In vivo vomeronasal stimulation reveals sensory encoding of conspecific and allospecific cues by the mouse accessory olfactory bulb

Abstract

The rodent vomeronasal system plays a critical role in mediating pheromone-evoked social and sexual behaviors. Recent studies of the anatomical and molecular architecture of the vomeronasal organ (VNO) and of its synaptic target, the accessory olfactory bulb (AOB), have suggested that unique features underlie vomeronasal sensory processing. However, the neuronal representation of pheromonal information leading to specific behavioral and endocrine responses has remained largely unexplored due to the experimental difficulty of precise stimulus delivery to the VNO. To determine the basic rules of information processing in the vomeronasal system, we developed a unique preparation that allows controlled and repeated stimulus delivery to the VNO and combined this approach with multisite recordings of neuronal activity in the AOB. We found that urine, a well-characterized pheromone source in mammals, as well as saliva, activates AOB neurons in a manner that reliably encodes the donor animal's sexual and genetic status. We also identified a significant fraction of AOB neurons that respond robustly and selectively to predator cues, suggesting an expanded role for the vomeronasal system in both conspecific and interspecific recognition. Further analysis reveals that mixed stimuli from distinct sources evoke synergistic responses in AOB neurons, thereby supporting the notion of integrative processing of chemosensory information.

Fig. 1.

Experimental setup. (A) Mouse nasal cavity and relevant structures. (Left Inset) Coronal section of the VNO showing uptake of DiI ipsilateral to nerve stimulation. BV, blood vessel; L, VNO lumen. (Right Inset) Sagittal AOB section showing a DiI-painted probe within the external cell layer (ECL). GL, glomerular layer; Gr, granule cell layer. (B) Sequence of events in a single trial showing the serial application of mouse urine followed by sympathetic stimulation and flushing. Each panel includes a cartoon of the presumed state of VNO, a raw electrode signal (stimulation artifacts clipped), and firing rates.

Fig. 2.

Sex specificity of responses and temporal response profiles. (A) Sympathetic stimulation-induced spike times in interleaved presentations of dilute male or female mouse urine from two simultaneously recorded neurons (orange and green). (Lower) Averaged responses (mean ± SEM). Time 0: sympathetic stimulation. (B) Histograms of start time (latency), peak time, and half time of individual responses relative to the onset of sympathetic stimulation (time 0).

Fig. 3.

Dependence of AOB responses on functional TRPC2 signaling. (A) Percentages of stimulus-induced rate changes of individual units to the most effective stimulus (stimuli were male, female, and predator urine). (B) Percentage of significant responses (P < 0.01) to mouse urine in wild-type (WT) and TRPC2^−/− mutant mice. Data in this analysis include multi-unit activity. (n = 1,195, WT; 221, TRPC^−/−).

Fig. 4.

Responses to urine and saliva. (A) Responses of three different single units to urine from mice of distinct sex and strain. Significant responses (P < 0.01, either positive or negative) are shown on a gray background. (BC, BalbC; C57, C57/Black6). (B) Same as A for saliva stimuli. (C) Responses to distinct urine samples. Samples ending with -1 and -1B denote different samples from the same individual whereas the sample ending with -2 is from a different individual. (D–F) Hierarchical clustering of population responses to the stimuli in A (n = 23), B (n = 13), and C (n = 16). A–C show responses of distinct units.

Fig. 5.

Responses to predator urine. (A) Percentages of units with significant responses (P < 0.01) to each of the stimulus combinations (n = 51 single units responding to at least one stimulus). Stimulus categories are male mouse urine, female mouse urine, and predator urine. Mouse urine was pooled from strains BalbC, C57Black6, and CBA. (B) Response magnitudes of these single units to mouse and predator urine. (C) Three single units with selective responses to female mouse, male mouse, or combined predator urine. (D) Single units responding selectively to different predators. The icons designate, from left to right, bobcat, fox, rat, and combined urine from these sources. (E) Single units responding across stimulus categories.

Fig. 6.

Responses to conflicting stimuli. (A) Relationship of population responses to the elemental stimuli and to their combinations using multidimensional scaling (MDS). Each circle represents the population response to one stimulus (see color legend).The “predator inhibition” simulation models the response were predator urine to entirely inhibit the response to mouse urine. (B) Responses of individual units to mixed stimuli. Each line represents the response of one single unit to one mix. The edges of the line are defined by the responses of a given unit to the two elemental stimuli (male or female mouse urine and predator urine) and the + indicates the response of the same unit to their mixture. Responses are categorized into those showing suppression or intermediate or synergistic interactions following mixing. n = 53 cases. Male and female mouse urine was pooled as described in Fig. 5.