Mating-induced differential coding of plant odour and sex pheromone in a male moth

Innate behaviours in animals can be influenced by several factors, such as the environment, experience, or physiological status. This behavioural plasticity originates from changes in the underlying neuronal substrate. A well-described form of plasticity is induced by mating. In both vertebrates and invertebrates, males experience a post-ejaculatory refractory period, during which they avoid new females. In the male moth Agrotis ipsilon, mating induces a transient inhibition of responses to the female-produced sex pheromone. To understand the neural bases of this inhibition and its possible odour specificity, we carried out a detailed analysis of the response characteristics of the different neuron types from the periphery to the central level. We examined the response patterns of pheromone-sensitive and plant volatile-sensitive neurons in virgin and mated male moths. By using intracellular recordings, we showed that mating changes the response characteristics of pheromone-sensitive antennal lobe (AL) neurons, and thus decreases their sensitivity to sex pheromone. Individual olfactory receptor neuron (ORN) recordings and calcium imaging experiments indicated that pheromone sensory input remains constant. On the other hand, calcium responses to non-pheromonal odours (plant volatiles) increased after mating, as reflected by increased firing frequencies of plant-sensitive AL neurons, although ORN responses to heptanal remained unchanged. We suggest that differential processing of pheromone and plant odours allows mated males to transiently block their central pheromone detection system, and increase non-pheromonal odour detection in order to efficiently locate food sources.     

Combined modulating effects of the general arousal and the specific hunger arousal on the olfactory bulb responses in the rat.

The combined modulating effects of the general level of arousal and specific hunger arousal on the olfactory bulb responses were investigated in the rat. Vigilance state parameters and multi-unit mitral cell activity were recorded in freely moving animals stimulated by control odours and by their usual food odour, either in the hungry or the satiated state. The nutritionally modulated bulb responses towards food odour were observed only for high arousal level (wakefulness). In rapid eye movement sleep (REMS), no olfactory response occurred. In slow wave sleep (SWS), one observed either a high bulb responsiveness to stimuli with neocortical arousal, or a general inexcitability. Each odorous stimulus in SWS elicited a higher neocortical arousal rate in the hungry than in the satiated state, as did food odour compared with control odours in both nutritional states. In SWS, a progressive alteration of the nutritionally modulated responses occurred at first at the bulb level and later for inner structures. Rats fed 2 h a day displayed a reversed circadian sleep-waking cycle and a lower SWS proportion compared with rats fed ad libitum. The hunger arousal could quantitatively and qualitatively modulate the activity of structures regulating the sleep-waking pattern.     

The coding of odour-intensity in the honeybee antennal lobe: local computation optimizes odour representation

We investigated strategies involved in odour intensity coding by the primary olfactory centre of insects, the antennal lobe (AL), the structural and functional analogue of the olfactory bulb. Using calcium imaging in the honeybee, we simultaneously measured the projection neuron output responses and a compound signal dominated by receptor neuron input in identified olfactory glomeruli to odours spanning seven log units of concentration. A comparison of the two processing levels indicates that the intercellular computation within the AL modulates and contrast-enhances the primary olfactory signals. As a result the AL network optimizes the olfactory code: odour representation is improved at lower concentrations, the relative activity of olfactory glomeruli allows encoding odour quality over up to four log-unit concentrations, and odour-intensity is reliably represented in the overall excitation across AL.     

Spontaneous and odour evoked activity in single avian olfactory bulb neurones

Extracellular single unit recordings were made from various depths in the left olfactory bulb of ten anaesthetised, freely breathing adult hens (Gallus domesticus) using glass insulated tungsten microelectrodes. The 66 spontaneously active neurons recorded had widely variable firing rates (mean 4.9 spikes/s, range 0.1-32.4 spikes/s) and variable temporal firing patterns (regular, bursting and random discharge). Interspike interval histograms were constructed for each unit and tested for goodness of fit to theoretical distributions. The activity of 23 units fitted gamma distributions, six units fitted lognormal distributions, three units fitted Weibull distributions, one unit fitted an exponential distribution and 11 units had bimodal distributions. Responses of some units to odour stimuli (Clove oil, Geraniol, Limonene and Ammonia all at 10% vapour saturation) delivered directly to the olfactory epithelium were examined. Odour stimulation modified spontaneous activity in 29 of the 44 units tested, causing either inhibition (56%) or excitation (44%) of firing. Of the 35 units stimulated with more than one odour, 15 responded to multiple stimuli, showing excitation or inhibition only or both inhibition and excitation, depending on the odour applied. Ammonia most commonly elicited a response (70% of applications) and all the odours were capable of inducing both inhibition and excitation of spontaneous firing. Examination of transverse sections of adult hen olfactory bulb in the region corresponding to the recording sites allowed unit recording depth to be related to cellular layers. There was some evidence of different neurone properties in each bulb layer. The spontaneous activity and odour responses observed resemble those seen in other vertebrate groups and the firing rates observed are between those previously reported for mammals and reptiles. This study provides detailed physiological evidence for a well-developed functional olfactory system in an avian species.     

Not all 'predator odours' are equal: cat odour but not 2,4,5 trimethylthiazoline (TMT; fox odour) elicits specific defensive behaviours in rats.

The behavioural responses to two commonly used 'predator odours' were assessed in male Wistar rats. Cat odour was presented to rats in the form of a piece of collar that had been worn by a domestic cat. Fox odour was presented in an equivalent piece of (unworn) collar that had been impregnated with 2,4,5 Trimethylthiazoline (TMT)-an extract of fox faeces. Other rats were exposed to collars containing Triethylamine (TEA), a putrid fishy smell, or formaldehyde, which has an acrid irritating smell. Experiment 1 showed that rats approached cat odour, TMT and TEA significantly less than they did an unworn collar. However, only cat odour increased retreat to the hide box, reduced locomotor activity and elicited 'head out' behaviour. When tested immediately after odour exposure, only cat odour exposed rats showed increased anxiety in the elevated plus maze and suppressed activity in a 90-min general activity test. When returned to the odour-paired environment 24 h later in the absence of test odours, only rats that had previously received cat odour showed evidence of conditioned fear. Experiment 2 showed that rats given the benzodiazepine drug midazolam (0.5 mg/kg) display increased approach and decreased defensiveness towards a cat odour impregnated collar. In contrast, midazolam accentuated the avoidance of TMT and formaldehyde containing collars. Experiment 3 showed that when cat odour was presented in a small, enclosed environment, rats display increased body immobility, decreased grooming and increased orientation towards the odour-exuding stimulus. These responses were not seen with TMT or TEA containing collars. Taken together, these results suggest that while cat odour strongly elicits specific defensive behaviours in rats, TMT has effects that are more characteristic of an aversive odour. We suggest that the results of some previous studies using TMT may need to be reassessed.     

Multiple memory traces after associative learning in the honey bee antennal lobe

We investigated the effect of associative learning on early sensory processing, by combining classical conditioning with in vivo calcium-imaging of secondary olfactory neurons, the projection neurons (PNs) in the honey bee antennal lobe (AL). We trained bees in a differential conditioning paradigm in which one odour (A+) was paired with a reward, while another odour (B-) was presented without a reward. Two to five hours after differential conditioning, the two odour-response patterns became more different in bees that learned to discriminate between A and B, but not in bees that did not discriminate. This learning-related change in neural odour representations can be traced back to glomerulus-specific neural plasticity, which depended on the response profile of the glomerulus before training. (i) Glomeruli responding to A but not to B generally increased in response strength. (ii) Glomeruli responding to B but not to A did not change in response strength. (iii) Glomeruli responding to A and B decreased in response strength. (iv) Glomeruli not responding to A or B increased in response strength. The data are consistent with a neural network model of the AL, which we based on two plastic synapse types and two well-known learning rules: associative, reinforcer-dependent Hebbian plasticity at synapses between olfactory receptor neurons (ORNs) and PNs; and reinforcer-independent Hebbian plasticity at synapses between local interneurons and ORNs. The observed changes strengthen the idea that odour learning optimizes odour representations, and facilitates the detection and discrimination of learned odours.     

Odour coding is bilaterally symmetrical in the antennal lobes of honeybees (Apis mellifera)

The primary olfactory neuropil, the antennal lobe (AL) in insects, is organized in glomeruli. Glomerular activity patterns are believed to represent the across-fibre pattern of the olfactory code. These patterns depend on an organized innervation from the afferent receptor cells, and interconnections of local interneurons. It is unclear how the complex organization of the AL is achieved ontogenetically. In this study, we measured the functional activity patterns elicited by stimulation with odours in the right and the left AL of the same honeybee (Apis mellifera) using optical imaging of the calcium-sensitive dye calcium green. We show here that these patterns are bilaterally symmetrical (n = 25 bees). This symmetry holds true for all odours tested, irrespective of their role as pheromones or as environmental odours, or whether they were pure substances or complex blends (n = 13 odours). Therefore, we exclude that activity dependent mechanisms local to one AL determine the functional glomerular activity. This identity is genetically predetermined. Alternatively, if activity dependent processes are involved, bilateral connections would have to shape symmetry, or, temporal constraints could lead to identical patterns on both sides due to their common history of odour exposure.     

Short‐lasting exposure to one odour decreases general reactivity in the olfactory bulb of adult rats

We investigated in adult rats whether a relatively short exposure to a novel odour can lead to changes in reactivity of olfactory bulb principal neurons. Naive rats were exposed to isoamyl acetate for 20 min per day either for 6 consecutive days or for a single 20-min exposure. Control group was non-exposed. Under anaesthesia, responsiveness of each recorded single mitral/tufted cell was tested towards isoamyl acetate and four other odours. Results show that the proportion of responding cells in the exposed groups decreased drastically when compared to controls. In the two experimental groups recorded 24 h following the last exposure, mitral/tufted cells show a significant decrease in the number of excitatory responses. In parallel, the number of non-responsive cells increased by at least a fourfold factor. This decrease in reactivity was not selective towards the odour used during the exposure but concerned any of the five test-odours presented during recordings. Finally, this lower responsiveness was long lasting as it was still observed 10 days after the end of the last exposure. This preliminary study points out the importance of even limited sensory experience in neural representation of odours.     

γ-Aminobutyric acid receptor A-mediated inhibition in the honeybee's antennal lobe is necessary for the formation of configural olfactory percepts

Complex odours often possess perceptual qualities that are distinct from their components. Previous studies in humans, rodents, and insects indicate that the perception of complex odour blends depends on the concentration of the components and the mixture's complexity. However, we know relatively little about the way that an odour mixture 'gestalt' is produced by the olfactory system. Here, using an assay for olfactory conditioning in the honeybee (Apis mellifera), we examine the role of γ-aminobutyric acid receptor A (GABA(A) )-ergic inhibition within the olfactory primary relay, the antennal lobe, in the formation of a unique odour percept for complex odours. We found that honeybees perceive odour mixtures as configural stimuli when the mixtures were of low concentration and when they were composed of more than two odorants. When GABA(A) receptors were disrupted using the antagonist, picrotoxin, injected directly into the antennal lobe, we observed that bees no longer perceived the mixture as a configural stimulus. Our results imply that synchronization of antennal lobe projection neurons mediated by GABA(A) receptors is the mechanism responsible for the formation of unique olfactory percepts for complex odours.     

Are oestrus odours species specific?

Adult male rats were exposed to faeces odours of three animal species (rat, fox and horse). They displayed erections in the presence of faeces from oestrous females (whatever the species). In addition, fox faeces (whatever the gender or hormonal status) elicited an expected freezing reaction. It is suggested that oestrous female faeces of these three species share common odorants which depend on the hormonal status and characterize female receptivity.     

A centrifugal pathway to the mouse accessory olfactory bulb from the medial amygdala conveys gender-specific volatile pheromonal signals

We previously found that female mice exhibited Fos responses in the accessory olfactory bulb (AOB) after exposure to volatile opposite-sex, but not same-sex, urinary odours. This effect was eliminated by lesioning the main olfactory epithelium, raising the possibility that the AOB receives information about gender via centrifugal inputs originating in the main olfactory system instead of from the vomeronasal organ. We asked which main olfactory forebrain targets send axonal projections to the AOB, and whether these input neurons express Fos in response to opposite-sex urinary volatiles. Female mice received bilateral injections of the retrograde tracer cholera toxin B (CTB), into the AOB, and were exposed to either same- or opposite-sex volatile urinary odours 1 week later. We found CTB-labeled cell bodies in several forebrain sites including the bed nucleus of the accessory olfactory tract, the rostral portion of the medial amygdala (MeA) and the posteromedial cortical nucleus of the amygdala. A significant increase in the percentage of CTB/Fos co-labeled cells was seen only in the MeA of female subjects exposed to male but not to female urinary volatiles. In Experiment 2, CTB-injected females were later exposed to volatile odours from male mouse urine, food, or cat urine. Again, a significant increase in the percentage of CTB/Fos co-labeled cells was seen in the MeA of females exposed to male mouse urinary volatiles but not to food or predator odours. Main olfactory–MeA–AOB signaling may motivate approach behaviour to opposite-sex pheromonal signals that ensure successful reproduction.     

Odour mixture suppression: evidence for a peripheral mechanism in human and rat

Rarely do we encounter a single odorant in our environment. Perception of odours, therefore, usually depends on the reception and neural processing of many components. However, little is known about how and where odour mixtures are processed. Evidence is presented here that suppression of one odour by another, a common result of mixing odours, is primarily a peripheral event. Having demonstrated with human subjects that perception of one or both odorants in two-component mixtures is dependent on the polarity and perceived intensity of the odorants, the same mixtures were presented to rats that had been injected with a metabolic marker, [3H]2-deoxyglucose (2-DG). By measuring the metabolic activity in the glomeruli of the rat olfactory bulb, where the axons of the receptor cells terminate, it was found that in a mixture where humans had perceived only one odour, there is a dramatic reduction in metabolic activity of glomeruli specific to the suppressed odour. In mixtures where both odorants were perceived, metabolic activity characteristic of both components was observed. These findings indicate that similar mechanisms underlie the perception of odour mixtures in the two species. Since metabolic activity revealed by 2-DG in glomeruli occurs predominantly in presynaptic receptor axons, the reduced activity seen after stimulation with odour mixtures indicates that a mechanism for mixture suppression begins at the receptor cells. Therefore, the ability of one odorant to suppress another in a mixture is probably determined by their relative chemical polarities, which effects access to and competition for membrane receptor sites in the olfactory epithelium.     

Expression of c-fos in the main olfactory bulb of neonatal rabbits in response to garlic as a novel and conditioned odour

Expression of c-Fos was examined in the olfactory bulbs of 3-day-old rabbits after they had been presented with the odour of garlic as a novel stimulus, as a learned odour, or during conditioning, and this expression compared with baseline levels in non-stimulated controls. Exposure to garlic odour resulted in substantial and widespread increases in c-Fos expression in the olfactory bulbs of all animals. However, although conditioned pups showed a specific behavioural response to the learned garlic odour, neither the amount nor pattern of c-Fos expression differed compared to pups exposed to garlic as a novel odour. The odour-induced expression of c-Fos was not well localised, although there was a significant increase in the number of granule cells expressing c-Fos in the ventrolateral region of the bulb. These results support previous reports that the response to odours in the olfactory bulb of new-born animals is not as spatially distinct as that in adults. Nevertheless, the immature olfactory system of these young animals is clearly capable of very specific odour learning.     

Existence of multiple receptors in single neurons: responses of single bullfrog olfactory neurons to many cAMP-dependent and independent odorants

The responses of single bullfrog olfactory neurons to various odorants were measured with the whole-cell patch clamp which offers direct information on cellular events and with the ciliary recording technique to obtain stable quantitative data from many neurons. A large portion of single olfactory neurons (about 64% and 79% in the whole-cell recording and in the ciliary recording, respectively) responded to many odorants with quite diverse molecular structures, including both odorants previously indicated to be cAMP-dependent (increasing) and independent odorants. One odorant elicited a response in many cells; e.g. hedione and citralva elicited the response in 100% and 92% of total neurons examined with the ciliary recording technique. To confirm that a single neuron carries different receptors or transduction pathways, the cross-adaptation technique was applied to single neurons. Application of hedione to a single neuron after desensitization of the current in response to lyral or citralva induced an inward current with a similar magnitude to that applied alone. It was suggested that most single olfactory neurons carry multiple receptors and at least dual transduction pathways.     

Maternal Odours Induce Fos in the Main But Not the Accessory Olfactory Bulbs of Neonatal Male and Female Ferrets

Previous research demonstrated that exposing gonadectomized adult ferrets to odours in oestrous female bedding induced nuclear Fos-immunoreactivity (Fos-IR; a marker of neuronal activity) in the main as opposed to the accessory olfactory system in a sexually dimorphic fashion, which was further augmented in both sexes by treatment with testosterone propionate. Ferrets are born in an altricial state and presumably use maternal odour cues to locate the nipples until the eyes open after postnatal (P) day 23. We investigated whether maternal odours augment neuronal Fos preferentially in the main versus accessory olfactory system of neonatal male and female ferret kits. Circulating testosterone levels peak in male ferrets on postnatal day P15, and mothers provide maximal anogenital stimulation (AGS) to males at this same age. Therefore, we assessed the ability of maternal odours to augment Fos-IR in the accessory olfactory bulb (AOB), the main olfactory bulb (MOB) and other forebrain regions of male and female ferret kits on P15 and investigated whether artificial AGS (provided with a paintbrush) would further enhance any effects of maternal odours. After separation from their mothers for 4 h, groups of male and female kits that were placed for 1.5 h with their anaesthetized mother had significantly more Fos-IR cells in the MOB granule cell layer and in the anterior-cortical amygdala, but not in the AOB cell layer, compared to control kits that were left on the heating pad. Artificial AGS failed to amplify these effects of maternal odours. Maternal odours (with or without concurrent AGS) failed to augment neuronal Fos-IR in medial amygdaloid and hypothalamic regions that are activated in adult ferrets by social odours. In neonatal ferrets of both sexes, as in adults, socially relevant odours are detected by the main olfactory epithelium and initially processed by the MOB and the anterior-cortical amygdala. In neonates, unlike adults, medial amygdaloid and hypothalamic neurones either do not respond to these inputs or respond in a manner that fails to induce Fos expression.     

Fine tuning of olfactory orientation behaviour by the interaction of oscillatory and single neuronal activity

We used a simple sensory and motor system to investigate the neuronal mechanisms of olfactory orientation behaviour. The main olfactory organs of terrestrial molluscs, the experimental animals used in this work, are located on the tips of their tentacles, which display complex movements when they explore a new environment. By reconstructing the trajectory of the tentacle tip ('nose') movements in three dimensions in freely moving snails, we showed that the protracted tentacles performed continuous scanning, both spontaneously and in response to odours. Odour applications elicited a brief startle-like quiver of the tentacle in a concentration-independent manner as well as a concentration-dependent contraction. Previous work showed that activation of an identified cerebral motoneuron, MtC3, produces tentacle contraction. Here we showed that in semi-intact preparations, MtC3 responded to odours in a concentration-dependent manner, similar to the tentacle contraction response to the same odours in intact animals. This observation suggests that MtC3 is involved in the central control of the scanning area by regulating the tentacle length. Using voltage-sensitive dyes and imaging, we demonstrated that during the hyperpolarizing phases of oscillations in the procerebral lobe, the main olfactory centre of the CNS of terrestrial molluscs, MtC3 spike frequency significantly decreased. We also showed that direct activation of the procerebral lobe resulted in the phasic inhibition of MtC3. This is therefore an example of an olfactory system in which the interaction of oscillatory and single neuronal activity plays an important role in the fine tuning of orientation behaviour to suit the particular odour environment.     

Cyto- and chemoarchitecture of the monotreme olfactory tubercle

This study was undertaken to determine whether the olfactory tubercles of two monotremes (platypus and echidna) showed cyto- or chemoarchitectural differences from the tubercles of therian mammals. Nissl staining was applied in conjunction with enzyme reactivity for NADPH diaphorase and acetylcholinesterase, and immunoreactivity for calcium binding proteins (parvalbumin, calbindin and calretinin) and tyrosine hydroxylase (echidna only). Golgi impregnations of the tubercle were also available for the echidna. The olfactory tubercle is a poorly laminated structure in the echidna, despite the pronounced development of other components of the echidna olfactory system, and the dense cell layer of the olfactory tubercle was found to be discontinuous and irregular. Granule cell clusters (islands of Calleja) were present, but were small, poorly defined and did not show the intense NADPH diaphorase activity seen in marsupial and placental mammals. A putative small island of Calleja magna was seen in only one echidna out of four. In Golgi impregnations of the echidna olfactory tubercle, the most abundant neuron type was a medium-sized densely spined neuron similar to that seen in the olfactory tubercle of some therians. Large spine-poor neurons were also seen in the polymorphic layer. In the platypus, the olfactory tubercle was very small but showed more pronounced lamination than the echidna, although no granule cell clusters were seen. In both monotremes, the development of the olfactory tubercle was poor relative to other components of the olfactory system (bulb and piriform cortex). The small olfactory tubercle region in the platypus is consistent with poor olfaction in that aquatic mammal, but the tubercle in the echidna is more like that of a microsmatic mammal than other placentals occupying a similar niche (e.g., insectivores).     

Central forebrain Fos responses to familiar male odours are attenuated in recently mated female mice

Exposure of recently mated female mice to the urinary odours of an unfamiliar male blocks pregnancy (the Bruce effect). The absence of a pregnancy block in response to the stud male's familiar odours depends on an olfactory memory that is formed in the accessory olfactory bulb (AOB) in response to vomeronasal organ (VNO) inputs during mating. Sexually naive Balb/c female mice in pro-oestrus/oestrus were either placed onto soiled bedding ('bedding-only' females) from, or allowed to mate with, a Balb/c male ('recently mated' females). After 42 h, females were placed for 90 min onto clean bedding (controls) or onto soiled bedding from either a C57BL/6 male (unfamiliar bedding) or a Balb/c male (familiar bedding). Significant increases in Fos-immunoreactivity (Fos-IR, a marker of neuronal activation) occurred in the medial amygdala and the medial preoptic area (MPA) of 'bedding only' females exposed to either unfamiliar or familiar bedding and in 'recently mated' females exposed to unfamiliar bedding but not to familiar bedding. This suggests that a mating-induced memory prevents the later activation by the familiar stud male's odours of neurons in forebrain regions that receive inputs from the VNO--AOB. 'Bedding-only' females later exposed to either familiar or unfamiliar bedding had increased Fos-IR in the G alpha(o) protein-expressing basal zone of the VNO whereas no such effect occurred in 'recently mated' females. Familiar, as well as unfamiliar, male odours augmented Fos-IR in significantly more rostral than caudal AOB granule cells in all groups, with the effect being strongest in 'recently mated' females exposed to familiar male bedding. This outcome is consistent with the absence of odour-induced Fos-IR in forebrain regions of these females and, presumably, the absence of a pregnancy block.     

Locus coeruleus activation modulates firing rate and temporal organization of odour-induced single-cell responses in rat piriform cortex

Piriform cortex (PCx) is the primary cortical projection region for olfactory information and has bidirectional monosynaptic connections with olfactory bulb and association cortices. PCx neurons display a complex receptive field, responding to odours rather than their molecular components, suggesting that these neurons are involved in higher order olfactory processing. Neuromodulators, especially noradrenaline (NA), have important influences on sensory processing in other cortical regions and might be responsible for the plasticity observed in PCx during learning. The present study is the first attempt to examine in vivo the actions of NA on sensory responses in the PCx. Stimulation of the noradrenergic nucleus locus coeruleus (LC) was used to induce release of NA in the forebrain in urethane-anaesthetized rats. Extracellular recording of single units was made simultaneously in anterior and posterior PCx. The responses to an odour stimulus were measured over 25 trials. Twenty-five subsequent odour presentations were preceded by stimulation of the ipsilateral LC through a bipolar electrode, previously placed in the LC under electrophysiological control. This priming stimulation modified the activity of 77 of the 135 recorded neurons. For most cells, LC stimulation enhanced cortical responses to odour in terms of both spike count and temporal organization, with some differential effects in anterior and posterior regions. These results are the first to show enhancement of sensory responses in the olfactory cortex by LC activation. Spontaneous activation of LC neurons such as occurs during learning could serve to enhance olfactory perception and promote learning.     

Collateral sprouting in central mesolimbic dopamine neurons: Biochemical and immunocytochemical evidence of changes in the activity and distribution of tyrosine hydroxylase in terminal fields and in cell bodies of A10 neurons

The olfactory tubercle of adult rats was examined for the development of collateral sprouts from intrinsic dopaminergic axons following unilateral olfactory bulbectomy. In the ipsilateral tubercle tyrosine hydroxylase (TH) activity began to increase by 10–14 days following the lesion, gradually reaching a maximum of 125% of control (P < 0.005) by 21 days where it remained permanently elevated. The rise of TH activity in the tubercle reflected changes of the dopaminergic innervation, since dopamine-β-hydroxylase (DBH) activity was unchanged, and lesions of the dorsal noradrenergic bundle reduced DBH but not TH activity in the tubercle. By immunocytochemical staining the elevation of TH reflected an increased number and altered distribution of TH-containing processes within the olfactory tubercle. By 30 days the uptake of [³H]dopamine into synaptosomes of the olfactory tubercle was also increased to 140% of control (P < 0.05). In the dopaminergic cell bodies of the ipsilateral A10 group (which innervate the tubercle) TH activity was transiently elevated to 121% (P < 0.05) by 4 days, returning to control levels by 10 days. Histologically no change in activity was detected. The results indicate that mesolimbic dopaminergic neurons of A10 which innervate the olfactory tubercle will sprout in response to removal of a major non-dopaminergic input, that the new innervation is sustained, and that during collateral sprouting there is a transient elevation of TH activity in the uninjured cell bodies which precedes the period of axonal growth. The findings suggest that (a) the increase of TH activity in the A10 cell bodies during collateral sprouting may be a reflection of an increase in the amount of enzyme protein required for transport into the enlarging terminal fields, (b) that as in development sprouts are in place before they reach biochemical maturity, (c) the biochemical mechanisms underlying collateral sprouting of uninjured neurons are not necessarily the same as those associated with regenerative sprouting in response to axonal injury, and (d) the development and the acquisition of biochemical maturation of collateral sprouts in the CNS involves complex two-way signalling between terminal field and cell bodies. The development of collateral sprouts of dopaminergic neurons may be the cellular substrate of the development of behavioral hyperactivity and aggression produced by bilateral olfactory bulbectomy in rat.