in odor discrimination tasks.

Olfactory performance of rats after selective deafferentation of the olfactory bulb by 3-methyl indole.

Rats trained to detect propyl acetate and valeric acid and to discriminate between propyl acetate and amyl acetate and between valeric acid and butyric acid were injected with a low dose of 3-methyl indole, a treatment that produces well-defined and selective deafferentation of the olfactory bulbs. Treatment completely deafferented most but not all bulbar loci for aliphatic acids and at least disrupted those for propyl and amyl acetate. In posttreatment tests, experimental rats performed somewhat but not significantly more poorly than controls and about as well on the acid detection and discrimination tasks as on the corresponding acetate tests.

Discrimination between the enantiomers of carvone and of terpinen-4-ol odorants in normal rats and those with lesions of the olfactory bulbs.

We assessed (1) whether the enantiomers of terpinen-4-ol, odorants that activate nearly identical areas of the olfactory bulb, are more difficult to discriminate than those of carvone, odorants that activate different areas of the olfactory bulb, and (2) whether olfactory bulb lesions that disrupt the pattern of bulbar activation produced by these enantiomers degraded the ability of rats to discriminate between them. In psychophysical tests, normal rats discriminated between the enantiomers of terpinen-4-ol and of carvone equally well. Surgical lesions that removed the majority of bulbar glomeruli activated by these odorants (as demonstrated in previous olfactory bulb studies using intrinsic optical imaging and 2-deoxyglucose) resulted in increased detection thresholds but few or no deficits in discriminating between suprathreshold concentrations of the enantiomers. These results fail to confirm predictions based on 2-deoxyglucose maps of bulbar activity that enantiomers of terpinen-4-ol should be more difficult to discriminate than those of carvone and that the ability to discriminate between enantiomers of an odorant are based on differences in patterns of bulbar activation revealed in such maps.

Olfactometry with mice.

A computer-controlled, multiple-channel odor generator, operant test chamber, and discrimination training procedure for mice are described. The odor generator allows controlled presentation of any one or combinations of eight odors to an odor-sampling port that contains a liquid reinforcement delivery tube that also serves to detect responses. A modified discrete trial operant conditioning procedure provides measures of both response accuracy and response rate during anticipation of stimulus delivery and in the presence of two different odor stimuli. Results from numerous experiments demonstrate that, with these methods, mice reliably and rapidly acquire odor detection and a variety of odor-discrimination tasks, and that response rate in the presence of the odor reflects the incentive motivation of stimuli that are or are not associated with reward.

Olfaction in olfactory bulbectomized rats.

Experimental rats had their right olfactory bulb removed on postnatal day 2 (P2) and their left olfactory bulb removed on P90. Control rats had one or both olfactory bulbs removed on P90. Before and after their adult-stage surgery, rats were trained using olfactometry and operant conditioning to detect and discriminate odors. Anterograde transport of horseradish peroxidase applied to the olfactory epithelium revealed numerous axons of olfactory sensory neurons in the right hemisphere of 27 experimental rats. These axons terminated in glomerular-like clusters within the frontal neocortex (n = 5) or anterior olfactory nucleus with some axons extending into the subventricular epithelium (n = 22). Seventeen of the experimental rats were able to detect a variety of odors and to discriminate between odors. Performance accuracy was related to the location and density of these anomalous inputs; experimental rats with inputs confined to frontal neocortex and those lacking any inputs to the forebrain were anosmic, as were adult-operated bilaterally bulbectomized rats. Our results provide strong support for the contention that, in the absence of the olfactory bulbs, olfactory connections to novel forebrain sites can support both odor detection and odor discrimination.

Zonal ablation of the olfactory sensory neuroepithelium of the mouse: effects on odorant detection.

Olfactory sensory neurons that express a specific odorant receptor, out of a thousand different, are unevenly distributed within, but restricted to one of four zones of the neuroepithelial sheet in the nasal cavity in the mouse. This zonal restriction of neurons expressing the same odorant receptor may have consequences, e.g. in case of localized injury. We found that the chemical dichlobenil can produce specific and permanent ablation of neurons in odorant receptor expression zone 1, while a higher dichlobenil dose causes reversible toxicity in neighboring zones. In behavior tests, mice lacking part of the olfactory epithelium had an increased detection threshold concentration of two-four orders of magnitude for some odorants but not others, resembling the phenomenon of specific hyposmia. This indicates that the broad tuning properties of single odorant receptors and their large number cannot fully compensate for loss of the receptor(s) with the highest sensitivity for a particular odorant.

Odors detected by mice deficient in cyclic nucleotide-gated channel subunit A2 stimulate the main olfactory system.

It is believed that odor transduction in the mammalian main olfactory system only involves the cAMP-signaling pathway. Here, we report on odor responsiveness in mice with a disrupted cyclic nucleotide-gated (CNG) channel subunit A2. Several odorants, including putative pheromones, can be detected and discriminated by these mice behaviorally. These odors elicit responses in the olfactory epithelium, main olfactory bulb, and olfactory (piriform) cortex of CNGA2 knock-out mice. In addition, responses to odors detected by CNGA2 knock-out mice are relatively insensitive to inhibitors of the cAMP pathway. These results provide strong evidence that cAMP-independent pathways in the main olfactory system of mammals participate in detecting a subset of odors.

Does intranasal application of zinc sulfate produce anosmia in the mouse? An olfactometric and anatomical study.

Mice pre-trained in an olfactometer were tested daily on odor detection and discrimination tasks after irrigation of their olfactory epithelium in each naris with 50 microl of 5% zinc sulfate or saline. Anterograde transport of a wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) conjugate from the epithelium to the olfactory bulb was used to assess anatomical connectivity in these and in mice that were used only for histological analyses. One day after treatment, saline controls performed at high levels of accuracy in detecting vapor from solutions of 5-0.01% ethyl acetate and in an odor discrimination task but most ZnSO4-treated mice performed at chance for 5-30 days before showing recovery. Although dense WGA-HRP reaction product was found in the accessory olfactory bulb, there was little or no evidence for axonal transport to glomeruli of the main olfactory bulb in the first 4-8 days after treatment. These results demonstrate that intranasal application of ZnSO4 to mice produces a brief but essentially total disruption of functional connections from the olfactory epithelium to the main olfactory bulb and a corresponding transient anosmia.