Neural mechanisms underlying the action of primer pheromones in mice

Our electrophysiological experiments in female mice have provided evidence that electrical stimulation of the accessory olfactory bulb orthodromically excites a subpopulation of tuberoinfundibular arcuate neurons by way of the amygdala. The present study shows that half of such neurons are identified as dopaminergic by examining the effectiveness of infusing 6-hydroxydopamine and 5,7-dihydroxytryptamine locally into the median eminence in blocking their antidromic response. Further attention is focused on excitatory amino acid receptors within the amygdala and the amygdaloid pathway that mediate the accessory bulb-induced excitation of tuberoinfundibular arcuate neurons. The excitatory transmission was reversibly blocked by intra-amygdala infusion (3 nmol) of the excitatory amino acid antagonists kynurenic acid, D,L-2-amino-5-phosphonovalerate, gamma-D-glutamylaminomethylsulphonate and D,L-2-amino-4-phosphonobutyrate. Intra-amygdala infusions (3 nmol) of N-methyl-D-aspartate and kainate markedly enhanced the firing activity of tuberoinfundibular arcuate neurons with excitatory inputs from the accessory bulb, whereas similar infusions of quisqualate were without effect Intra-stria terminalis infusions of the local anaesthetic lignocaine completely abolished the excitatory transmission in all the cells tested. Furthermore, tuberoinfundibular arcuate neurons stimulated from the accessory bulb were also orthodromically stimulated from the stria terminalis with a shorter latency. These studies demonstrate that the projections of the accessory olfactory bulb activate excitatory amino acid receptors within the amygdala and subsequently the stria terminalis route, thereby causing excitation of tuberoinfundibular dopaminergic arcuate neurons. This functional pathway can account for the reproductive effects so far described as a consequence of vomeronasal chemoreception.     

Oestrogen infusions into the amygdala potentiate excitatory transmission from the accessory olfactory bulb to tuberoinfundibular arcuate neurones in the mouse.

We have previously shown that oestrogen increases the percentage of tuberoinfundibular (TI) arcuate neurones that respond to electrical stimulation of the accessory olfactory bulb (AOB). This study focuses on the amygdala as a possible site for the hormonal modulation of AOB input to TI arcuate neurones. Local infusions of 17 beta-oestradiol (30 pmol) into the amygdala of ovariectomized female mice significantly potentiated excitatory responses of TI arcuate neurones to AOB stimulation. This effect appeared rapidly (less than 10 min) after infusion. The inactive oestrogen isomer, 17 alpha-oestradiol, infused in the same manner, was without effect. These results suggest that oestrogen acts directly on amygdala neurones, thereby modulating olfactory information relayed along the vomeronasal pathway to TI arcuate neurones.     

An electrophysiological study of the accessory olfactory bulb in the rabbit--II. Input-output relations as assessed from analysis of intra- and extracellular unit recordings

The input-output relations of the rabbit accessory olfactory bulb were studied by intra- and extracellular single unit recordings following electrical stimulation of the vomeronasal nerves, the lateral olfactory tract and the corticomedial amygdala. Cellular activity of accessory bulb mitral cells evoked by stimulation of the vomeronasal nerves consisted of a brief excitation with a latency of 16 ms. This initial response was followed by a period of reduced firing probability which was due to an inhibitory postsynaptic potential. In many cases this secondary response was followed by a second excitatory postsynaptic potential on which action potentials were generated at higher stimulus intensities. Deeper cells in the granule cell layer responded with a long latency, long duration, excitation, often consisting of bursts of 2-3 spikes. The majority of mitral cells were antidromically invaded by amygdala stimulation. The latencies of the antidromic spikes showed a wide range of variation (12-80 ms). Due to this great variation in antidromic latency the inhibitory postsynaptic potential following the antidromic action potential was rather modest but prolonged in duration. In many cases the onset of the inhibitory postsynaptic potential preceded the antidromic response. The majority of cells did not respond to lateral olfactory tract stimulation. Only 10% of the tested cells were invaded antidromically by stimulation at this site. These neurons were also driven antidromically by amygdala stimulation. We conclude that, although the physiological characteristics of mitral cells of the main and accessory olfactory bulb are very similar, there are important differences. The efferent fibres of the accessory bulb conduct at very slow and variable rates and project directly to the corticomedial amygdala.     

Cholecystokinin: critical role in mediating olfactory influences on reproduction.

Our electrophysiological studies in female mice have demonstrated that electrical stimulation of the accessory olfactory bulb excites tuberoinfundibular dopaminergic arcuate neurons via the amygdala-stria terminalis route. This study shows that the medial preoptic area is identified as an additional relay for the excitatory transmission by examining the effectiveness of locally infused lignocaine anaesthetic in blocking the transmission and that of electrical stimulation in evoking a shorter latency response. Based on the known immunohistochemical findings, further attention is focused on a transmitter mediating synaptic transmission in the medial preoptic area. The cholecystokinin-B type receptor antagonist L-365,260 (0.3, 0.6, 0.9 pmol), but not the A type receptor antagonist L-364,718 (0.9 pmol), infused into the medial preoptic area, blocked the excitation of tuberoinfundibular arcuate neurons in a dose-dependent manner. Conversely, cholecystokinin octapeptide (0.6 pmol) increased firing activity in such neurons. The antagonizing effect of L-365,260 was reproduced in the context of the olfactory block to pregnancy: bilateral infusions of this drug into the medical preoptic area of recently mated females immediately before exposures to strange males' pheromones prevented them from inducing pregnancy block. These findings implicate cholecystokinin acting on cholecystokinin-B receptors in the medial preoptic area as a mediator of olfactory influences on reproductive physiology.     

GABAergic mechanisms are involved in the control of tuberoinfundibular arcuate neurons by the accessory olfactory bulb

In this study we examined electrophysiologically the involvement of the intrinsic GABAergic system of the accessory olfactory bulb (AOB) in controlling the activity of tuberoinfundibular (TI) arcuate neurons in anaesthetized female mice. Local infusions of the γ-aminobutyric acid-A (GABA_A) receptor antagonist, bicuculline into the AOB enhanced the spontaneous firing activity of TI arcuate neurons with excitatory inputs from the AOB. This finding reveals a neural mechanism responsible for the pregnancy blocking effect of this drug in freely behaving female mice and, taken together with the cytoarchitecture of the AOB, suggests that the reciprocal dendrodendritic interaction between mitral cells and GABAergic granule cells in the AOB is critical to control of AOB output to TI arcuate neurons as part of the final common pathway of the accessory olfactory system.     

An electrophysiological study of the accessory olfactory bulb in the rabbit--I. Analysis of electrically evoked potential fields

Following electrical stimulation of the vomeronasal nerves, the primary olfactory nerves, the lateral olfactory tract and the corticomedial amygdala, we have made a study of evoked potentials in the rabbit accessory olfactory bulb. Vomeronasal nerve stimulation evoked a complex field potential consisting of a compound action potential followed by 4 negative waves (N1, N2, N3, N4). In contrast to the field potential elicited in the main olfactory bulb following primary olfactory nerve stimulation, there was either no evoked wave or only a weak positive component of the field in the accessory bulb. Amygdala stimulation caused a long latency, long duration negative-positive dipolar field potential in the accessory olfactory bulb. Both antidromic and orthodromic field potentials showed sign reversal when the electrode penetrated the bulb at a point corresponding to the lower border of the mitral cell band. Stimulation of the lateral olfactory tract elicited a weak, short-latency wave which did not show any sign reversal when the electrode was lowered into the accessory bulb. This wave was presumably due to fibres arising in the main bulb and projecting through the accessory bulb into the lateral olfactory tract. Electrical stimulation of the primary olfactory nerves did not induce any response in the accessory bulb neither did vomeronasal nerve stimulation evoke a response in the main olfactory bulb. The origin of these potential fields is discussed and it is concluded that the synaptic organization of the accessory olfactory bulb resembles that of the main olfactory bulb in lower vertebrates. There is no detectable communication between the two olfactory systems.     

Influence of amygdala stimulation on the activity of identified tuberoinfundibular neurones in the rat hypothalamus.

1. Extracellular action potentials were recorded from 1246 neurones in the mediobasal hypothalamus of pentobarbitone or urethane anaesthetized male rats. Antidromic invasion from the surface of the median eminence identified 165 cells, located in the arcuate and ventromedial nuclei and the periventricular area, as tuberoinfundibular neurones. The majority (65%) of these cells displayed no spontaneous activity. 2. Latencies for antidromic invasion from median eminence ranged from 0-5 to 14-0 msec (mean 4-3 +/- 2-9 msec, S.D.). Conduction velocities for axons of tuberoinfundibular neurones were under 1-0 m/sec, and were slowest (under 0-2 m/sec) for those tuberoinfundibular neurones located in the arcuate nucleus. 3. Single 1 HZ stimulation of amygdala evoked short latency (mean 18-8 +/- 7-0 msec; n = 30) excitation of tuberoinfundibular neurones in the ventromedial nucleus. Stria terminalis stimulation evoked similar responses at a shorter latency (mean 10-2 +/- 3-5 msec; n = 12) from other ventromedial tuberoinfundibular neurones. Three of these neurones were also excited by amygdala stimulation at comparably longer latencies. In spontaneously active tuberoinfundibular cells, the initial excitation was followed by a decrease in excitability lasting 70-150 msec. Tuberoinfundibular neurones soldom followed orthodromic activation at frequencies beyond 30 HZ. 4. An initial decrease in activity at latencies of 18-40 msec (mean 29-2 +/- 10-2 msec) characterized the amygdala evoked responses from nine tuberoinfundibular neurones. A similar response from one other tuberoinfundibular neurone followed stria terminalis stimulation at a latency of 11 msec. Most of these tuberoinfundibular neurones were located in the dorsal part of the ventromedial nucleus. 5. Two ventromedial tuberoinfundibular neurones also displayed antidromic invasion from the amygdala; interaction studies suggested an axon collateral pathway that originated close to the origin of the axon. 6. Tuberoinfundibular neurones unresponsive to amygdala stimulation were usually located in the arcuate nucleus or periventricular area. 7. These results provide electrophysiological evidence for a direct influence of the amygdala on the activity of tuberoinfundibular neurones in the ventromedial hypothalamic nucleus. There are also data to indicate that some ventromedial tuberoinfundibular neurones have axon collaterals that return to the amygdala. These reciprocal connexions between the amygdala and ventromedial tuberoinfundibular neurones may indicate neural circuits important for extrahypothalamic modulation of adenohypophyseal secretion.     

Functional synapse formation between cultured rat accessory olfactory bulb neurons and vomeronasal pockets

To investigate the interaction between vomeronasal receptor neurons and accessory olfactory bulb neurons during pheromonal signal processing and specific synapse formation, partially dissociated rat vomeronasal receptor neurons were co-cultured with accessory olfactory bulb neurons. Between 7 and 14 days in co-culture, a few bundles of fibers from a spherical structure, termed the vomeronasal pocket, of cultured vomeronasal receptor neurons extended to the accessory olfactory bulb neurons. An optical recording of the intracellular Ca(2+) concentration was used to monitor the synaptic activation of cultured accessory olfactory bulb neurons. Electrical stimulation of the vomeronasal pocket between 7 and 14 days in co-culture had no effects on most of the cultured neurons tested, although it occasionally evoked weak responses in a small number of neurons. In contrast, vomeronasal pocket stimulation after 21 days in co-culture evoked clear calcium transients in a substantial number of cultured accessory olfactory bulb neurons. These responses of accessory olfactory bulb neurons were reversibly suppressed by the application of 6-cyano-7-nitroquinoxaline-2,3-dione; the calcium transients disappeared in most of the neurons and were diminished in the others. The application of d-2-amino-5-phosphonopentanoic acid partially affected the calcium transients, but blocked spontaneous calcium increases, which were observed repeatedly in accessory olfactory bulb-alone cultures. The application of both 6-cyano-7-nitroquinoxaline-2,3-dione and d-2-amino-5-phosphonopentanoic acid completely blocked the evoked calcium transients. These results suggest that functional glutamatergic synapses between vomeronasal receptor neurons and accessory olfactory bulb neurons were formed at around 21 days in co-culture.     

Multiple connections of medial hypothalamic neurons in the rat

Summary The responses of 700 single neurons in the hypothalamus to electrical stimulation of the preoptic area, limbic structures, and midbrain were studied to determine the location of neurons with multiple inputs and to identify by antidromic activation the projection areas of those neurons.Converging excitatory inputs, observed in 134 responsive hypothalamic neurons, were principally derived from the preoptic, limbic, and midbrain areas. Inputs from separate nuclei of the amygdala were noted in the response of individual hypothalamic neurons. Two classes of short latency transsynaptic responses to amygdala stimulation were defined, indicating either separate pathways from the amygdala to the medial hypothalamus or two types of fibers conducting at different velocities. Stimulation of single or multiple sites in the preoptic and limbic areas, as well as in the arcuate nucleus and medial forebrain bundle produced inhibition of hypothalamic neuronal activity.Most antidromically identified medial hypothalamic neurons projected to the preoptic area, median eminence (tuberoinfundibular neurons), or midbrain. Evidence is presented for collateral projections of tuberoinfundibular neurons to the preoptic area and reticular formation. Medial hypothalamic neurons received inputs from the preoptic area, lateral septal nucleus, amygdala, ventral hippocampus (subiculum), and fornix. These findings illustrate a pattern of reciprocal connections between the medial hypothalamus and limbic and midbrain structures.It was concluded that the hypothalamus contains a type of neuron that is equipped to perform complex integrations and to coordinate directly the behavior of neurons in a diversity of anatomical regions.Abbreviations ABL basolateral nucleus of the amygdala - ACO cotical nucleus of the amygdala - AHA anterior area of the hypothalamus - ARH arcuate nucleus of the hypothalamus - DMH dorsomedial nucleus of the hypothalamus - FX fornix - HPC ventral hippocampus (subiculum) - LS lateral septal nucleus - ME median eminence - MH medial hypothalamus - MFB medial forebrain bundle - MP posterior mamillary nucleus - PH posterior nucleus of the hypothalamus - PMD dorsal premamillary nucleus - PMV ventral premamillary nucleus - POA preoptic area - PVG periventricular gray - PVH paraventricular nucleus of the hypothalamus - RF reticular formation of the mesencephalon - RT reticular nucleus of the thalamus - SUM supramamillary nucleus - VMH ventromedial nucleus of the hypothalamus Performed with financial support from the National Institutes of Health (Grants NS 09688 and RR 00165)     

An experimental study of the projection of the amygdala to the accessory olfactory bulb and its relationship to the concept of a dual olfactory system

Summary The present anatomical findings point to the existence of a separate subdivision of the olfactory system whose connections are quite different from the principal part. The main olfactory bulb has olfactory afferents from the receptors of the general olfactory mucosa, while the accessory bulb has afferents from receptors in the vomeronasal organ. The main bulb projects to the olfactory tubercle and pyriform cortex, while the accessory bulb projects to the amygdala. In turn these areas are further related with the medial forebrain bundle in the case of the pyriform cortex and olfactory tubercle, and with the medial preoptic area and medial hypothalamus in the case of the amygdala. The main and accessory olfactory bulbs are further distinguished by their centrifugal connections, the main bulb receiving fibres from the olfactory tubercle passing through the lateral olfactory tract, and the accessory olfactory bulb receiving fibres from the amygdala through the stria terminalis. The centrifugals to the accessory olfactory bulb resemble those to the main bulb in that both appear to terminate upon granule cells, although further projections to the external plexiform layer or to the periglomerular region have not been demonstrated for the accessory bulb. By virtue of its neural connections the accessory olfactory system is ideally placed to mediate the effects of olfactory stimuli on reproduction.     

Increase in the number of tyrosine hydroxylase-containing neurons in a primary culture system of the rat accessory olfactory bulb by co-culture with vomeronasal pockets

Previously, we established a culture system of the accessory olfactory bulb in order to investigate the functional role of each accessory olfactory bulb neurons in pheromonal signal processing. In the present study, we developed a co-culture system of cultured accessory olfactory bulb neurons with partially dissociated cells of the vomeronasal organ. The dissociated cells of the vomeronasal organ form spherical structures surrounding a central cavity in culture, referred to as the vomeronasal pockets. The projection and activity of olfactory receptor neurons affect the differentiation and maturation of main olfactory bulb neurons. It was also reported induction of tyrosine hydroxylase expression in main olfactory bulb neurons when they were co-cultured with explants of the olfactory epithelium. Thus, we investigated the effects of co-culture with vomeronasal pockets on the differentiation and/or maturation of cultured accessory olfactory bulb neurons in relation to tyrosine hydroxylase expression. The number of tyrosine hydroxylase-containing neurons developmentally increased over time in the accessory olfactory bulb culture. This increase was significantly enhanced by coculture with vomeronasal pockets. Interestingly, a significant change in tyrosine hydroxylase expression was not observed when main olfactory bulb neurons were co-cultured with vomeronasal pockets. Moreover, significant changes in tyrosine hydroxylase expression were not observed when accessory olfactory bulb neurons were co-cultured with olfactory epithelium explants, as was previously observed in co-culture of main olfactory bulb neurons and olfactory epithelium explants. These results suggest that the differentiation and/or maturation of accessory olfactory bulb neurons is modified by vomeronasal organ neurons via specific interactions between the sensory organ and its target.     

3H-thymidine-radiographic studies of neurogenesis in the rat olfactory bulb

Neurogenesis in the rat olfactory bulb was examined with 3H-thymidine-radiography. For the animals in the prenatal groups, the initial 3H-thymidine exposures were separated by 24 h; they were the offspring of pregnant females given two injections on consecutive embryonic (E) days (E12-E13, E13-E14, . . . E21-E22). For the animals in the postnatal (P) groups, the initial 3H-thymidine injections were separated by 48 h, each group receiving either four (PO-P3, P2-P4, . . . P6-P9) or two (P8-P9, P10-P11, . . . P20-P21) consecutive daily injections. On P60, the percentage of labeled cells and the proportion of cells added during either 24 h or 48 h periods were quantified at several anatomical levels for each neuronal population in the main olfactory bulb (mitral cells, tufted cells, granule cells, interneurons in the external plexiform layer, periglomerular granule cells) and accessory olfactory bulb (output neurons, granule cells, periglomerular granule cells). The total time span of neurogenesis extends from E12 to beyond P20. Output neurons are prenatally generated over 5-9 day periods (with most neurogenesis occurring over 2-4 days) in a strict sequential order beginning with the accessory bulb output neurons (E13-E14) and ending with the interstitial tufted cells lying between the glomeruli in the main bulb (E20-E22). These data are correlated with the main and accessory bulb projection fields in the amygdala and with the chronology of amygdala neurogenesis. With the exception of the granule cells in the accessory bulb (88% generated between E15-E22), the rest of the interneuronal populations are generated postnatally and nearly simultaneously. While most neurons (75-80%) originate during the first three weeks of life, all interneuronal populations, including accessory bulb granule cells, show some neurogenesis beyond P20. Injections of 3H-thymidine in juvenile and adult rats indicates neurogenesis up to P60 in the accessory bulb and up to P180 in the main bulb, especially in the main bulb granule cell population. There is circumstantial evidence for turnover of main bulb granule cells during adult life.     

Learning-induced enhancement of postsynaptic potentials in pyramidal neurons

We studied the effect of olfactory learning-induced modifications in piriform (olfactory) cortex pyramidal neurons on the propagation of postsynaptic potentials (PSPs). Rats were trained to distinguish between odors in pairs, in an olfactory discrimination task. Three days after training completion, PSPs were evoked in layer II pyramidal cells in piriform cortex brain slices by electrical stimulation of two pathways. Stimulation of layer Ib activated the intra-cortical fibers that terminate on the proximal region of the apical and basal dendrites. Stimulation of layer Ia activated the afferent axons that originate from the olfactory bulb and terminate on the distal apical dendrites. We have previously shown that olfactory training is accompanied by enhanced synaptic transmission in the intrinsic pathway, but not in the afferent pathway at 3 days after training. Here we show that at this stage, in both pathways PSPs evoked in neurons from trained rats had significantly faster rise time measured at the soma compared with PSPs in neurons from pseudo-trained and naive rats. Activation of the slow afterhyperpolarization (AHP), which is generated by potassium channels probably located at the proximal region of both apical and basal dendrites, reduced the amplitude measured at the soma of the proximal intrinsic pathway PSPs more effectively than PSPs that were generated distally by the afferent fibers. Thus the amount of reduction by AHP was used as a measure for the relative distance of PSP-generating sites from the soma. In neurons from trained rats, despite the previously reported reduction in AHP amplitude, AHP conductance shunted the PSPs from both synaptic pathways more efficiently compared with neurons from the control rats. We suggest that in neurons from trained rats PSPs are electrotonicly closer to the soma.     

Delayed deficits in behavior after transection of the olfactory tracts in hamsters

This study compared the effects of transection of the lateral olfactory tracts (LOT) and the accessory olfactory tracts (AOT) in male hamsters on nest building, food piling, and sexual behavior. Autoradiographic tracing of amino acids injected into the olfactory bulbs allowed accurate determination of the location and extent of the transections. Animals with complete bilateral transections of the projections to the amygdaloid targets of the accessory olfactory bulbs and to the main olfactory targets posterior to the olfactory tubercle showed no sexual behavior postoperatively; they did not exhibit extensive genital investigation and did not mount females. In contrast, most of the animals with partial sparing of accessory olfactory bulb efferents to the amygdala did exhibit investigatory and copulatory behaviors postoperatively, although half of the animals with this partial sparing developed delayed deficits in these sexual behaviors. Almost all animals without detectable main olfactory bulb efferents to posterior targets showed delayed deficits in nest building and food piling. This was true whether or not there was partial sparing of accessory olfactory bulb efferents to the amygdala. The animals with LOT transections typically built nests and piled food during the first postoperative week, but stopped building nests and piling food by the fourth postoperative week. Cold stress enhanced these two behaviors in control animals but did not obviate the deficits in experimental animals. Caudally placed transections, which spared a larger portion of the main olfactory projections than rostally placed transections, did not spare more behavior. In fact, the caudally placed transections produced shorter delays in the appearance of deficits in nest building and food piling. These results indicate that the accessory olfactory bulb efferents to the amygdala are more important for sexual behavior than for nest building and food piling in male hamsters. Nest building and food piling are not directly dependent on normal ongoing or sensory evoked activity in the main or accessory olfactory bulb efferents which project through the LOT and AOT. The deficits in nest building and food piling may represent a deterioration in the ability of the animals to organize their living space. The observed delays in the appearance of deficits in behavior may also reflect slow degenerative processes or humoral changes associated with loss of input from the main olfactory bulbs to posterior olfactory target areas, and possibly with interruptions of projections to targets of the accessory olfactory system.     

Nicotine-induced enhancement of glutamatergic and GABAergic synaptic transmission in the mouse amygdala.

Presynaptic nicotinic acetylcholine receptors (nAChRs) are thought to mediate some of the cognitive and behavioral effects of nicotine. The olfactory projection to the amygdala, and intra-amygdaloid projections, are limbic relays involved in behavioral reinforcement, a property influenced by nicotine. Co-cultures consisting of murine olfactory bulb (OB) explants and dispersed amygdala neurons were developed to reconstruct this pathway in vitro. Whole cell patch-clamp recordings were obtained from amygdala neurons contacted by OB explant neurites, and spontaneous and evoked synaptic currents were characterized. The majority of the 108 innervated amygdala neurons exhibited glutamatergic spontaneous postsynaptic currents (PSCs), 20% exhibited GABAergic spontaneous PSCs, and 17% exhibited both. Direct extracellular stimulation of OB explants elicited glutamatergic synaptic currents in amygdala neurons. Antibodies to nAChR subunits co-localized with an antibody to synapsin I, a presynaptic marker, along OB explant processes, consistent with the targeting of nAChR protein to presynaptic sites of the mitral cell projections. Hence, we examined the role of presynaptic nAChRs in modulating synaptic transmission in the OB-amygdala co-cultures. Focal application of 500 nM to 1 microM nicotine for 5-60 s markedly increased the frequency of spontaneous PSCs, without a change in the amplitude, in 39% of neurons that exhibited glutamatergic spontaneous PSCs (average peak fold increase = 125.2 +/- 33.3). Nicotine also enhanced evoked glutamatergic currents elicited by direct stimulation of OB explant fibers. Nicotine increased the frequency of spontaneous PSCs, without a change in the amplitude, in 35% of neurons that exhibited GABAergic spontaneous PSCs (average peak fold increase = 63.9 +/- 34.3). Thus activation of presynaptic nAChRs can modulate glutamatergic as well as GABAergic synaptic transmission in the amygdala. These results suggest that behaviors mediated by olfactory projections may be modulated by presynaptic nAChRs in the amygdala, where integration of olfactory and pheromonal input is thought to occur.     

Effects of odor stimulation on antidromic spikes in olfactory sensory neurons

Spikes were evoked in rat olfactory sensory neuron (OSN) populations by electrical stimulation of the olfactory bulb nerve layer in pentobarbital anesthetized rats. The latencies and recording positions for these compound spikes showed that they originated in olfactory epithelium. Dual simultaneous recordings indicated conduction velocities in the C-fiber range, around 0.5 m/s. These spikes are concluded to arise from antidromically activated olfactory sensory neurons. Electrical stimulation at 5 Hz was used to track changes in the size and latency of the antidromic compound population spike during the odor response. Strong odorant stimuli suppressed the spike size and prolonged its latency. The latency was prolonged throughout long odor stimuli, indicating continued activation of olfactory receptor neuron axons. The amounts of spike suppression and latency change were strongly correlated with the electroolfactogram (EOG) peak size evoked at the same site across odorants and across stimulus intensities. We conclude that the curve of antidromic spike suppression gives a reasonable representation of spiking activity in olfactory sensory neurons driven by odorants and that the correlation of peak spike suppression with the peak EOG shows the accuracy of the EOG as an estimate of intracellular potential in the population of olfactory sensory neurons. In addition, these results have important implications about traffic in olfactory nerve bundles. We did not observe multiple peaks corresponding to stimulated and unstimulated receptor neurons. This suggests synchronization of spikes in olfactory nerve, perhaps by ephaptic interactions. The long-lasting effect on spike latency shows that action potentials continue in the nerve throughout the duration of an odor stimulus in spite of many reports of depolarization block in olfactory receptor neuron cell bodies. Finally, strong odor stimulation caused almost complete block of antidromic spikes. This indicates that a very large proportion of olfactory axons was activated by single strong odor stimuli.     

Ventral hippocampal neurons project axons simultaneously to the medial prefrontal cortex and amygdala in the rat

The ventral hippocampus (VH) may have an important role in spatial memory processes and emotional behaviors through connections with the medial prefrontal cortex (mPFC) and amygdala. Although the mPFC and amygdala receive afferent projections from the VH, it has not been determined whether the individual VH neurons project to both the mPFC and the amygdala. In this study, antidromic responses to the mPFC and amygdala stimulation were evoked in single VH neurons. In addition, VH neurons were retrogradely double-labeled with fluorescent tracers injected in the mPFC and amygdala. VH neurons projecting to both the mPFC and amygdala were predominantly located in the subiculum and CA1 and bifurcated near or at the soma. Our anatomical and electrophysiological evidence for the presence of VH neurons projecting to both the mPFC and amygdala provides a previously unrecognized pathway from the hippocampus that simultaneously activates the mPFC and amygdala.     

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat main and accessory olfactory bulbs

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat olfactory bulb was obtained with sheep antiserum to glutamate decarboxylase (GAD) and rabbit antiserum to tyrosine hydroxylase (TH). GAD-positive neurons include periglomerular cells, granule cells, superficial and deep short axon cells. TH-positive neurons represent periglomerular cells. Two-color immunocytochemistry shows that GABA and dopamine periglomerular cells are separate populations. The accessory olfactory bulb has rare dopamine cells and few superficial short axon cells. Radial gradients of GAD-immunostaining are evident in the main but not in the accessory olfactory bulb.     

Neural basis of olfactory memory in the context of pregnancy block

In mice, only strange male pheromones block pregnancy; pheromones of the familiar male with which the female has mated have the capacity to block pregnancy but are ineffective with the consort female. Hence, some form of recognition/memory to the stud male is formed at mating. By infusing lignocaine locally into the accessory olfactory bulb and second order olfactory synapses in the medial nucleus of the amygdala, this study localizes changes that occur in the accessory olfactory bulb at mating to be subsequently important in preventing the stud male's pheromones from blocking pregnancy. Further attention is focused on the dendrodendritic synapses between mitral and granule cells in the accessory olfactory bulb. Blockade of the GABA receptors (granule to mitral cell synapse) in the accessory bulb without mating, but in the presence of male pheromones, prevents any male from blocking pregnancy. Conversely inhibition of protein kinase C, a second messenger system activated by excitatory amino acids (mitral to granule cell synapse), in the accessory bulb during a 4-h period after mating permits all male pheromones including the stud's to activate pregnancy block. While blockade of protein kinase C activity during the critical exposure time for memory formation prevents memory formation, infusions of a protein synthesis inhibitor (anisomycin) are without effect. However, protein synthesis inhibition in the accessory olfactory bulb in the late phase of the critical exposure time (3-6 h after mating) does prevent memory formation. These studies show that changes in synaptic plasticity in the accessory olfactory bulb following mating are critical to recognition of the stud male's pheromones, hence preventing these from subsequently blocking pregnancy.     

Entorhinal cortex stimulation modulates amygdala and piriform cortex responses to olfactory bulb inputs in the rat

The rodent olfactory bulb sends direct projections to the piriform cortex and to two structures intimately implicated in memory processes, the entorhinal cortex and the amygdala. The piriform cortex has monosynaptic projections with the amygdala and the piriform cortex and is therefore in a position to modulate olfactory input either directly in the piriform cortex, or via the amygdala. In order to investigate this hypothesis, field potential signals induced in anesthetized rats by electrical stimulation of the olfactory bulb or the entorhinal cortex were recorded simultaneously in the piriform cortex (anterior part and posterior part) and the amygdala (basolateral nucleus and cortical nucleus). Single-site paired-pulse stimulation was used to assess the time courses of short-term inhibition and facilitation in each recording site in response to electrical stimulation of the olfactory bulb and entorhinal cortex. Paired-pulse stimulation of the olfactory bulb induced homosynaptic inhibition for short interpulse interpulse intervals (20-30 ms) in all the recording sites, with a significantly lower degree of inhibition in the anterior piriform cortex than in the other structures. At longer intervals (40-80 ms), paired-pulse facilitation was observed in all the structures. Paired-pulse stimulation of the entorhinal cortex mainly resulted in inhibition for the shortest interval duration (20 ms) in anterior piriform cortex, posterior piriform cortex and amygdala basolateral but not cortical nucleus. Double-site paired-pulse stimulation was then applied to determine if stimulation of the entorhinal cortex can modulate responses to olfactory bulb stimulation. For short interpulse intervals (20 ms) heterosynaptic inhibition was observed in anterior piriform cortex, posterior piriform cortex and amygdala basolateral but not cortical nucleus. The level of inhibition was greater in the basolateral nucleus than in the other structures. Taken together these data suggest that the entorhinal cortex exerts a main inhibitory effect on the olfactory input via the amygdala basolateral nucleus and to a lesser extent the piriform cortex. The potential role of these effects on the processing of olfactory information is discussed.