High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.     

Specific Binding of Insulin to Membranes from Dendrodendritic Synaptosomes of Rat Olfactory Bulb

The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I-insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin-receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5-1,000 nM, revealed binding site heterogeneity consistent with a two-site model for insulin binding to a high-affinity (KD = 6 nM), low-capacity (Bmax = 110 fmol/mg of protein) site and a low-affinity (KD = 190 nM), high-capacity (Bmax = 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.     

A biophysical model of vertebrate olfactory epithelium and bulb exhibiting gap junction dependent odor-evoked spatiotemporal patterns of activity

This work describes a biophysical model of the initial stages of vertebrate olfactory system containing structures representing the olfactory epithelium and bulb. Its main novelty is the introduction of gap junctions connecting neurons both in the epithelium and bulb, and of biologically detailed dendrodendritic synapses between granule and mitral cells in the bulb. The model was used to simulate the effect of an odor presentation on the neural activity pattern in the epithelium and bulb. During the time for which an odor is presented with a constant concentration, there are spatiotemporal patterns in the epithelium and bulb generated by the couplings due to the gap junctions and/or dendrodendritic synapses. A study varying the strength of the gap junction coupling shows that the spatiotemporal patterns, both in the epithelium and bulb, are dependent of the coupling strength. It is also shown that the olfactory bulb's spatiotemporal pattern depends on the existence of the dendrodendritic connections between mitral and granule cells. If these spatiotemporal patterns really exist in the early processing stages of the olfactory system they may be used for odor coding and the gap junctions and dendrodendritic synapses might have a role on it.     

Mitral cell differentiation and synaptogenesis of rat presumptive olfactory bulb in organ culture

Summary The ultrastructure of differentiating rat presumptive olfactory bulb in organ culture was investigated with particular reference to mitral cell differentiation and formation of synapses. The presumptive olfactory bulb and olfactory mucosa were dissected en bloc from rat embryos on the fifteenth day of gestation and cultured for 7 days, after which the expiants were examined by electron microscopy. The presumptive olfactory bulb had differentiated into a laminated structure with layers corresponding to the glomerular, external plexiform and mitral cell layers. Mitral-like cells were identified by their location and large cell size. Ultrastructural observations indicated that they were relatively well-differentiated. Their dendrites extended into the glomerular layer in which they were postsynaptic to incoming olfactory axons. The distal part of these dendrites frequently contained coated vesicles. Both asymmetrical and symmetrical synapses were found. The symmetrical synapses involved dendrodendritic contacts between periglomerular cells. Synapses in reciprocal arrangements were not observed in the organ cultures.     

Dynamic gating of spike propagation in the mitral cell lateral dendrites

A unique feature of the olfactory bulb circuit is the long projection of the mitral cell lateral dendrites. Through dendrodendritic reciprocal synapses, these dendrites connect one olfactory glomerular module to hundreds of others; but the functional principles governing these extensive lateral interactions remain largely unknown. Here we report that the spatial extent of action potential propagation in these dendrites is dynamically regulated by inhibitory synapses distributed along the dendrites. The extent of propagation determines the spatial pattern of Ca(2+) influx and thus the range and number of dendrodendritic synapses to be activated. Accordingly, network control of spike traffic in the mitral cell lateral dendrites can mediate dynamic interaction with different combinations of glomerular modules in response to different odorants.     

Long-term potentiation and olfactory memory formation in the carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) olfactory bulb

Long-term potentiation of synaptic transmission is considered to be an elementary process underlying the cellular mechanism of memory formation. In the present study we aimed to examine whether or not the dendrodendritic mitral-to-granule cell synapses in the carp olfactory bulb show plastic changes after their repeated activation. It was found that: (1) the dendrodendritic mitral-to-granule cell synapses showed three types of plasticity after tetanic electrical stimulation applied to the olfactory tract—long-term potentiation (potentiation lasting >1 h), short-term potentiation (potentiation lasting <1 h) and post-tetanic potentiation (potentiation lasting <10 min); (2) Long-term potentiation was generally induced when both the dendrodendritic mitral-to-granule cell synapses and centrifugal fiber-to-granule cell synapses were repeatedly and simultaneously activated; (3) long-term enhancement (>1 h) of the odor-evoked bulbar response accompanied the electrically-induced LTP, and; (4) repeated olfactory stimulation enhanced dendrodendritic mitral-to-granule cell transmission. Based on these results, it was proposed that long-term potentiation (as well as olfactory memory) occurs at the dendrodendritic mitral-to-granule cell synapses after strong and long-lasting depolarization of granule cells, which follows repeated and simultaneous synaptic activation of both the peripheral and deep dendrites (or somata).     

GABA IN THE OLFACTORY BULB AND OLFACTORY NUCLEUS OF THE RAT: THE DISTRIBUTION OF GAMMA-AMINOBUTYRIC ACID, GLUTAMIC ACID DECARBOXYLASE, GABA TRANSAMINASE AND SUCCINATE SEMIALDEHYDE DEHYDROGENASE

Abstract— The distribution of GABA and enzymes involved in its metabolism was investigated in the different regions of the olfactory bulb and olfactory nucleus. The highest levels of GABA in the olfactory bulb were found in the layers rich in nerve terminals (31 μmol/g dry wt.). A similar distribution was found in the olfactory nucleus although the overall level of GABA was only a quarter of that measured in the bulb. Glutamic acid decarboxylase (GAD) levels in the various layers of the olfactory nucleus were similar in distribution to those of GABA. However, the correlation between GAD and GABA did not hold for the olfactory bulb, particularly in the granule cell layer and the medulla. The activities of GAD and the levels of GABA are significantly higher in the bulb than in the nucleus but succinic acid scmialdehyde dehydrogenase and GABA aminotransaminase activities are almost identical in both regions.     

mtDNA m.3635G>A may be classified as a common primary mutation for Leber hereditary optic neuropathy in the Chinese population

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via ionotropic (GABA_A and GABA_C) and metabotropic (GABA_B) receptors. The GABA_B receptor is a dimer composed of R1 and R2 components. In addition to their location on neurons, GABA and functional GABA_B receptors also have been detected in some peripheral tissues. In the present study, we combined immunohistochemistry, immunoblot and tension recording to determine if the human fallopian tube express glutamic acid decarboxylase (GAD65/67), two isoforms for synthesis of GABA and functional GABA_B receptors. Immunoblots showed that the human fallopian tube tissue contained GABA_BR1 protein which was localized in the epithelial cells and smooth muscle cells by immunohistochemistry. In addition, epithelial cells also expressed GAD65/67. Tension recording found that both GABA and baclofen, a GABA_B receptor agonist increased the spontaneous activity of human fallopian tube. The expressions of GABA_BR and GAD65/67 were significantly upregulated in the ectopic pregnancy group than in the intrauterine pregnancy group. We conclude that the human fallopian tube is capable of synthesizing GABA and expresses functionally active GABA_B receptors. An upregulation of GABA synthesis and corresponding GABA_B receptors may involve in ectopic pregnancy.     

A novel local circuit in the olfactory bulb involving an old short-axon cell

Many local circuit interactions in the olfactory bulb involve atypical dendrodendritic synapses. In this issue of Neuron, Pressler and Strowbridge report a functional analysis of a class of short-axon interneurons in the bulb called Blanes cells. Blanes cells make GABAergic axonal contacts onto granule cells and may mediate a form of local feedforward disinhibition.     

Synaptic mechanisms underlying pheromonal memory in vomeronasal system

When female mice are mated, they form a memory of the pheromonal signal of the male with which they mated. Our research objective was to determine the neural mechanisms underlying learning and memory by employing a convenient model of pheromone-induced olfactory memory (pheromonal memory). Formation of pheromonal memory depends on the association between mating and exposure to pheromones. Synaptic plasticity involving this memory occurs in the accessory olfactory bulb (AOB), depending on vaginocervical stimulation at mating. The vaginocervical stimulation at mating reduces the dendrodendritic feedback inhibition of principal neurons (mitral/tufted (MT) cells) in the AOB and enhances their cell activity. The enhancement of activity induces on these plastic changes in dendrodendritic synapses, which in turn enhance GABA-mediated inhibition of MT cell activity. This "self-inhibition" of MT cells activity in response to pheromonal signals of the partner can disrupt its signals at the AOB thereby preventing the signals from reaching the central brain. The formation and maintenance of pheromonal memory is based on this inhibition mechanism.     

Dendritic action potentials connect distributed dendrodendritic microcircuits

Lateral inhibition of cells surrounding an excited area is a key property of sensory systems, sharpening the preferential tuning of individual cells in the presence of closely related input signals. In the olfactory pathway, a dendrodendritic synaptic microcircuit between mitral and granule cells in the olfactory bulb has been proposed to mediate this type of interaction through granule cell inhibition of surrounding mitral cells. However, it is becoming evident that odor inputs result in broad activation of the olfactory bulb with interactions that go beyond neighboring cells. Using a realistic modeling approach we show how backpropagating action potentials in the long lateral dendrites of mitral cells, together with granule cell actions on mitral cells within narrow columns forming glomerular units, can provide a mechanism to activate strong local inhibition between arbitrarily distant mitral cells. The simulations predict a new role for the dendrodendritic synapses in the multicolumnar organization of the granule cells. This new paradigm gives insight into the functional significance of the patterns of connectivity revealed by recent viral tracing studies. Together they suggest a functional wiring of the olfactory bulb that could greatly expand the computational roles of the mitral-granule cell network.     

An in vitro study of long-term potentiation in the carp (Cyprinus carpio L.) olfactory bulb

Long-term potentiation (LTP) of synaptic transmission is considered a cellular mechanism for neural plasticity and memory formation. Previously, we showed that in the carp olfactory bulb, LTP occurs at the dendrodendritic mitral-to-granule cell synapse following tetanic electrical stimulation applied to the olfactory tract, and suggested that it is involved in the process of olfactory memory formation. As a first step towards understanding mechanisms underlying plasticity at this synapse, we examined the effects of various drugs (glutamate and GABA receptor agonists and antagonists, noradrenaline, and drugs affecting cAMP signaling) on dendrodendritic mitral-to-granule cell synaptic transmission in an in vitro preparation. Two forms of LTP are involved: a postsynaptic form (tetanus-evoked LTP) and a presynaptic form. The postsynaptic form is evoked at the granule cell dendrite following tetanic olfactory tract stimulation and is suppressed by the NMDA receptor antagonist, D-AP5, enhanced by noradrenaline, and occluded by the metabotropic glutamate receptor agonist, trans-ACPD. The presynaptic form occurs at the mitral cell dendrite following blockade of the GABA_A receptor by picrotoxin and bicuculline, or via activation of cAMP signaling by forskolin and 8-Br-cAMP.     

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABA_A) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl⁻conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.     

Double-labeling techniques demonstrate that rod bipolar cells are under GABAergic control in the inner plexiform layer of the rat retina

The synaptic connectivity between rod bipolar cells and GABAergic neurons in the inner plexiform layer (IPL) of the rat retina was studied using two immunocytochemical markers. Rod bipolar cells were stained with an antibody specific for protein kinase C (PKC, α isoenzyme), and GABAergic neurons were stained with an antiserum specific for glutamic-acid decarboxylase (GAD). Some amacrine cells were also labeled with the anti-PKC antiserum. All PKC-labeled amacrine cells examined showed GABA immunoreactivity, indicating that PKC-labeled amacrine cells constitute a subpopulation of GABAergic amacrine cells in the rat retina. A total of 150 ribbon synapses established by rod bipolar cells were observed in the IPL. One member of the postsynaptic dyads was always an unlabeled AII amacrine cell process, and the other belonged to an amacrine-cell process showing GAD immunoreactivity. The majority (n=92) (61.3%) of these processes made reciprocal synapses back to the axon terminals of rod bipolar cells. In addition, 78 conventional synapses onto rod bipolar axons were observed, and among them 52 (66.7%) were GAD-immunoreactive. Thus GABA provides the major inhibitory input to rod bipolar cells.     

Analysis of relations between NMDA receptors and GABA release at olfactory bulb reciprocal synapses

In the mammalian olfactory bulb, signal processing is mediated by synaptic interactions between dendrites. Glutamate released from mitral cell dendrites excites dendritic spines of granule cells, which in turn release GABA back onto the mitral cell dendrites, forming a reciprocal synaptic pair. This feedback synaptic circuit was shown to be mediated predominantly by NMDA receptors. We further utilized caged Ca2+ compounds to obtain insight into the mechanism that couples NMDA receptor activation to GABA release. Feedback inhibition elicited by photo-release of caged Ca2+ in mitral cell secondary dendrites persisted when voltage-gated Ca2+ channels were blocked by cadmium (Cd2+) and nickel (Ni2+). These results indicate that Ca2+ influx through NMDA receptors can directly trigger presynaptic GABA release for local dendrodendritic feedback inhibition.     

Sparse incomplete representations: a potential role of olfactory granule cells

Mitral/tufted cells of the olfactory bulb receive odorant information from receptor neurons and transmit this information to the cortex. Studies in awake behaving animals have found that sustained responses of mitral cells to odorants are rare, suggesting sparse combinatorial representation of the odorants. Careful alignment of mitral cell firing with the phase of the respiration cycle revealed brief transient activity in the larger population of mitral cells, which respond to odorants during a small fraction of the respiration cycle. Responses of these cells are therefore temporally sparse. Here, we propose a mathematical model for the olfactory bulb network that can reproduce both combinatorially and temporally sparse mitral cell codes. We argue that sparse codes emerge as a result of the balance between mitral cells' excitatory inputs and inhibition provided by the granule cells. Our model suggests functional significance for the dendrodendritic synapses mediating interactions between mitral and granule cells.     

Inhibition in the olfactory bulb of the lizard

Inhibition in neurons of the lizard olfactory bulb was investigated by intracellular recording. The hyperpolarization arising in the neurons after the spike in the response to orthodromic and antidromic activation is similar in composition and reflects the development of early and late IPSPs, differing from one another in latency, duration, and mechanism of generation. The early IPSP is evidently generated by the functioning of dendrodendritic synapses, formed by dendrites of the interglomerular cell on the membrane of the apical dendrites of the secondary neurons, whereas synapses generating the late IPSP are located on the basal dendrites and are formed by endings of the granular cells. The mechanisms of generation of the early and late IPSPs in the secondary neurons are discussed. A classification of neurons of the lizard olfactory bulb is given on the basis of analysis of their intracellular activity.     

Regional distributions of γ-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and γ-aminobutyrate transaminase (GABA-T) in the central nervous brains of C57/BR,C3H/He,and F1 hybrid mice

The distributions of -aminobutyric acid (GABA), glutamate decarboxylase (GAD), and -aminobutyrate transaminase (GABA-T) have been studied in various brain areas of mice. These neurochemical markers, which are related to inhibitory neurotransmission, were investigated in different inbred strains of mice (C3H/He, C57/BR, and their F₁ hybrids). The regional distributions of GABA, GAD activity, and GABA-T activity in adult mice of these three strains were quite similar. No significant differences were found in any brain area for GAD or GABA-T activity. However, significant differences in GABA level were found in several brain areas among these strains of mice, especially in hypothalamus, hippcampus, olfactory bulb, and occipital cortex. These results provide further information to the possible influence of the GABAergic system in these brain areas.     

The Role of Inhibition in an Associative Memory Model of the Olfactory Bulb

The external plexiform layer is where theinteractions between the mitral (excitatory) and granule (inhibitory)cells of the olfactory bulb (OB) take place. Two outstanding features ofthese interactions are that they aredendrodendritic and that there seem to be nonebetween excitatory cells. The latter are usually credited with the role of forming Hebbian cell assemblies.Hence, it would seem that this structure lacks the necessaryingredients for an associative memory system.In this article we show that in spite of these two properties thissystem can serve as an associative memory. Our model incorporates theessential anatomical characteristics of the OB. The memories in oursystem, defined by Hebbian mitral assemblies, are activated viathe interactions with the inhibitory granule cells. The nonlinearityis introduced in our model via a sigmoid function that describesneurotransmitter release in reciprocal dendrodendritic synapses. Thecapacity (maximal number of odors that can be memorized) depends onthe sparseness of coding that is being used. For very low memoryactivities, the capacity grows as a fractional power of the number ofneurons. We validate the theoretical results by numericalsimulations. An interesting result of our model is that its capacityincreases as a function of the ratio of inhibitory to excitatorypopulations. This may provide an explanation for the dominance ofinhibitory cells in the olfactory bulb.