Chronic recording of vomeronasal pump activation in awake behaving hamsters

Physiology& Behavior,Vol. 56, No. 2, pp. 345-354, 1994 Copyright © 1994ElsevierScienceLtd Printedin the USA. All rights reserved 0031-9384/94 $6.00 + .00

Pergamon 0031-9384(94)E0087-K

Chronic Recording of Vomeronasal Pump Activation in Awake Behaving Hamsters MICHAEL

MEI~DITH

Neuroscience Program, Florida State University, Tallahassee, FL 32306-4075 R e c e i v e d 20 D e c e m b e r 1993 MEREDITH, M. Chronicrecording of vomeronasalpump activation in awakebehavinghamsters. PHYSIOL BEHAV 56(2) 345354, 1994.--The vomeronasal organs, the receptor organs of the accessory olfactory system, are important in chemical communication. Each organ contains receptor neurons sequestered inside a blind-ending tube with a narrow access duct. Large blood vessels surrounding the vomeronasal lumen act as a pump to draw substances into the lumen, under the control of vasomotor fibers in the nasopalatine nerve. Stimulation of the superior cervical sympathetic ganglion or nasopalatine nerve operates the pump (24) but its schedule of activation in awake behaving animals is unknown. Electrodes, implanted inside the vomemnasal organ capsule of male hamsters, recorded changes in electrical properties accompanying vomeronasal pump activation. Recorded signals were validated by anesthetizing the animals and recording from the same electrodes while driving the pump by nasopalatine nerve stimulation. Recordings in awake behaving animals show that the pump does not operate only in situations where the vomeronasal organ is known to be important. It appears to operate in response to any novel situation where the animal's attention is attracted. The signals recorded suggest that blood vessels axe constricted repetitively by bursts of activity in the vasomotor sympathetic nerves each time the pump is triggered, while the underlying level of arousal is reflected in the ongoing sympathetic tone. The low selectivity in operation of the pump may require a greater degree of selectivity in the receptors than previously thought. The ready activation of the pump also suggests that the vomeronasal system may have other functions than the communication of reproductive events. Vomeronasal organ

Pump

Electrophysiology

Arousal

THE vomeronasal organ is a chemoreceptor organ located anterior and ventral to the main olfactory epithelium in the nose of most vertebrates. The bipolar receptor neurons resemble primary olfactory neurons but have apical microvili rather than cilia and project to the accessory olfactory bulb [reviews (5,21,38)]. We have previously shown (24) that there is a mechanism for delivering stimuli to the sequestered sensory epithelium in the hamster. Evidence is presented here that this pumping mechanism operates in response to novel situations and stimuli that catch the animal's attention, rather than strictly in those situations where the system is presently known to be important. This finding provides new information about the animal's use of its vomeronasal organ. It has some consequences for theories about vomeronasal receptor specificity and also implies that the system may function in other contexts than those presently known. The vomeronasal system appears to function in many species to detect chemical signals from other animals, especially signals involved in ensuring reproductive synchrony and arousal (19,38). The central connections of the system are to the corticomedial amygdala and to basal forebrain areas important in reproductive physiology and behavior. Removal of the organ or other damage to the system is associated with impairment of mating behavior in male mice and hamsters (20,23,29,35) and possibly in rats (13). In female rodents, vomeronasal damage is associated with failure of chemosignal-associated hormonal regulation underlying acceleration of puberty, estrous synchrony, and pregnancy block in mice (9,14,15,30), and induction of reproductive state

in voles (37), among many other phenomena. In all other orders of mammals that have been examined, except aquatic mammals, most members have well-developed vomeronasal organs. An atypical version of the organ has recently been reported to exist in many human adults (4,6), possibly containing bipolar neurons (33,34), and possibly responsive to some chemicals (26). The best evidence for vomeronasal function in nonmammalian vertebrates is in snakes, where prey trailing and attack as well as reproductive communication are largely dependent on vomeronasal input in some species (5). Stimulus access in snakes appears to depend on delivery of substances by tongue flicking (2,22), a mechanism totally different from that in mammals. One of the early debates about vomeronasal organ function in mammals concerned the difficulties of stimulus access. In mammals, the receptor neurons are sequestered inside a blindending tube at the base of the nasal septum that opens through a narrow duct into the nasal cavity (typical of rodents) or into the nasopalatine canal, which connects nasal and oral cavities (typical of carnivores, ungulates, and lower primates). In the hamster, the duct often appears narrow and flattened in histological sections but has a circumference approximately equivalent to a circular duct 5 0 - 9 0 / z m wide (assuming 20% shrinkage in histological preparation--unpublished observations). This duct extends for approx. 1/3 mm before any sensory neurons appear and then gradually widens out into a crescent-section lumen with a receptor epithelium that expands to a width of about 7 0 0 - 9 0 0 #m. The lumen continues for 3 - 4 mm, giving a total sensory 345

346 epithelial area of about 2.5-3 mm 2 (unpublished observations). In other species, the duct may be considerably longer, and in almost all species it presents a considerable diffusion barrier. In most species, the organ is enclosed by a bony or cartilaginous capsule, within which are large blood vessels and cavernous sinuses with the appearance of erectile tissue. This tissue constitutes a pump in the hamster (24) and cat (3) that is controlled by autonomic sympathetic, and probably parasympathetic, vasomotor systems. In the hamster, the vasomotor fibers enter the caudal end of the organ in the nasopalatine nerve, a branch of the maxillary trigeminal distribution. Sympathetic fibers join the nerve from the internal carotid plexus and parasympathetic fibers join from the sphenopalatine ganglion. Electrical stimulation of the superior cervical sympathetic ganglion or the nasopalatine nerve causes constriction of blood vessels within the VNO capsule. The volume of blood in the cavernous tissue is reduced, creating a pressure differential that expands the VNO lumen and draws in fluid from the region around the duct opening. In surgically exposed preparations, a dramatic influx of mucus into the duct follows this stimulation. Stimuli probably reach the vomeronasal organ naturally in solution in the mucus stream that passes from the anterior nasal cavity past the vomeronasal duct. It is likely, therefore, that effective vomeronasal stimuli would be mucus soluble substances, or substances that become mucus soluble when bound to carrier molecules that may be released from nasal glands (10,28). If this is the case, nonvolatile stimuli might reach the nostrils by nasal contact with stimulus sources. Volatile, mucus-soluble stimuli might be drawn into the nose to dissolve in mucus. In many of the examples of known vomeronasal function there appears to be a necessity for physical contact between the stimulus and the animal detecting it and, in some casesm, the stimulus itself appears to be nonvolatile. An example is the active principle of female hamster vaginal fluid (HVF) which, in appropriate conditions, appears to induce mating behavior in males. This active ingredient has been identified as a protein, aphrodisin (31), possibly with an attendant small molecule (32). There are other examples of vomeronasal stimuli that appear to be nonvolatile. However, there is no evidence that only nonvolatile stimuli can stimulate the vomeronasal system nor, indeed, that nonvolatile stimuli are incapable of stimulating the main olfactory system [see discussion in (25)]. In the hamster, we have presented evidence that the vascular pump is important in mating behavior. Male hamster mating behavior is dependent on chemosensory input from females (17,23,27,29), especially from the vomeronasal system, although experienced animals can use olfactory input to compensate for a loss of vomeronasal input (20). Sexually experienced animals with bilateral lesions to vomeronasal sensory neurons failed to mate after main olfactory damage by intranasal zinc sulfate lava g e - - a treatment that has no effect on mating behavior when given alone. In parallel experiments, animals with bilateral lesions of the nasopalatine nerves, that control the pump, showed deficits in mating behavior similar to those shown by animals with vomeronasal sensory lesions (23), suggesting that the pump is necessary for effective stimulus access to the sensory receptors. Although a pumping mechanism adequate for stimulus access is present and appears to be functional and necessary in behaving animals, its schedule of operation during normal behavior is not known. One possibility is that it might operate only in situations where there were other indications that vomeronasal stimuli might be available. For, example the pump might only be activated during mating. Such a restricted operation could give extra selectivity to vomeronasal receptors because they would only be exposed to a subset of possible stimuli. Such a system could also prevent inappropriate behavior if vomeronasal input were espe-

M I:,REDIq H

cially potent in inducing mating behavior. On the other hand, the pump might operate reflexly in response to particular stimuli m riving over other sensory pathways, or might be operated continuously, perhaps entrained by respiration. In the experimems described here, electrodes were implanted into the w~meronasal capsule and chronic recordings made of changes in the electrical properties of the tissues constituting the pump in awake behaving animals. The results suggest that the pump is operated in response to any novel situation but is not active continuously and is not driven by respiration. METHOD

Surgery Male hamsters, 3 months of age and approximately 100-120 g, were individually housed with ad lib food (Purina rat chow) and water, in a room with partially reversed 14D:10L cycle. Animals were implanted through the palate with a pair of VNO electrodes made from sterile, Teflon-coated silver wire (178/am, Medwire, NY). The animal was anesthetized with pentobarbital sodium (Nembutal 90 mg/kg) and arranged inverted with mouth held open by an upper tooth bar and a rubber band holding back the lower jaw. A midline incision from approximately the second palatal ridge nearly to the incisors allowed the vomeronasal capsules and the anterior palatal bone to be exposed by pulling back the palatal mucosa on each side. A small hole was made with a dental drill in the palatal bone to one side of the midline, just rostral to the natural palatal foramen and extending through the VNO capsule. A second hole was made similarly in the capsule on the same side, about 3/4 of its length from the rostral end. The electrode wires were twisted together and, using a trochar, inserted between the bone and soft tissue of the nose leading from the palatal incision to emerge on the top surface of the nose. At the palatal end the twisted pair were separated just enough to insert the free ends approximately 1 mm into the holes in the VNO capsule. Before insertion, the wires were bared for the terminal 0.5 mm and bent so that they could be hooked into the holes, extending a short distance under the capsule. The wires were arranged to present the minimal bulk overlying the palatal bone and cemented in place with cyanoacrylate adhesive (Fig. 1). The palatal mucosa was then sutured closed along the midline using 5-0 silk suture. The wires emerging from the dorsal skin on the nose were reinserted in the same hole and pulled under the skin, with the trochar, to reemerge through a midline incision extending approximately from bregma to lambda on the dorsal skull. The wires were attached to gold-pin connectors and inserted into a head plug made from plastic strip-connector blanks (Amphenol, Newark electronics, Chicago, IL). The head plug was attached via a small metal bracket with 0-80 skull screws and dental acrylic. The skin of the head was brought back around the base of the acrylic pedestal holding the head plug and the edge sealed to it with more acrylic. All surgery was done with sterile instruments and gloves. After surgery, the animals were observed continuously until they had recovered from anesthesia and then hourly for the next several hours and at least daily thereafter until the end of the experiment. They showed no sign of distress from the surgical procedures and would readily eat, drink, sleep, and mate, even when attached to the recording wires (see below). Animals were allowed to recover for 2 days after surgery before recordings were made from the implanted electrodes.

Recording VNO Movements The electrical properties (resistance, capacitance) of the tissue between the pair of electrodes was recorded using a phase-lock

RECORDING VOMERONASAL PUMP ACTIVATION

t~/~.

~,~,

~ f- ] ~"

347

<-',,-"'~/I¢",f JI ~ • ~" All

/ ~~ -

] level phase + ~ ; I reference II Phase-lock AmplifierI~ T output

\

STIMULATOR t 500hz5oxourv ot

-I_/ FM DigitalTapeJ

FIG. 1. Electrode connections and recording circuit. The tips of twisted insulated wire electrodes were inserted into the VNO capsule between the VNO itself and the bone capsule. Wires were led under the skin to a head plug, allowing connection via a flexible shielded cable to the recording system. A lowvoltage AC input signal from a stimulus isolation unit (SIU), passed between the electrodes and back to the input of the phase-lock amplifier.The DC output of the amplifieris proportionalto the amplitude of the AC signal at the frequency and phase of the injected signal, as indicated by the reference input. See text for further details. (PL) amplifier (Keithley Instruments, Cleveland, OH) connected via the head-plug as illustrated in Fig 1. A small AC signal was delivered to one VNO electrode through a stimulus isolation unit (SIU, Grass Instruments). The other side of the SIU was connected to the positive input of the PL amplifier and the circuit completed by a connection from the second VNO electrode via the head-plugto the negative input of the PL amplifier (Fig. 1). The AC signal was a positive-going square wave of 20 or 40 mV amplitude at 500 Hz and 50% duty cycle. The animals showed no reaction to the onset or offset of this signal and apparently were not conscious of it. The PL amplifier selectively amplifies signals with the same frequency and a selectable phase relationship to a reference signal which, in this case, was the same as the signal delivered to the VNO. The output of the PL amplifier is a DC signal whose amplitude reflects the amplitude of the matched-phase AC signal passing through the VNO. The output has an adjustable low-pass filter, here set to 100 ms time constant. The phase and polarity of the reference signal were adjusted to match the incoming signal, with the wires connected and the animal at rest. The equivalent resistance of the circuit through the VNO could be measured by plugging the recording wires into a potentiometer, substituting for the animal, and returning the oscilloscope trace to its original position. The potentiometer resistance is then the same as the equivalent resistance of the animal. Adjusting the potentiometer to move the trace up shows that with this circuit, an increasing PL amplifier signal is equivalent to an increase in resistance. In the normal recording condition, changes in resistance or capacitance in the VNO, that would change the amplitude or phase of the signal at the PL amplifier input, were reflected in a change of amplitude in the PL amplifier's DC output signal. Other signals coming from the VNO electrodes, due to muscle potentials, movement artifacts, etc., that are not at the same frequency as the reference signal or do not have the selected phase relationship with it, will not be amplified and, thus, will not interfere with the VNO signal. The result of these processes is a relatively noise-free recording of a signal reflecting the resistance/capacitance of the tissues between the VNO electrodes,

primarily the vascular tissue, and the VNO itself. Respiratory-, pulse- and movement-related artifacts were not a problem unless the glue holding the electrode wires came loose from the palatal bones. Presumably, the artifactual signals observed under these circumstances were due to changing resistance between wires and tissue as the implant moved. Occasionally, artifacts due to poor connections in the head plug appeared superimposed on a normal appearing recording and could be corrected by restoring a good connection. In some animals, similar erratic signals, here attributed also to poor electrical connections, totally obscured the recordings, and data from these animals were discarded. In several animals, electrode wires were arranged in the same way as described above but the bared ends were left exposed beneath the palatal mucosa, not inserted into the VNO capsule. Several of these animals also had VNO intracapsular implants on the other side--to provide a direct comparison. Unfortunately, the extra mass of wires and glue did not adhere securely to the palatal bones and most of these double implants came loose before significant data could be recorded. However, in those animals where stable recordings could be obtained from the dummy implants, the signals produced were not similar to those with implants in the capsule and there was no response to NP nerve stimulation. Signals from the PL amplifier were observed on a storage oscilloscope at low sweep speed and continuouslyrecorded on a 4-channel FM tape recorder. Other channels carried stimulus marker signals and a voice commentary. The PL amplifier signals illustrated here were photographed from the storage oscilloscope either during the experiment or after playback from the tape recorder. The vertical scale varies from recording to recording with the baseline resistance between the electrodes, so is essentially arbitrary. Regardless of the absolute size, the pattern of change reflects VNO pump activation (see verification, below) and can indicate those situations where vomeronasal stimulation might occur. Behavioral Observations

Animals were momentarily restrained with one hand while connecting the direct-wire recording leads to the head plug, and

348

were then observed continuously for the duration of the recording session. Observations were made with the animals in 42 × 20 × 20 cm shoebox-type clear plastic cages containing ground corn cob bedding and covered with a ventilated clear plastic lid for ease of observation. The recording wires were suspended above the cage, entering through a hole in the lid. No food or water was available during the observation period except that when animals were observed in their home cage (which was of the same type). They would usually have small pieces of food pellet available in the bedding. During long observation sessions the wires were disconnected periodically and a wire-grid cage lid carrying food pellets and a water bottle was placed over the cage to allow the animals to eat and drink if they wished. Animals were generally observed first in their home cages where they soon settled down to apparently normal activities (see below), most of which were associated with a relatively stable pattern in the PL amplifier signal. A stimulus was introduced into the cage or some change was made that could have been detected by the animal. Any behavioral reactions were indicated on the voice commentary and/or by hand-triggered signal markers, concurrently with the recording of the VNO signal trace. Stimuli included the introduction of various types of intruder animal, objects, puffs of air or odor, or auditory or visual stimuli (outside the cage). Simply raising the lid also constituted a stimulus that generally attracted the animals attention and often led to a change in the VNO signal. After a stimulus, the animal was allowed to settle down again until the VNO signal had again resumed a relatively stable pattern, after which another stimulus of the same or different type was presented. Three stimuli having different potential for arousing the animals, were used with virtually all the animals. These were a) opening the cage lid without presenting any other stimulus, b) opening the lid and introducing a receptive female hamster; and c) opening the lid and transferring the animal (and lid) to a clean cage containing clean bedding. Other stimuli used with many animals included nonreceptive females, males, anesthetized animals, vaginal fluid from receptive females on a glass slide, a clean slide, food pellets, corks, and wooden blocks. Auditory stimuli were generated by a variable frequency sine-wave generator. Visual stimuli consisted of a bright light directed into the cage through the transparent wall. Tests were conducted in a dimly lit room generally during the dark phase of the animal's light dark cycle.

Verification of VNO Signals Although the theory of PL amplifier operation suggests that artifacts should not interfere with the recording of electrical properties within the VNO, it was important to demonstrate that the recorded signals did, indeed, reflect VNO pump operation. To do this, signals were recorded from the same electrodes during stimulation of the nasopalatine nerve that carries vasomotor fibers to the VNO (24). After VNO signals had been recorded from the awake animal, it was anesthetized with Nembutal (90 mg/kg), placed inverted in a stereotaxic holder with the mouth held open with a rubber band, and the nasopharynx was surgically opened through the posterior palate. The nasopalatine nerves were then visible where they run along the free border of the nasal septum dorsal to the septal window. The mucosa was opened, the nerve on the side of the implant was cut, drawn into a suction electrode, and stimulated using the methods of Meredith and O'Connell (24). Various patterns of electrical stimulation were then imposed on the nerve and the resulting VNO electrode signals recorded. Patterns similar to those observed during recordings in awake animals could be produced by appropriate patterns of stimulation. In initial experiments of this type, a small window was cut into

MLREDITH

the vomeronasal capsule to observe the movements ~I the ~,t~ tissues within, when the nasopalatine nerve was stimulated, a'. the same time as the signal from the VNO electrodes was re. corded. The VNO electrode signal coincided with observed movement within the capsule. Previous experiments (24) had established that these movements were due to constriction of the blood vessels within the capsule that constitute the vomeronasal pump, and that these movements were associated with the suction of mucus (and any dissolved stimulus substances) into the VN lumen. Thus, the signals recorded here should reflect the operation of the VN pump and indicate when chemical stimuli were actively delivered to the VN lumen.

Histology After collection of data in the awake animal and in response to NP nerve stimulation, animals were euthanized while still under anesthesia by perfusion through the heart with saline followed by Bouin's fluid. The head was decalcified in RDO (Apex Engineering, Plainfield, IL) and embedded in paraffin. The VNO region was sectioned in the coronal plane at 15 #m, usually after removing the electrode wires but in some cases with the soft silver wires in place. The location of the wire tips could be seen. even when the wires had been removed, by the indentations they had made in the soft tissues. The region of the head plug was also sectioned and it was confirmed that the skull screw-holes had not encroached on the brain. RESULTS

Signals Recorded from VNO The DC level of the PL amplifier signal appeared to reflect the degree of arousal of the animal and tended to drift in one direction (upwards on the oscilloscope and in the figures) as the animal settled down after being connected to the recording wires and returned to its home cage. The direction corresponds to an increase in resistance, as confirmed by substituting a potentiometer in the circuit in place of the animal. The trace drifted in the same direction when the animal relaxed or went to sleep and when it was anesthetized (see below). This upward drift of the signal is interpreted as a relaxation of vascular tone, as explained in more detail below. In contrast, when the animal was aroused by the introduction of a novel stimulus, the trace moved, often sharply, in the opposite direction (downward in the figures), frequently with the appearance of faster oscillations having a period of one to a few seconds.

Typical Response Figure 2A illustrates a characteristic result with the introduction of a novel stimulus, in this case, a receptive female. In A, the implanted male was asleep in its nest, the trace being high and flat, drifting slowly higher. At A1, the cage lid was opened and the female was placed in the cage. The implanted male was aroused, sniffing the air, and approached the female. At the same time, the trace dropped rapidly and oscillations at about 1 per 2 s appeared. The male contacted the perineal area of the female at A2, and at A3 began to mount and make pelvic thrusts. Figure 2 also illustrates the relationship between the state of alertness of the animal and the DC level of the signal. In Fig, 2B, recorded a few minutes after A, the same animal was moderately active and the trace was at an intermediate level, with large slow oscillations of about 4 s period. At B1, the cage lid was opened and the animal was lifted out into a clean cage with clean bedding, At B2, the lid was replaced on the new cage. The DC level of the trace dropped fairly rapidly and the oscillations increased in

RECORDING VOMERONASAL PUMP ACTIVATION

A

349

B

C

0.2v lOs

"'~WwV~WVV~WWV~

2

A

A

I

2

ww~w"

3

/ FIG. 2. Typical responses to novel stimuli as reflected in the VNO signals recorded in different situations; illustrated by the line drawings below. (A1) The cage-lid was opened and a receptive female placed in the cage of a resting male (at arrowhead 1). The high, flat trace characteristic of a resting animal fell rapidly (DC shift) and oscillations increased as the male left the nest to investigate. (A2) Male contacted female's perineal area. (A3) Male mounted. (B1) The same animal, now more active, showed a lower trace with large slow oscillations (4 s period) before the cage lid was opened (at arrowhead); oscillations increased in frequency and a small DC shift began. (B2) The animal was lifted into a clean unfamiliar cage. The oscillations slowed after about 10 s of intense investigation. (B3) The animal was moved back to its home cage. Oscillations increased as the animal was lifted and transfered (arrowhead) back to its home cage where oscillations rapidly decreased in frequency as the animal settled down. (C1) The cage lid was opened and a receptive female introduced while the male was actively exploring. The already-low trace did not show a DC shift, but the oscillations increased in frequency and amplitude as the male followed the female, making contact at C2. Scale bars 10 s; 0.2 V. frequency as the animal moved rapidly about the new cage sniffing the walls and bedding. The animal was then moved back to its home cage, arriving there at B3, when it quickly settled down, showing none of the frantic investigation characteristic of its behavior in the clean (unfamiliar) cage. The oscillations also slowed in frequency and, more slowly, the DC level drifted back to a higher level. At the far right of Fig. 2, in C, the pattern of response of a fully alert male to a receptive female is illustrated. This is, again, the same male as in A and B, shortly after the beginning of the session, actually preceding parts A and B. The animal was actively investigating the home cage and the trace was relatively low with oscillations of 2 - 5 s period. The receptive female was introduced at C1. The male followed the female making contact at C2 and investigating the female's perineal area. The trace did not drop, being already at a low level, but the oscillations increased in frequency and amplitude as the male followed and investigated the female. A persistently low level of the trace as seen in this recording was characteristic of an already alert animal. The precipitous drop in the DC level of the trace, as seen in A, was characteristic of a relaxed or sleeping animal that was aroused by a novel stimulus. In this particular case, the animal retired to its nest after a number of stimuli including the mating bout initiated in C, in which the female was removed after the first intromission. When the female was reintroduced, the male was sleeping in its nest but was rapidly aroused (illustrated in A) and mating ensued after minimal delay.

Characteristic Signals from VNO These patterns of response; a dramatic DC shift with oscillations in quiescent animals and less or no shift but with promi-

nent oscillations in alert animals; was characteristic of the data recorded here, each being observed in many animals. Figure 3A,B shows responses of three animals in each situation (Fig. 3A1 and 3B3 are from the same animal). Both types of response were observed in the same recording session in several animals. In some cases, the two types of response alternated more than once in the same session as animals varied in their level of alertness throughout the session. Thus, both types of VNO signal, and the intermediate types which appeared to form a continuum, seem to be characteristic of changes in the V N O electrical properties in behaving animals responding to the introduction of receptive females. They are interpreted below as due to autonomic vasomotor changes in the vascular tissue constituting the V N O pump. In 3A1 and 3A2, the full trace preceding the introduction of the female is also shown, illustrating the near-fiat or slightly rising signal characteristic of quiescent animals.

Signals Are Elicited by Novelty, Not Reproductive Context Although these tests were initiated with the idea that the VN pump might only operate in reproductive contexts and, thus, protect the VN system from inadvertent stimulation, the characteristic V N O signals described above were not restricted to appropriate reproductive contexts. Anesthetized male hamsters, scented on their anogenital area with female vaginal fluid, were also used here. In earlier behavioral tests, such animals were treated by intact males as though they were receptive females. In these experiments, the VNO signals that appeared when implanted animals investigated scented anesthetized males were similar to those elicited by receptive females (Fig. 4A). Some implanted males did attempt to mate with the scented anestbe-

350

Mi:REDITH

A Quiescent A1

L

A Scented

B Active A1

10s

"

~

B Unscented B1

~

A2

r...

B2

10s

A3

~

~

B3

A2

B2

•

FIG. 3. Characteristic responses to the introduction of a receptive female. (Column A) Resposes, from animals in a quiescent state (similar to that shown in 2A), for three different animals. (Column B) Responses from an active state for three animals. Traces A1 and B3 were from the same animal. The nearly flat traces superimposed on the responses in A1 and A2 show the DC signal for approximately 45 s before the beginning of the response trace. The gaps in trace A2 are where rapid vertical contactartefacts were erased. Scale bars: 10 s; A1, A3, B3:1.0 V; B2:0.4 V; A2, BI: 0.2V.

tized males. However, similar VNO signals also appeared when implanted males investigated unscented anesthetized males (Fig. 4B). No mating attempts were directed towards the unscented animals, consistent with previous observations (16,23). Reinforcing the impression that the characteristic VNO signals were produced in response to arousal by novel stimuli rather than in reproductively appropriate contexts, were the results of tests with female vaginal fluid, which is very attractive to males. However, the initial stages of the VNO signals elicited by female vaginal fluid, introduced into the cage on a glass slide (Fig. 5A1,A2), were not reliably distinguishable from the signals elicited by a clean slide with no vaginal fluid (Fig. 5B 1,5B2). The implanted animals were intensely interested in the vaginal fluid, sniffing, licking, and consuming it in short order. They investigated the clean slide less intensely, but this difference in intensity of investigation was not always reflected in the VNO signals recorded. This pattern characteristic of arousal was also elicited by simply raising the cage lid, probably attracting the animal's attention largely by the entry of outside air into the familiar smelling cage (Fig. 5C1,5C2). As illustrated in Figs. 1 and 5, moving the animal to a clean cage with clean bedding was a potent stimulus for triggering significant changes in the VNO signal. The animal's intense, almost frantic investigation of the walls and bedding in the new cage suggested a high degree of arousal and was accompanied by approximately equivalent changes in the VNO signals to those elicited by a receptive female. A similarly intense arousal pattern in behavior or in VNO signals was not seen when the animal was lifted from, but then returned to, its home cage. Both the DC level and the frequency of oscillations were related to level of arousal, the latter being more of a short-term indication of the current or immediately preceding stimulus, while the former reflected the longer term history of stimulation, modulated by the present state of the animal. When the animal

FIG. 4. Responses to anesthetised males scented with hamster vaginal fluid (A1,A2), or unscented (B1,B2), placed into the experimental animal's cage (at arrowhead). Before introduction of the stimulus animal in A1 and B1, the experimental animal was quiescent with a high slowly rising trace and little oscillation. Response shows a large DC shift and increasing oscillation frequency. In A2 and B2, the experimental animal was already active with large slow oscillations, which increased in frequency as the experimental animal investigated the anesthetised stimulus animal. Scale bars: 10 s; 0.2 V. was quiescent and was presented with a novel stimulus, the DC level could change by as much as 2 - 3 volts over a period of 10 s, as shown by the large DC shifts illustrated in the figures (down-

A1

B1

lOs A2

B2

A3

B3

FIG. 5. Responses in two animals (columns A and B) to inanimate stimuli placed in the cage (arrowhead). (Row 1) A glass microscope slide smeared with hamster vaginal fluid was placed in the cage of a quiescent (A1) or active (B1) animal. (Row 2) A clean glass slide was placed in the cage of a quiescent (A2) or more active (B2) animal. (Row 3) The cage lid was opened and closed but nothing was placed in the cage of a moderately active (A3) or more quiescent (B3) animal. Scale bars: i0 s; A I - A 3 : 2 V; B3:0.4 V; B1,B2:1.0 V.

RECORDING VOMERONASAL PUMP ACTIVATION

ward shifts are in the direction of decreased resistance when the amplifier is set up as in the examples shown here). The baseline level would then generally remain steady, or drift slowly up or down, while the animal investigated. The stimulus was generally removed after a few minutes, or after 1 intromission in the case of a receptive female, and the subsequent change in DC level would depend on the animal's behavior. If it began actively foraging through the bedding, the DC level generally changed only slowly. In these cases, a further novel stimulus would not elicit a large DC shift, as illustrated in several cases here for alert animals. In the absence of further overt stimulation, the DC level would usually show a slow trend upward (in the direction of increased resistance and in the opposite direction to the shift on arousal). On the other hand, if the animal became quiescent following overt stimulation, either resting on the bedding floor or returning to its nest and lying down, the DC level drifted more rapidly upward, as occurred between the traces in Fig. 1C and A (which actually occurred later than C). Over the course of a single recording session, the level might drift more than 4 volts. Absolute DC levels were not monitored systematically and the possibility of drift over time due to electrode polarization was also not investigated systematically. However, the direction of drift was always in one direction (upward) in unstimulated animals, as expected for a process of recovery from stimulation that produced the opposite (downward) shift. Moreover, when two levels of voltage applied to the electrodes were used, either 20 or 40 mV, there did not appear to be a greater or a noticeably changed drift when the voltage was switched to the higher level--as might have been expected if polarization were the cause of drift. The oscillations that typically appeared when the animal investigated a novel stimulus generally continued at a somewhat lower frequency, at least as long as the animal remained active. When the animal was resting, the oscillations were less frequent. When sleeping, only an occasional displacement similar to those forming the continuous oscillations in aroused animals could be seen. In alert animals, the oscillations had the appearance of a continuous waveform varying in wavelength (period) and frequency as the animal encountered different stimuli. In sleeping (or anesthetized) animals, the short displacements of the trace became separated by periods of time with no sharp displacements, as though they were discrete events. Occasionally, during stereotyped behavior, the oscillations disappeared from signals recorded from an active animal that otherwise gave clear signals as described here. These behaviors included some types of grooming, and the stereotyped scrabbling behavior where animals stand against the cage wall and make rhythmic leg movements as though trying to walk up the wall. In these circumstances, the trace would occasionally go flat, with no change of DC level but no oscillations for a few seconds. The type of grooming during which this happened was the type where the animal licks its paws and washes its face. Other types of grooming, of the body or behind the ears, were not accompanied by these pauses in ongoing oscillations. These pauses did not occur in all animals, but where they did there was the appearance that some ongoing process was suspended, either voluntarily or reflexly.

Verification That VNO Signals Indicate Pump Operation Electrical stimulation of the nasopalatine nerve that carries vasomotor fibers to the VNO was used to produce authentic VN pump movements (24) so that the VNO signals generated by such movements could be recorded. These authentic signals could then be compared with the signals recorded from awake behaving animals. After recording VNO signals in the behaving animal, it

351

was anesthetized. The NP nerve was exposed through the palate and stimulated with trains of shocks through a suction electrode. The VNO signals were recorded with the same implanted electrodes through which signals had been recorded in that animal while awake. In two animals, NP nerve stimulation with single short trains of pulses resulted in a DC shift in the VN signal trace somewhat comparable to that seen in the behaving animal, but with no oscillations. Examples from two animals are shown in Fig. 6. In Fig. 6B1, a single train of 10 pulses, 1 ms in duration, 30 ms apart, and 500 #A current, elicited a large DC shift that returned slowly to the original level. The DC shift is similar to that seen in the same animal responding to a receptive female (Fig. 6A1; same response as Fig. 2A). Oscillations in the electrically induced signal could be produced by repeated short trains of stimulus pulses. In Fig. 6B2 and B3, a single 1 s long train of pulses followed by repeated short trains (100 ms) produced both a DC shift and oscillations, a response that is remarkably similar in general outline to recordings from awake animals investigating novel stimuli. These recordings are from a second animal, and Figs. 6A2 and A3 show signals from the same animal recorded in previous awake-recording sessions. Various patterns of stimulation were used for the short bursts following the 1 s initial

Awake A1

Anesthetized B1

lOs A2

B2

A3

B3

Stir..

IIIII!1111111 II1'

FIG 0 Verificationthat VNO signals indicate pump operation. Column A shows signals recorded in awake animals responding to novel stimuli. Column B shows signals recorded from the same electrodes in the same (anesthetised) animals, in response to electrical stimulation of the nasaopalatine (NP) nerve to drive the vomeronasal pump. (A1) Response to receptive female (same response as Fig. 2A). (B2) Responseto a 1 s train of pulses (1 ms, 33 Hz at 500 #A) consisted of a DC shift but no oscillations. (A2,A3) Responses of another animal to natural stimuli. (B2,B3) Signals similar to those recorded in behaving animals, produced here in the second animal by NP nerve stimulation; using an initial 1 s train of pulses (arrowhead in B2) followedby repeated short trains of pulses (100 ms at 33 Hz before upward phase of oscillations). The train used in B3 is shown in a lower trace. Scale bars: 10 s; B2, B3:1.0 V; others 0.2 V.

352

train, generating corresponding patterns of oscillations. The stimulation pattern for Fig. 6B3 is shown below the VNO signal trace. The major difference between natural and electrically induced signals was the presence, in the artificially generated signals, of a distinct positive peak before any negative-going DC shift. In the initial experiments on the vomeronasal pump (24), such a positive peak was attributed to stimulation of the parasympathetic fibers in the NP nerve. Selective activation of sympathetic fibers may occur when the animal operates the VNO pump naturally but cannot be achieved with simple electrical stimulation. In another animal, where none of the characteristic signals could be recorded during behavioral response, no signals appeared when the nasopalatine nerve was stimulated to produce VN pump movements. Possibly the VN wires were short circuited somewhere within the animal. Exposing the glue-encapsulated implant and drying the surrounding bone did affect the recorded trace, suggesting a current path outside the VNO. In any case, the results from this animal are consistent with those from other animals, in that the lack of signals recorded during NP nerve stimulation reflected a similar lack of signals seen in the behaving animal.

Anesthesia and Epinephrine Injection Several animals were anesthetized during the course of a recording session and allowed to recover while recording continued. Recordings were also made during the induction of anesthesia before attempting to stimulate the nasopalatine nerve. In all cases where any signals could be recorded from the awake animal, anesthesia caused the DC level of the signal to drift in the direction of increased resistance (upward in figures) as the animal relaxed. When an animal was anesthetized, the DC level drifted up to considerably higher levels than was characteristic of the unanesthetized animal, even when sleeping. Rhythmic oscillations disappeared rapidly to be replaced by occasional single sharp displacements similar to single cycles of the oscillations seen in alert animals but separated by long periods. In animals allowed to recover, the trace drifted back in the opposite direction during recovery. In two cases, animals were injected with Adrenalin (epinephrine) while anesthetized. In both cases, the DC signal shifted back towards the awake level in concert with an acceleration of heartrate. Oscillations did not appear with Adrenalin injection, however. Rhythmic oscillations appeared to be characteristic of awake nonresting animals.

Potential Artifacts The oscillations described above are repetitive signals as are respiration and beartrate. However, there was no indication that they were associated with either of these potential generators of artifactual signals. When animals were not too aroused, and during the initial stages of anesthesia, the rate of respiration could be measured and compared with the rate of oscillations. Oscillations rarely exceeded 4 in 10 s, even in aroused animals, a rate of 24/min. Respiration was never that slow except in deeply anesthetized animals, but in these circumstances there were also essentially no oscillations. In awake animals, respiration was generally above 100/min. Heartrate, which might also generate artifactual signals from pulse-related movements in the tissue, was also at a totally different frequency from the VNO signal oscillations, faster even than respiration. Recordings were also made during eating, drinking, grooming, mating, digging, and other active behaviors. None of the oscillations characteristic of VNO signals were associated with any of the rhythms characteristic of those behaviors. In particular, chewing did not produce oscillations, suggesting that they are unlikely to be due to a rhyth-

M ~iREI)IT~I

mic grinding of the teeth or to tongue movement agmnst thc palatal mucosa overlying the electrodes. In some animals, the normal VNO signals were interrupted occasionally bv brief bursts of large amplitude noise, characteristic of a poor connection in the input circuit of the phase-lock amplifier. In a few such cases, these noise-bursts could be reduced or eliminated by repairs to the head-plug to improve electrical continuity between connectors and wires, and these problems were attributed to poor connections. Three such animals gave good data between noise bursts, and a few examples are included in the data analyzed and reported here. The noise bursts were simply erased from the trace. leaving a short gap, and necessary measurements made on stretches of uninterrupted data. Three further animals had almost continuous noise of the same type, attributed to bad connections in the electrode circuits, which could not be corrected. Data from these animals were discarded. Six animals gave good signals of the type described here, and the conclusions from these experiments are based on their data. Examples from several animals are shown here but most of the illustrations are taken from two animals, both because these gave consistently clear signals in multiple recording sessions and also to show that all of the phenomena described could be seen in an individual animal. All of the phenomena described here were also seen in other animals DISCUSSION

These experiments have demonstrated that signals, recorded from the VNO, correlate with the operation of the VNO pump and can indicate when the animal chooses to operate the pump in behavioral situations. Surprisingly, the pump is not operated only in reproductive contexts, as we might have expected from the known functions of the vomeronasal system. Instead, the pump is operated whenever the animal encounters a novel stimulus or situation. The signal recorded from the VNO appears to indicate quite well the animal's ongoing level of arousal, especially in its DC level. The oscillations are seen most often for a minute or several minutes following the introduction of a novel stimulus but may also be seen continuously while the animal is investigating an object, a new environment, as when moved into a new cage, or while actively foraging through the bedding.

The DC Level The DC level of the signal appears to correspond to the animal's sympathetic tone, the level of ongoing sympathetic outflow. When the recording circuit is set up so that decreases in resistance are indicated by downward movement, arousal, and Adrenalin injection produce downward movement, while rest, sleep, and anesthesia all produce upward movement. In previous experiments on the vomeronasal pump (24), increased sympathetic activity produced by superior cervical ganglion stimulation produced constriction in VNO blood vessels and inflow of mucus into the VN duct. In the same series of experiments stimulation of the nasopalatine nerve also produced a predominant vasoconstriction as measured by movements of the sidewall of the organ. In the present experiments, the time course of the DC electrical signal recorded from the VNO electrodes during nasopalatine nerve stimulation resembled the mechanical signal recorded in the previous experiments, all of which supports the proposal that the DC electrical signal indicates vasomotor tone in the organ.

The Oscillations The oscillations, enhanced following a novel stimulus presentation in awake animals, could be mimicked by repetitive trains of electrical stimuli in anesthetized animals. These electrical

RECORDING VOMERONASAL PUMP ACTIVATION

stimuli must produce volleys of action potentials in nasopalatine nerve axons, including the vasomotor fibers. In awake animals, the oscillations could be caused by corresponding bursts of action potentials in sympathetic fibers serving the blood vessels of the pump. It is not clear whether the rapid upsweep of the oscillations might indicate a periodic activation of vasodilator fibers, out of phase with the vasoconstrictor sympathetic activity. If vasodilator fibers are activated, there may be an expulsion of VNO contents before each rapid influx.

Relation of Electrical Signals to VNO Stimulation The picture of VNO pump operation outlined by this interpretation of the electrical signals, suggests the following. In quiescent animals, there would be a dramatic vasoconstriction when the pump is operated in response to novel stimuli; presumably resulting in a rapid influx of fluid through the VNO duct. In alert animals, a smaller net influx on initial operation of the pump is suggested by the form of the electrical signal. In both cases, the oscillations suggest a rapid bidirectional movement of fluid through the duct while the animal actively investigates the stimulus. As reported previously (24), VN stimuli are likely to be carried into the organ following their solution in mucus, although direct influx of airborne stimuli from the nasal cavity was not ruled out (17). The bidirectional flow suggested by the oscillations in the VNO signal would result in a continuous sampling of the mucus and/or air at the entrance of the VN duct during investigation of stimulus objects. Chemical stimuli from stimulus objects could reach the entrance to the duct in the mucus stream along the ventral groove of the nasal cavity, which passes the opening of the duct. This stream almost certainly includes the products of the lateral nasal glands and other large glands that open near the nostril, anterior to the VNO duct (1). Nasal gland secretions contain binding proteins (10,28) which may be involved in carrying some stimulus chemicals to the organ. The mucus secreted inside the organ may also contain binding proteins (10) which may function to deliver stimuli to the receptors, to stimulate receptors when bound to stimuli, or to scavenge stimulus chemicals after stimulation. There is also the possibility of competition between binding proteins of different affinity. Although there seems little doubt that the VNO electrodes record a signal that changes on VNO pump operation, the quantitative relation between electrical signals and vasomotor movements has not been measured. The phase lock amplifier output appears to vary approximately linearly with simple changes in resistance, but the VN signals may involve changes in both resistance and capacitance and these changes may not be linearly related to pressure changes in the organ or to the flow of fluids into or out of the organ. These considerations, together with the complications of mucus flow and of binding protein dynamics, make it impossible to predict in detail the time course and spatial distribution of stimuli arriving at receptor sites distributed along the length of the VN sensory epithelium. Nevertheless, it seems likely that the receptors (cells and sites) located near the entrance to the organ would experience greater changes in stimulus concentration than those at the caudal end of the organ, especially during the later stages of investigation involving oscillations with little DC level change. The initial large DC level change in response to a novel stimulus presented to a quiescent animal might signal the delivery of a large amount of mucus to the organ. In anesthetized animals, a dramatic influx of mucus into the VN pore (duct entrance) could be observed coincident with the vasoconstrictor activity during nasopalatine nerve stimulation (17,24). The correspondence between VNO signals produced by nasopalatine nerve stimulation and those during natural pump

353 operation in awake animals, suggests a similar dramatic influx during a large change in the DC signal. The flow produced during oscillations alone is likely to be less dramatic. Nevertheless, this lower flow appears to be sufficient for effective stimulation of the system. In standard mating behavior tests the animals are introduced into clean cages 1 min before the female. Behaviorally, the animals quieted down considerably during that 1 min but the vomeronasal arousal, as indicated here by VNO recordings, would probably not be much reduced and such animals would be using the oscillations-alone mode of pump operation. Data from standard tests, with animals whose nasopalatine nerves were cauterized to prevent pump operation (23), suggest that the pump is important even in such tests.

Necessity for Active Sampling It may seem unusual for an exteroceptor system to be dependent on active delivery of stimuli for its function. However, other systems may have similar limitations in some circumstances. The olfactory system can be activated by stimuli in the respiratory airflow through the nose, but a deliberate sniff should be more effective in humans (12). Tactile identification of surfaces and objects requires movement (7), usually achieved by active exploration, and much of the echo-location/echo-identificationperformance by bats requires an actively produced outgoing signal (8). In each case, as with vomeronasal systems, the trigger for active sampling may be sensory input through other systems, and is likely to be a response to novelty. Active sampling of vomeronasal stimuli, including use of a pump, is probably involved in stimulus access in other species. For example, Ladewig and Hart (11) showed that delivery of a labeled stimulus (urine) into the vomeronasal organ in goats was associated with Flehmen, a lip-curling facial grimace made by ungulates and some other species in response to social chemosignals. However, they also point out that the goat VNO contains large blood vessels and suggest that a pump is involved in the final delivery of stimuli into the organ. Wysocki et al. (36) do suggest that an active pump may be unnecessary for VNO access in voles. Their experiments with stimuli placed on the philtrum suggest that some stimulus reaches the vomeronasal organ in dead animals, but it is possible in these experiments that some tracer movement occurs during rapid freezing of the head [see discussion in (25)].

Implications for Other Aspects of Vomeronasal Function One of the puzzles concerning the vomeronasal system is why the receptor epithelium should be sequestered in such a way that an active sampling system, separate from that serving olfactory receptors, becomes necessary. The advantage to species with sequestered vomeronasal receptor epithelia is not clear. One possibility is that the necessity for active sampling provides additional selectivity in stimulating the system, possibly preventing inappropriate behavior driven by inadvertent vomeronasal stimulation (18,21). However, the results presented here suggest that there is no selectivity in operation of the pump, except in that it is operated in response to novelty. Thus, this argument is greatly weakened. Other possibilities include a greater vulnerability of vomeronasal receptors (molecules or cells) to damage by environmental exposure, or a necessity for special components in the mucus overlying vomeronasal receptors. The existence of special glands that drain into the vomeronasal lumen, and a variety of binding proteins in mucus-producing glands, including the vomeronasal glands, supports this notion. Difficulties in recording chemosensory responses to stimuli perfused into the VNO lumen in situ or onto the excised epithelium (Meredith, unpublished) also support this notion, as either method would displace the

354

M I R EI) ITt!

normal mucus covering. The selectivity of vomeronasal receptor neurons has not been critically tested [see (21)]. Because the delivery system (pump) now appears not to be very selective, it seems more likely that the molecular receptors would need to be selective in their responses. Finally, the active sampling that occurs in response to novelty suggests that important stimuli not related to reproduction may be sampled. The potential exists for

other, as yet undiscovered, vomeronasal function functions un.. related to reproduction. ACKNOWLEDGEMENTS I thank Gay Howard for technical assistance and Charles Badland fi~r assistance with photography. This work was supported by NIH Grant # DC00906.

REFERENCES 1. Bojsen-Moller, F. Topography of the nasal glands in rats and some other mammals. Anatom. Rec. 150:11-24; 1964. 2. Burghardt, G. M.; Pruitt, C. H. Role of the toungue and senses in feeding of naive and experienced garter snakes. Physiol. Behav. 14:185-194; 1975. 3. Eccles, R. Autonomic irmervation of the vomeronasal organ of the cat. Physiol. Behav. 28:1011-1015; 1982. 4. Garcia-Velasco, J.; Mondragon, M. The incidence of the vomeronasal organ in 1000 human subjects and its possible clinical significance. J. Steroid Biochem. Mol. Biol. 39:561-563; 1991. 5. Halpern, M. The organization and function of the vomeronasal system. Annu. Rev. Neurosci. 10:325-362; 1987. 6. Johnson, A.; Josephson, R.; Hawke, M. Clinical and histological evidence for the presence of the vomeronasal (Jacobson' s) organ in adult humans. J. Otolaryngol. 14:71-79; 1985. 7. Johnson, K. O.; Hsiao, S. S. Neural mechanisms of tactile form and texture perception. Annu. Rev. Neurosci. 15:227-250; 1992. 8. Kawasaki, M.; Margoliash, D.; Suga, N. Delay-tuned combinationsensitive neurons in the auditory cortex of the vocalizing mustached bat. J. Neurophysiol. 59:623; 1988. 9. Keveme, E. B.; de la Riva, C. Pheromones in mice: Reciprocal interaction netween the nose and brain. Nature 296:148-150; 1982. 10. Khew-Goodall, Y.; Grillo, M.; Getchell, M. L.; Dartho, W.; Getchell, T. V.; Margolis, F. L. Vomeromodulin, a putative pheromone transporter: Cloning, characterization, and cellular localization of a novel glycoprotein of lateral nasal gland. FASEB J. 5:2976-2982; 1991. 11. Ladewig, J.; Hart, B. L. Flehmen and vomeronasal organ function in male goats. Physiol. Behav. 24:1067-1071; 1980. 12. Laing, D. G. Identification of single dissimilar odors is achieved by humans with a single sniff. Physiol. Behav. 37:163-170; 1986. 13. Larsson, K. Sexual impairment of inexperienced male rats following pre and postpubertal olfactory bulbectomy. Physiol. Behav. 14:195199; 1993. 14. Lloyd-Thomas, A.; Keverne, E. B. Role of the brain and accessory olfactory system in the block to pregnancy in mice. Neuroscience 7:907-912; 1982. 15. Lomas, D. E.; Keverne, E. B. Role of the vomeronasal organ and prolactin in the acceleration of puberty in female mice. J. Reprod. Fertil. 66:101-107; 1982. 16. Macrides, F.; Johnson, P. A.; Schneider, S. P. Responses of the male golden hamster to vaginal secretion and dimethyl disulfide: Attraction versus sexual behavior. Behav. Biol. 20:377-386; 1977. 17. Meredith, M. The vomeronasal organ and accessory olfactory system in the hamster. In: Muller-Schwarze, D.; Silverstein, R. M., eds. Chemical signals in vertebrates and aquatic animals. New York: Plenum Press; 1980:303-326. 18. Meredith, M. Stimulus access and other processes involved in nasal chemosensory function: Potential substrates for neural and hormonal influence. In: Breipohl, W., ed. Olfaction and endocrine regulation. London: IRL Press; 1982:223-236. 19. Meredith, M. Sensory physiology of pheromone communication. In: Vandenbergh, J. G., ed. Pheromones and reproduction in mammals. New York: Academic Press; 1983:199-252. 20. Meredith, M. Vomeronasal organ removal before sexual experience impairs male hamster mating behavior. Physiol. Behav. 36:737743; 1986.

21. Meredith, M. Sensory processing in the main and accessory olfactory systems: Comparisons and contrasts. J. Steroid Biochem. Mol. Biol. 39:601-614; 1991. 22. Meredith, M.; Burghardt, G. M. Electrophysiological studies of the tongue and accessory olfactory bulb in garter snakes. Physiol. Behav. 21:1001-1008; 1978. 23. Meredith, M.; Marques, D. M.; O'Connell, R. J.; Stern, F. L. Vomeronasal pump: Significance for male hamster sexual behavior. Science 207:1224-1226; 1980. 24. Meredith, M.; O'Connell, R. J. Efferent control of stimulus access to the hamster vomeronasal organ. J. Physiol. 286:301-316; 1979. 25. Meredith, M.; O'Connell, R. J. HRP uptake by olfactory and vomeronasal receptor neurons: Use as an indicator of incomplete lesions and relevance for non-volatile chemoreception. Chem. Senses 13:487-515; 1988. 26. Monti-Bloch, L.; Grosser, B. I. Effect of putative pheromones on the electrical activity of the human vomeronasal organ and olfactory epithelium. J. Steroid Biochem. Mol. Biol. 39:573582; 1991. 27. Murphy, M. R.; Schneider, G. E. Olfactory bulb removal eliminates mating behavior in the male golden hamster. Science 167:302 - 303; 1970. 28. Pevsner, J.; Snyder, S. H. Odorant-binding protein: Odorant transport function in vertebrate nasal epithelium. Chem. Senses 15:217222; 1990. 29. Powers, J. B.; Winans, S. S. Vomeronasal organ: Critical role in mediating sexual behavior of the male hamster. Science 187:961963; 1975. 30. Reynolds, J.; Keverne, E. B. The accessory olfactory system and its role in the pheromonally mediated suppression of oestrus in grouped mice. J. Reprod. Fertil. 57:31-35; 1979. 31. Singer, A. G.; Macrides, F. Aphrodisin: Pheromone or transducer? Chem. Senses 15:199-204; 1990. 32. Singer, A. G.; Macrides, F. Composition of an aphrodisiac pheromone. Chem. Senses 18: (in press). 33. Stensaas, L. J.; Lavker, R. M.; Monti-Bloch, L.; Grosser, B. 1.; Berliner, D. L. Ultrastructure of the human vomeronasal organ. J. Steroid Biochem. Mol. Biol. 39:553-560; 1991. 34. Takami, S.; Getchell, M.; Chen Y.; Monti-Bloch, L.; Berliner, D. L.; Stensaas, L.; Getchell, T. V. Vomeronasal epithelial cells of the adult human express neuron-specific molecules. Neuroreport 4:375-378; 1993. 35. Winans, S. S.; Powers, J. B. Olfactory and vomeronasal deafferentation of male hamsters: Histological and behavioral analyses. Brain Res. 126:325-344; 1977. 36. Wysocki, C. J.; Beauchamp, G. K.; Redinger, R. F.; Wellington, J. L. Access of large and non-volatile molecules to the vomeronasal organ of mammals during social and feeding behaviors. J. Chem. Ecol. 11:1147-1159; 1985. 37. Wysocki, C. J.; Kruczek, M.; Wysocki, L. M.; Lepri, J. J. Activation of reproduction in nulliparous and primiparous voles is blocked by vomeronasal organ removal. Biol. Reprod. 45:611 616; 1991. 38. Wysocki, C. J.; Meredith, M. The vomeronasal system. In: Finger, T. E.; Silver, W. L., eds. Neurobiology of taste and smell. New York: Wiley; 1987:125-150.