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The standard protocol for studying the spiking properties of single neurons is the application of current steps while monitoring the voltage response. Although this is informative, the jump in applied current is artificial. A more physiological input is where the applied current is ramped up, reflecting chemosensory input. Unsurprisingly, neurons can respond differently to the two protocols, since ion channel activation and inactivation are affected differently. Understanding the effects of current ramps, and changes in their slopes, is facilitated by mathematical models. However, techniques for analyzing current ramps are under-developed. In this article, we demonstrate how current ramps can be analyzed in single neuron models. The primary issue is the presence of gating variables that activate on slow time scales and are therefore far from equilibrium throughout the ramp. The use of an appropriate fast-slow analysis technique allows one to fully understand the neural response to ramps of different slopes. This study is motivated by data from olfactory bulb dopamine neurons, where both fast ramp (tens of milliseconds) and slow ramp (tens of seconds) protocols are used to understand the spiking profiles of the cells. The slow ramps generate experimental bifurcation diagrams with the applied current as a bifurcation parameter, thereby establishing asymptotic spiking activity patterns. The faster ramps elicit purely transient behavior that is of relevance to most physiological inputs, which are short in duration. The two protocols together provide a broader understanding of the neuron's spiking profile and the role that slowly activating ion channels can play., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In the central nervous system, dopamine is well-known as the neuromodulator that is involved with regulating reward, addiction, motivation, and fine motor control. Yet, decades of findings are revealing another crucial function of dopamine: modulating sensory systems. Dopamine is endogenous to subsets of neurons in the retina and olfactory bulb (OB), where it sharpens sensory processing of visual and olfactory information. For example, dopamine modulation allows the neural circuity in the retina to transition from processing dim light to daylight and the neural circuity in the OB to regulate odor discrimination and detection. Dopamine accomplishes these tasks through numerous, complex mechanisms in both neural structures. In this review, we provide an overview of the established and emerging research on these mechanisms and describe similarities and differences in dopamine expression and modulation of synaptic transmission in the retinas and OBs of various vertebrate organisms. This includes discussion of dopamine neurons' morphologies, potential identities, and biophysical properties along with their contributions to circadian rhythms and stimulus-driven synthesis, activation, and release of dopamine. As dysregulation of some of these mechanisms may occur in patients with Parkinson's disease, these symptoms are also discussed. The exploration and comparison of these two separate dopamine populations shows just how remarkably similar the retina and OB are, even though they are functionally distinct. It also shows that the modulatory properties of dopamine neurons are just as important to vision and olfaction as they are to motor coordination and neuropsychiatric/neurodegenerative conditions, thus, we hope this review encourages further research to elucidate these mechanisms., (Copyright © 2020 Korshunov, Blakemore and Trombley.)

The mammalian olfactory bulb (OB) has a vast population of dopamine (DA) neurons, whose function is to increase odor discrimination through mostly inhibitory synaptic mechanisms. However, it is not well understood whether there is more than one neuronal type of OB DA neuron, how these neurons respond to different stimuli, and the ionic mechanisms behind those responses. In this study, we used a transgenic rat line (hTH-GFP) to identify fluorescent OB DA neurons for recording via whole-cell electrophysiology. These neurons were grouped based on their localization in the glomerular layer ("Top" vs. "Bottom") with these largest and smallest neurons grouped by neuronal area ("Large" vs. "Small," in μm 2 ). We found that some membrane properties could be distinguished based on a neuron's area, but not by its glomerular localization. All OB DA neurons produced a single action potential when receiving a sufficiently depolarizing stimulus, while some could also spike multiple times when receiving weaker stimuli, an activity that was more likely in Large than Small neurons. This single spiking activity is likely driven by the Na + current, which showed a sensitivity to inactivation by depolarization and a relatively long time constant for the removal of inactivation. These recordings showed that Small neurons were more sensitive to inactivation of Na + current at membrane potentials of -70 and -60 mV than Large neurons. The hyperpolarization-activated H-current (identified by voltage sags) was more pronounced in Small than Large DA neurons across hyperpolarized membrane potentials. Lastly, to mimic a more physiological stimulus, these neurons received ramp stimuli of various durations and current amplitudes. When stimulated with weaker/shallow ramps, the neurons needed less current to begin and end firing and they produced more action potentials at a slower frequency. These spiking properties were further analyzed between the four groups of neurons, and these analyses support the difference in spiking induced with current step stimuli. Thus, there may be more than one type of OB DA neuron, and these neurons' activities may support a possible role of being high-pass filters in the OB by allowing the transmission of stronger odor signals while inhibiting weaker ones., (Copyright © 2020 Korshunov, Blakemore, Bertram and Trombley.)

Kainate receptors (KARs) are glutamate receptors with ionotropic and metabotropic activity composed of the GluK1-GluK5 subunits. We previously reported that KARs modulate excitatory and inhibitory transmission in the olfactory bulb (OB). Zinc, which is highly concentrated in the OB, also appears to modulate OB synaptic transmission via actions at other ionotropic glutamate receptors (i.e., AMPA, NMDA). However, few reports of effects of zinc on recombinant and/or native KARs exist and none have involved the OB. In the present study, we investigated the effects of exogenously applied zinc on OB KARs expressed by mitral/tufted (M/T) cells. We found that 100 µM zinc inhibits currents evoked by various combinations of KAR agonists (kainate or SYM 2081) and the AMPA receptor antagonist SYM 2206. The greatest degree of zinc-mediated inhibition was observed with coapplication of zinc with the GluK1- and GluK2-preferring agonist SYM 2081 plus SYM 2206. This finding is consistent with prior reports of zinc's inhibitory effects on some recombinant (homomeric GluK1 and GluK2 and heteromeric GluK2/GluK4 and GluK2/GluK5) KARs, although potentiation of other (GluK3, GluK2/3) KARs has also been described. It is also of potential importance given our previously reported molecular data suggesting that OB neurons express relatively high levels of GluK1 and GluK2. Our present findings suggest that a physiologically relevant concentration of zinc modulates KARs expressed by M/T cells. As M/T cells are targets of zinc-containing olfactory sensory neurons, synaptically released zinc may influence odor information-encoding synaptic circuits in the OB via actions at KARs., (Published by Elsevier Ltd.)

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors mediates most fast excitatory transmission. Glutamate binding to AMPA receptors (AMPARs) causes most AMPARs to rapidly and completely desensitize, and their desensitization kinetics influence synaptic timing. Thus, factors that alter AMPAR desensitization influence synaptic transmission. Synaptically released zinc is such a factor. Zinc is a neuromodulator with effects on amino acid receptors and synaptic transmission in many brain regions, including the olfactory bulb (OB). We have previously shown in the OB that zinc potentiates AMPAR-mediated currents at low concentrations (30 μM, 100 μM) and inhibits them at a higher concentration (1 mM). It has been hypothesized that zinc potentiates AMPARs by decreasing receptor desensitization. Here, we used cyclothiazide (CTZ), a drug that blocks AMPAR desensitization, to determine whether zinc-mediated potentiation and/or inhibition of AMPA-evoked currents reflect(s) changes in AMPAR desensitization. Zinc largely had biphasic concentration-dependent effects at OB AMPARs. CTZ completely blocked potentiation by zinc but had no significant effect on inhibition. There was a significant negative correlation between the degree of potentiation of AMPAR-mediated currents by 100 μM zinc and a quantitative measure of the degree of AMPAR desensitization (the steady-state to peak [S:P] ratio of AMPA-evoked currents), but no correlation between the degree of current inhibition by 1 mM zinc and the S:P ratio. Together, these findings suggest that low zinc concentrations potentiate rat OB AMPARs by decreasing receptor desensitization, but that the inhibitory effects of higher zinc concentrations are mediated by a separate mechanism., (Published by Elsevier Ltd.)

Glutamate is the neurotransmitter used at most excitatory synapses in the mammalian brain, including those in the olfactory bulb (OB). There, ionotropic glutamate receptors including N-methyl-d-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) play a role in processes such as reciprocal inhibition and glomerular synchronization. Kainate receptors (KARs) represent another type of ionotropic glutamate receptor, which are composed of five (GluK1-GluK5) subunits. Whereas KARs appear to be heterogeneously expressed in the OB, evidence as to whether these KARs are functional, found at synapses, or modify synaptic transmission is limited. In the present study, coapplication of KAR agonists (kainate, SYM 2081) and AMPAR antagonists (GYKI 52466, SYM 2206) demonstrated that functional KARs are expressed by OB neurons, with a subset of receptors located at synapses. Application of kainate and the GluK1-selective agonist ATPA had modulatory effects on excitatory postsynaptic currents (EPSCs) evoked by stimulation of the olfactory nerve layer. Application of kainate and ATPA also had modulatory effects on reciprocal inhibitory postsynaptic currents (IPSCs) evoked using a protocol that evokes dendrodendritic inhibition. The latter finding suggests that KARs, with relatively slow kinetics, may play a role in circuits in which the relatively brief duration of AMPAR-mediated currents limits the role of AMPARs in synaptic transmission (e.g., reciprocal inhibition at dendrodendritic synapses). Collectively, our findings suggest that KARs, including those containing the GluK1 subunit, modulate excitatory and inhibitory transmission in the OB. These data further suggest that KARs participate in the regulation of synaptic circuits that encode odor information., (Published by Elsevier Ltd.)

The olfactory bulb (OB) is central to the sense of smell, as it is the site of the first synaptic relay involved in the processing of odor information. Odor sensations are first transduced by olfactory sensory neurons (OSNs) before being transmitted, by way of the OB, to higher olfactory centers that mediate olfactory discrimination and perception. Zinc is a common trace element, and it is highly concentrated in the synaptic vesicles of subsets of glutamatergic neurons in some brain regions including the hippocampus and OB. In addition, zinc is contained in the synaptic vesicles of some glycinergic and GABAergic neurons. Thus, zinc released from synaptic vesicles is available to modulate synaptic transmission mediated by excitatory (e.g., N-methyl-D aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)) and inhibitory (e.g., gamma-aminobutyric acid (GABA), glycine) amino acid receptors. Furthermore, extracellular zinc can alter the excitability of neurons through effects on a variety of voltage-gated ion channels. Consistent with the notion that zinc acts as a regulator of neuronal activity, we and others have shown zinc modulation (inhibition and/or potentiation) of amino acid receptors and voltage-gated ion channels expressed by OB neurons. This review summarizes the locations and release of vesicular zinc in the central nervous system (CNS), including in the OB. It also summarizes the effects of zinc on various amino acid receptors and ion channels involved in regulating synaptic transmission and neuronal excitability, with a special emphasis on the actions of zinc as a neuromodulator in the OB. An understanding of how neuroactive substances such as zinc modulate receptors and ion channels expressed by OB neurons will increase our understanding of the roles that synaptic circuits in the OB play in odor information processing and transmission.

Circadian rhythms are daily rhythms that regulate many biological processes - from gene transcription to behavior - and a disruption of these rhythms can lead to a myriad of health risks. Circadian rhythms are entrained by light, and their 24-h oscillation is maintained by a core molecular feedback loop composed of canonical circadian ("clock") genes and proteins. Different modulators help to maintain the proper rhythmicity of these genes and proteins, and one emerging modulator is dopamine. Dopamine has been shown to have circadian-like activities in the retina, olfactory bulb, striatum, midbrain, and hypothalamus, where it regulates, and is regulated by, clock genes in some of these areas. Thus, it is likely that dopamine is essential to mechanisms that maintain proper rhythmicity of these five brain areas. This review discusses studies that showcase different dopaminergic mechanisms that may be involved with the regulation of these brain areas' circadian rhythms. Mechanisms include how dopamine and dopamine receptor activity directly and indirectly influence clock genes and proteins, how dopamine's interactions with gap junctions influence daily neuronal excitability, and how dopamine's release and effects are gated by low- and high-pass filters. Because the dopamine neurons described in this review also release the inhibitory neurotransmitter GABA which influences clock protein expression in the retina, we discuss articles that explore how GABA may contribute to the actions of dopamine neurons on circadian rhythms. Finally, to understand how the loss of function of dopamine neurons could influence circadian rhythms, we review studies linking the neurodegenerative disease Parkinson's Disease to disruptions of circadian rhythms in these five brain areas. The purpose of this review is to summarize growing evidence that dopamine is involved in regulating circadian rhythms, either directly or indirectly, in the brain areas discussed here. An appreciation of the growing evidence of dopamine's influence on circadian rhythms may lead to new treatments including pharmacological agents directed at alleviating the various symptoms of circadian rhythm disruption.

Melatonin is a neurohormone associated with circadian rhythms. A diurnal rhythm in olfactory sensitivity has been previously reported and melatonin receptor mRNAs have been observed in the olfactory bulb, but the effects of melatonin in the olfactory bulb have not been explored. First, we corroborated data from a previous study that identified melatonin receptor messenger RNAs in the olfactory bulb. We then investigated whether melatonin treatment would affect cells in the olfactory bulbs of rats. Using a combination of polymerase chain reaction (PCR), quantitative PCR (qPCR), cell culture, and electrophysiology, we discovered that melatonin receptors and melatonin synthesis enzymes were present in the olfactory bulb and we observed changes in connexin43 protein, GluR1 mRNA, GluR2 mRNA, Per1 mRNA, Cry2 mRNA, and K(+) currents in response to 2-iodomelatonin. Via qPCR, we observed that messenger RNAs encoding melatonin receptors and melatonin biosynthesis enzymes fluctuated in the olfactory bulb across 24h. Together, these data show that melatonin receptors are present in the olfactory bulb and likely affect olfactory function. Additionally, these data suggest that melatonin may be locally synthesized in the olfactory bulb., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The olfactory bulb (OB) has been recently identified as a circadian oscillator capable of operating independently of the master circadian pacemaker, the suprachiasmatic nuclei of the hypothalamus. OB oscillations manifest as rhythms in clock genes, electrical activity, and odor sensitivity. Dopamine, norepinephrine, and serotonin have been shown to modulate olfactory information processing by the OB and may be part of the mechanism that underlies diurnal changes in olfactory sensitivity. Rhythmic release of these neurotransmitters could generate OB rhythms in electrical activity and olfactory sensitivity. We hypothesized that these monoamines were rhythmically released in the OB. To test our hypotheses, we examined monoamine levels in the OB, over the course of a day, by high-performance liquid chromatography coupled to electrochemical detection. We observed that dopamine and its metabolite, 3-4-dihydroxyphenylacetic acid, rhythmically fluctuate over the day. In contrast, norepinephrine is arrhythmic. Serotonin and its metabolite hydroxyindoleacetic acid appear to rhythmically fluctuate. Each of these monoamines has been shown to alter OB circuit behavior and influence odor processing. Rhythmic release of serotonin may be a mechanism by which the suprachiasmatic nuclei communicate, indirectly, with the OB., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Zinc is a trace element with a multitude of roles in biological systems including structural and cofactor functions for proteins. Although most zinc in the central nervous system (CNS) is protein bound, the CNS contains a pool of mobile zinc housed in synaptic vesicles within a subset of neurons. Such mobile zinc occurs in many brain regions, such as the hippocampus, hypothalamus, and cortex, but the olfactory bulb (OB) contains one of the highest such concentrations in the CNS. Zinc is distributed throughout the OB, with the glomerular and granule cell layers containing the highest levels. Here, we visualize vesicular zinc in the OB using zinc-responsive fluorescent probes developed by one of us. Moreover, we provide the first demonstration that vesicular pools of zinc can be released from olfactory nerve terminals within individual glomeruli by patterned electrical stimulation of the olfactory nerve designed to mimic the breathing cycle in rats. We also provide electrophysiological evidence that elevated extracellular zinc potentiates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic events. AMPA receptors are required for the synchronous activation of neurons within individual OB glomeruli, and zinc-mediated potentiation leads to enhanced synaptic summation.

Circadian rhythms affect olfaction by an unknown molecular mechanism. Independent of the suprachiasmatic nuclei, the mammalian olfactory bulb (OB) has recently been identified as a circadian oscillator. The electrical activity in the OB was reported to be synchronized to a daily rhythm and the clock gene, Period1, was oscillatory in its expression pattern. Because gap junctions composed of connexin36 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) have been reported to work together to synchronize firing of action potentials in the OB, we hypothesized that circadian electrical oscillations could be synchronized by daily changes in the expression of connexins and AMPAR subunits (GluR1-4). We examined the OB for the presence of clock genes by polymerase chain reaction (PCR) and whether Period2, connexins, and AMPARs fluctuated across the light/dark cycle by quantitative PCR or SDS-PAGE/Western blot analysis. We observed significant changes in the messenger RNA and protein expression of our targets across 24 or 48 h. Whereas most targets were rhythmic by some measures, only GluR1 mRNA and protein were both rhythmic by the majority of our tests of rhythmicity across all time scales. Differential expression of these synaptic proteins over the light/dark cycle may underlie circadian synchronization of action potential firing in the OB or modify synaptic interactions that would be predicted to impact olfactory coding, such as alteration of granule cell inhibition, increased number of available AMPARs to bind glutamate, or an increased gap junction conductance between mitral/tufted cells., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Glycine receptors are widely expressed in the mammalian central nervous system, and previous studies have demonstrated that glycine receptors are modulated by endogenous zinc. Zinc is concentrated in synaptic vesicles in several brain regions but is particularly abundant in the hippocampus and olfactory bulb. In the present study, we used patch-clamp electrophysiology of rat hippocampal and olfactory bulb neurons in primary culture to examine the effects of zinc on glycine receptors. Although glycine has been reported to reach millimolar concentrations during synaptic transmission, most previous studies on the effects of zinc on glycine receptors have used relatively low concentrations of glycine. High concentrations of glycine cause receptor desensitization. Our current results extend our previous demonstration that the modulatory actions of zinc are largely prevented when co-applied with desensitizing concentrations of glycine (300 μM), suggesting that the effects of zinc are dependent on the state of the receptor. In contrast, pre-application of 300 μM zinc, prior to glycine (300 μM) application, causes a slowly developing inhibition with a slow rate of recovery, suggesting that the timing of zinc and glycine release also influences the effects of zinc. Furthermore, previous evidence suggests that synaptically released zinc can gain intracellular access, and we provide the first demonstration that low concentrations of intracellular zinc can potentiate glycine receptors. These results support the notion that zinc has complex effects on glycine receptors and multiple factors may interact to influence the efficacy of glycinergic transmission., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Although recent results suggest roles for NMDA and AMPA receptors in odor encoding, little is known about kainate receptors (KARs) in the olfactory bulb (OB). Molecular, immunological, and electrophysiological techniques were used to provide a functional analysis of KARs in the OB. Reverse transcriptase-polymerase chain reaction revealed that the relative level of expression of KAR subunits was GluR5 approximately GluR6 approximately KA2 > KA1 >> GluR7. In situ hybridization data imply that mitral/tufted cells express mostly GluR5 and KA2, whereas interneurons express mostly GluR6 and KA2. Immunohistochemical double-labeling experiments (GluR5/6/7 or GluR5 + synapsin) suggest that KARs are expressed at both synaptic and extrasynaptic loci. This heterogeneous expression of KAR subunits suggests that KARs may play a multitude of roles in odor processing, each tailored to the function of specific OB circuits. A functional analysis, using whole-cell electrophysiology, suggests that one such role is to increase the frequency of glutamate transmission while attenuating the amplitude of individual events, likely via a presynaptic depolarizing mechanism. Such effects would be important to odor processing particularly by OB glomeruli. In these highly compartmentalized structures, an increase in the frequency of glutamate release and the high density of extrasynaptic KARs, activated by spillover, could enhance glomerular synchronization and thus the transfer of more specific sensory information to cortical structures.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), a subtype of glutamate receptor, contribute to olfactory processing in the olfactory bulb (OB). These ion channels consist of various combinations of the subunits GluR1-GluR4, which bestow certain properties. For example, AMPARs that lack GluR2 are highly permeable to Ca(2+) and generate inwardly rectifying currents. Because increased intracellular Ca(2+) could trigger a host of Ca(2+)-dependent odor-encoding processes, we used whole cell recording as well as histological and immunocytochemical (ICC) techniques to investigate whether AMPARs on rat OB neurons flux Ca(2+). Application of 1-naphthylacetyl spermine (NAS), a selective antagonist of Ca(2+)-permeable AMPARs (CP-AMPARs), inhibited AMPAR-mediated currents in subsets of interneurons and principal cells in cultures and slices. The addition of spermine to the electrode yielded inwardly rectifying current-voltage plots in some cells. In OB slices, olfactory nerve stimulation elicited excitatory responses in juxtaglomerular and mitral cells. Bath application of NAS with d,l-2-amino-5-phosphonovaleric acid (AP5) to isolate AMPARs suppressed the amplitudes of these synaptic responses compared with responses obtained using AP5 alone. Co(2+) staining, which involves the kainate-stimulated influx of Co(2+) through CP-AMPARs, produced diverse patterns of labeling in cultures and slices as did ICC techniques used with a GluR2-selective antibody. These results suggest that subsets of OB neurons express CP-AMPARs, including functional CP-AMPARs at synapses. Ca(2+) entry into cells via these receptors could influence odor encoding by modulating K(+) channels, N-methyl-d-aspartate receptors, and Ca(2+)-binding proteins, or it could facilitate synaptic vesicle fusion.

Although the olfactory bulb contains one of the highest concentrations of neuropeptide Y in the CNS, its function in the bulb remains unclear. In this study, we used whole-cell electrophysiological, molecular, and primary culture techniques to investigate neuropeptide Y gene expression and neuromodulatory actions of neuropeptide Y on rat olfactory bulb neurons. Northern analysis showed that neuropeptide Y mRNA increases with animal age or time in culture, in a parallel manner. In electrophysiology experiments, agonists that activate neuropeptide Y receptors (whole neuropeptide Y) and the Y2 receptor subtype (neuropeptide Y 13-36) reduced spontaneous excitatory activity in bulb interneurons. In investigating potential presynaptic effects, both agonists reduced the amplitude of calcium channel currents in the presynaptic (mitral/tufted) cell. Also consistent with a presynaptic effect, both agonists reduced the frequency but not the amplitude of miniature excitatory postsynaptic currents (or "minis") in interneurons. In examining potential postsynaptic effects, both agonists slightly increased membrane resistance but had no effect on currents evoked by glutamate. Together, these data suggest that neuropeptide Y inhibits excitatory neurotransmission between olfactory bulb neurons via a presynaptic effect on transmitter (glutamate) release.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs) mediate rapid responses at most central excitatory synapses, including those in the olfactory bulb (OB). These receptors are composed of the glutamate subunits GluR1-4, which each has two splice variant (flip/flop) forms. We recently showed that AMPARs on OB neurons are kinetically and pharmacologically diverse. Here, we explored whether this functional heterogeneity reflects a diverse expression of AMPAR subunits and/or splice variants. Total RNA from rat OBs was amplified by RT-PCR. Digestion of the panGluR PCR product with subunit-specific restriction enzymes revealed that the OB expresses mRNAs for GluR1-4 but in different relative amounts i.e., GluR2 (61 +/- 2.4%), GluR1 (31 +/- 3.5%), GluR4 (6.3 +/- 1.4%), GluR3 (1.4 +/- 0.7%). Furthermore, GluR2 and GluR4 transcripts were composed of similar amounts of flip and flop, whereas GluR1 and GluR3 transcripts consisted mostly of flip. If similar to other brain regions, this heterogeneity in patterns of expression may facilitate information processing.

Increasing evidence suggests that zinc modulates synaptic transmission in the olfactory bulb and other brain regions. We investigated the sensitivity of AMPA receptors on the bulb's two primary neuronal populations to several concentrations of zinc. Zinc (30-1000 microM) was coapplied to mitral/tufted cells and interneurons during AMPA-evoked currents, and current responses (potentiation, inhibition, no effect) were analyzed. Both neuronal populations expressed zinc-sensitive and zinc-insensitive AMPA receptors. However, the frequency and magnitude of zinc's effects varied with cell type. In addition, zinc did not always have biphasic effects at AMPA receptors (potentiation at low concentrations; inhibition at high concentrations), as reported in other brain regions. Zinc's diverse effects suggest that zinc may alter odor information processing by differential modulation of excitatory circuits.

The glomerular layer of the olfactory bulb (OB) contains synaptic connections between olfactory sensory neurons and OB neurons as well as connections among OB neurons. A subpopulation of external tufted cells and periglomerular cells (juxtaglomerular neurons) expresses dopamine, and recent reports suggest that dopamine can inhibit olfactory sensory neuron activation of OB neurons. In this study, whole cell electrophysiological and primary culture techniques were employed to characterize the neuromodulatory properties of dopamine on glutamatergic transmission between rat OB mitral/tufted (M/T) cells and interneurons. Immunocytochemical analysis confirmed the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in a subpopulation of cultured neurons. D2 receptor immunoreactivity was also observed in cultured M/T cells. Dopamine reduced spontaneous excitatory synaptic events recorded in interneurons. Although the D1 receptor agonist SKF38393 and the D2 receptor agonist bromocriptine mesylate mimicked this effect, evoked excitatory postsynaptic potentials (EPSPs) recorded from monosynaptically coupled neuron pairs were attenuated by dopamine and bromocriptine but not by SKF38393. Neither glutamate-evoked currents nor the membrane resistance of the postsynaptic interneuron were affected by dopamine. However, evoked calcium channel currents in the presynaptic M/T cell were diminished during the application of either dopamine or bromocriptine, but not SKF38393. Dopamine suppressed calcium channel currents even after nifedipine blockade of L-type channels, suggesting that inhibition of the dihydropyridine-resistant high-voltage activated calcium channels implicated in transmitter release may mediate dopamine's effects on spontaneous and evoked synaptic transmission. Together, these data suggest that dopamine inhibits excitatory neurotransmission between M/T cells and interneurons via a presynaptic mechanism.

AMPA receptors, which are composed of four subunits (GluR1-4), help mediate synaptic transmission at most excitatory synapses in the brain. Their subunit composition influences the kinetics of AMPA receptor deactivation and desensitization, thus, the efficacy of synaptic transmission. Immunohistochemical data suggest that AMPA receptor subunit expression in the olfactory bulb (OB) follows a distinct laminar and cellular distribution. However, little is known about the kinetic properties of AMPA receptors on OB neurons. In this study, we used kinetic analysis and pharmacologic methods to demonstrate that the rate and extent of desensitization of OB AMPA receptors vary within and between neuronal subtypes. Such variability suggests that a broad functional diversity of AMPA receptors contributes to olfactory information processing.

Zinc and copper are highly concentrated in several mammalian brain regions, including the olfactory bulb and hippocampus. Whole cell electrophysiological recordings were made from rat olfactory bulb neurons in primary culture to compare the effects of zinc and copper on synaptic transmission and voltage-gated ion channels. Application of either zinc or copper eliminated GABA-mediated spontaneous inhibitory postsynaptic potentials. However, in contrast to the similarity of their effects on inhibitory transmission, spontaneous glutamate-mediated excitatory synaptic activity was completely blocked by copper but only inhibited by zinc. Among voltage-gated ion channels, zinc or copper inhibited TTX-sensitive sodium channels and delayed rectifier-type potassium channels but did not prevent the firing of evoked single action potentials or dramatically alter their kinetics. Zinc and copper had distinct effects on transient A-type potassium currents. Whereas copper only inhibited the A-type current, zinc modulation of A-type currents resulted in either potentiation or inhibition of the current depending on the membrane potential. The effects of zinc and copper on potassium channels likely underlie their effects on repetitive firing in response to long-duration step depolarizations. Copper reduced repetitive firing independent of the initial membrane voltage. In contrast, whereas zinc reduced repetitive firing at membrane potentials associated with zinc-mediated enhancement of the A-type current (-50 mV), in a significant proportion of neurons, zinc increased repetitive firing at membrane potentials associated with zinc-mediated inhibition of the A-type current (-90 mV). Application of zinc or copper also inhibited voltage-gated Ca(2+) channels, suggesting a possible role for presynaptic modulation of neurotransmitter release. Despite similarities between the effects of zinc and copper on some ligand- and voltage-gated ion channels, these data suggest that their net effects likely contribute to differential modulation of neuronal excitability.

This review examines interactions in the mammalian central nervous system (CNS) between carnosine and the endogenous transition metals zinc and copper. Although the relationship between these substances may be applicable to other brain regions, the focus is on the olfactory system where these substances may have special significance. Carnosine is not only highly concentrated in the olfactory system, but it is also contained in neurons (in contrast to glia cells in most of the brain) and has many features of a neurotransmitter. Whereas the function of carnosine in the CNS is not well understood, we review evidence that suggests that it may act as both a neuromodulator and a neuroprotective agent. Although zinc and/or copper are found in many neuronal pathways in the brain, the concentrations of zinc and copper in the olfactory bulb (the target of afferent input from sensory neurons in the nose) are among the highest in the CNS. Included in the multitude of physiological roles that zinc and copper play in the CNS is modulation of neuronal excitability. However, zinc and copper also have been implicated in a variety of neurologic conditions including Alzheimer's disease, Parkinson's disease, stroke, and seizures. Here we review the modulatory effects that carnosine can have on zinc and copper's abilities to influence neuronal excitability and to exert neurotoxic effects in the olfactory system. Other aspects of carnosine in the CNS are reviewed elsewhere in this issue.

Dopamine can change the membrane potential, regulate cyclic nucleotides, and modulate transmitter release in central neurons. In the olfactory bulb (OB), the dopamine synthetic enzyme, tyrosine hydroxylase, is largely confined to neurons in the glomerular layer. After demonstrating dopamine D2 receptors in the glomerular and olfactory nerve (ON) layers, Nickell et al. [W.T. Nickell, A.B. Norman, L.M. Wyatt, M.T. Shipley, Olfactory bulb DA receptors may be located on terminals of the olfactory nerve, NeuroReport, 2 (1991) 9-12.] proposed that these receptors may reduce transmitter release due to their localization to ON presynaptic boutons. We have previously demonstrated that olfactory receptor neurons use glutamate to excite OB neurons through activation of glutamate receptors subtypes, NMDA and AMPA/kainate [D.A. Berkowicz, P.Q. Trombley, G.M. Shepherd, Evidence for glutamate as the olfactory receptor cell neurotransmitter. J. Neurophysiol., 71 (1994) 2557-2561]. Here, we used a hemisected turtle OB preparation and patch-clamp recording techniques to assess dopamine modulation of the ON/OB neuron synapse. We found that dopamine (10-300 microM) reversibly decreased the excitatory postsynaptic response to ON stimulation. This effect could be overcome by recruiting additional nerve fibers by increasing the intensity of ON stimulation. Quinpirole (10 microM), a D2 agonist, mimicked the effects of dopamine. Conversely, sulpiride (300 microM), a D2 antagonist, prevented the inhibitory effects of dopamine on synaptic transmission. Whereas dopamine appeared to equally affect the NMDA and AMPA/kainate receptor-mediated components of the synaptically evoked response, it had no direct effect on membrane currents evoked by exogenous glutamate, kainate or NMDA applied to cultured OB neurons. Our data, therefore, support the notion that dopamine modulates synaptic transmission between olfactory receptor neurons and OB neurons via a presynaptic mechanism involving D2 receptor activation. Our abstract (Berkowicz et al. (1994) Neuroscience Abs. 20:328) is the first report of these results.

Zinc and copper are endogenous transition metals that can be synaptically released during neuronal activity. Synaptically released zinc and copper probably function to modulate neuronal excitability under normal conditions. However, zinc and copper also can be neurotoxic, and it has been proposed that they may contribute to the neuropathology associated with a variety of conditions, such as Alzheimer's disease, stroke, and seizures. Recently, we demonstrated that carnosine, a dipeptide expressed in glial cells throughout the brain as well as in neuronal pathways of the visual and olfactory systems, can modulate the effects of zinc and copper on neuronal excitability. This result led us to hypothesize that carnosine may modulate the neurotoxic effects of zinc and copper as well. Our results demonstrate that carnosine can rescue neurons from zinc- and copper-mediated neurotoxicity and suggest that one function of carnosine may be as an endogenous neuroprotective agent.

Whole cell voltage-clamp electrophysiology was used to examine interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons in primary culture. Membrane currents evoked by GABA and glycine were selectively inhibited by low concentrations of bicuculline and strychnine, respectively, suggesting that they activate pharmacologically distinct receptors. However, GABA- and glycine-mediated currents showed cross-inhibition when the two amino acids were applied sequentially. Application of one amino acid inhibited the response to immediate subsequent application of the other. In the majority of neurons, GABA inhibited subsequent glycine-evoked currents and glycine inhibited subsequent GABA-evoked currents. In a small proportion of neurons, however, GABA inhibited glycine-evoked currents but glycine had little effect on GABA-evoked currents. The reverse was true in other neurons, suggesting that alterations in chloride gradients alone did not account for the cross-inhibition. Furthermore, no cross-inhibition was observed between GABA- and glycine-evoked currents in some neurons. The amplitude of the current evoked by the coapplication of saturating concentrations of GABA and glycine in these neurons was nearly the sum of the currents evoked by GABA and glycine alone. In contrast, the currents were not additive in neurons demonstrating cross-inhibition. These results suggest that olfactory bulb neurons heterogeneously express a population of inhibitory amino acid receptors that can bind either GABA or glycine. Interactions between GABA and glycine at inhibitory amino acid receptors may provide a mechanism to modulate inhibitory synaptic transmission.

Neuropeptide Y is highly abundant in both the peripheral and central nervous systems and is known to have diverse functions including regulation of feeding behavior, blood pressure, circadian rhythms, reproductive behavior and the response to stress. Northern analysis showed that copper deficiency increased brain NPY mRNA abundance particularly in the olfactory bulb (OB). These increases were not accompanied by alterations in food intake or blood pressure. After 4 weeks of a copper-restricted diet, OB copper concentrations decreased to 44% of control and NPY mRNA increased 1.5-fold. Addition of a copper chelator to the restricted diet, resulted in a two-fold increase in OB NPY mRNA over copper adequate controls. These results were confirmed in primary cultures of OB neurons suggesting that the regulation of NPY mRNA is at the level of the bulb rather than by a hormonal or other copper-regulated factor external to the OB. Immunoreactive NPY (IR-NPY) levels were not, however, increased following the 4 weeks of copper deficiency. Addition of the chelator resulted in a 1.4-fold increase in IR-NPY that, while statistically significant, was not proportional to the two-fold increase in NPY mRNA in the same study. This may suggest that copper deficiency inhibits the translational mechanisms responsible for the synthesis of NPY or that NPY is exported from the bulb in copper deficiency., (Copyright 1999 Elsevier Science B.V.)

Carnosine is a dipeptide which is highly concentrated in mammalian olfactory sensory neurons along with zinc and/or copper, and glutamate. Although carnosine has been proposed as a neurotransmitter or neuromodulator, no specific function for carnosine has been identified. We used whole-cell current- and voltage-clamp recording to examine the direct effects and neuromodulatory actions of carnosine on rat olfactory bulb neurons in primary culture. Carnosine did not evoke a membrane current or affect currents evoked by glutamate, GABA or glycine. Copper and zinc inhibited NMDA and GABA receptor-mediated currents and inhibited synaptic transmission. Carnosine prevented the actions of copper and reduced the effects of zinc. These results suggest that carnosine may indirectly influence neuronal excitability by modulating the effects of zinc and copper.

Aluminum has been implicated in several neurodegenerative conditions including Alzheimer's disease. Because the mammalian olfactory system has an unusual capacity for the uptake and transneuronal spread of inhaled substances such as aluminum, whole cell recording techniques were used to examine the actions of aluminum on basic membrane properties and amino acid receptors on rat olfactory bulb mitral/tufted (M/T) neurons in culture. Aluminum had little direct effects on M/T neurons. Aluminum (100 microM) did not evoke a membrane current or alter action-potential shape or duration. Aluminum also had no marked effects on the family of voltage-gated membrane currents evoked by a series of 10-mV, 50-ms depolarizing steps. However, aluminum dramatically potentiated the current evoked by 30 microM gamma-aminobutyric acid (GABA) at concentrations <100 microM. Conversely, higher concentrations of aluminum blocked the GABA-evoked current. The effects of aluminum on GABA-evoked currents were not voltage dependent. Aluminum (100 microM) equally potentiated both inward currents at -30 mV and outward currents at + 30 mV. At 300 microM, aluminum blocked both inward and outward currents to a similar extent. In some neurons, aluminum only blocked the current and potentiation was not observed. The biphasic action of aluminum on GABA-evoked currents suggests separate binding sites: a high-affinity potentiating site and a low-affinity inhibiting site. Despite its effects on GABA-evoked currents, aluminum did not alter membrane currents evoked by glutamate, N-methyl-D-aspartate, kainate, or glycine. Aluminum also did not reduce spontaneous excitatory synaptic activity, suggesting little, if any, effect on glutamate release. Although a causal role for aluminum in Alzheimer's disease and other neuropathological conditions remains controversial, it is clear that elevated aluminum concentrations in the brain are associated with a variety of cognitive impairments. The present results indicate that aluminum can alter the function of GABAA receptors and may suggest that aluminum can contribute to cognitive impairment through disruption of inhibitory circuits.

1. The olfactory bulb contains high concentrations of zinc and copper. Whole cell recording techniques were used to examine the modulatory actions of zinc and copper on N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA), and glycine receptors on rat olfactory bulb neurons in culture and acutely isolated from adult animals. 2. Zinc and copper were effective antagonists of both NMDA-and GABA-mediated currents. The median inhibiting concentrations (IC50s) for zinc were 19 microM for NMDA receptors and 17 microM for GABA receptors. The IC50s for copper were 22 microM for NMDA receptors and 18 microM for GABA receptors. 3. Zinc and copper (100 microM) had no effect on the steady-state, desensitized component of currents evoked by high concentrations of glycine (300 microM). In contrast, when low, nondesensitizing concentrations of glycine (30 microM) were used, 100 microM zinc dramatically potentiated the current and 100 microM copper blocked the current. 4. The effects of zinc and copper on NMDA-, GABA-, or glycine-mediated currents were not voltage dependent, irrespective of whether the effect was potentiation or inhibition. 5. These results provide the first evidence for an inhibitory effect of copper on NMDA receptors, and the first evidence that the effects of zinc and copper on glycine receptors are dependent on the state of the receptor. These results suggest that endogenous zinc and copper may act as allosteric neuromodulators of amino acid receptors on olfactory bulb neurons. Furthermore, zinc and copper may provide a mechanism for differential modulation of inhibitory transmission because of their distinct effects on glycine versus GABA receptors.

1. Gramicidin-perforated patch clamp recording was employed to study GABA-mediated responses in rat hypothalamic neurones (n = 102) with an intracellular Cl- concentration unaltered by the pipette solution. 2. In young cultures after 1-7 days in vitro (DIV), GABA induced depolarizing membrane potentials (+16.5 +/- 1.3 mV) that often surpassed the threshold for the firing of action potentials (-42 +/- 1 mV) and resulted in an increase in neuronal activity. The depolarizing responses to GABA in young cultures were dose dependent. The concentration of GABA necessary to evoke the half-maximal depolarization (EC50) was 2.8 microM. In contrast, GABA induced hyperpolarizing membrane potentials (-12.0 +/- 1.4 mV) and a decrease in neuronal activity in older neurones (20-33 DIV). Both the depolarization and the hyperpolarization induced by GABA were blocked by bicuculline, indicating a mediation by GABAA receptors. 3. The reversal potentials of the GABA-evoked currents were between -40 to -50 mV during the first week of culture, and shifted to below -70 mV after 3 weeks of culture. In parallel, neurones that were dissociated from older animals (postnatal day 5) had a more negative reversal potential for the GABA-evoked currents than cells from younger animals (embryonic day 15), suggesting that the negative shift of the reversal potential occurs both in vitro and in vivo. Our data suggest that the mechanism for GABA-induced depolarization is the depolarized Cl- reversal potential found in young but not older neurones. 4. Consistent with the depolarizing response to exogenous application of GABA, some spontaneous depolarizing postsynaptic potentials in young cultures were insensitive to AP5-CNQX, but were eliminated by bicuculline, indicating that synaptically released GABA mediated excitatory synaptic transmission in early development. 5. By combining a rapid computer-controlled delivery of GABA with subthreshold positive current injections into recorded neurones, we found in young cultures that the GABA-evoked depolarization could directly trigger action potentials, facilitate some depolarizing input to fire action potentials, and shunt other depolarizing input. Whether the GABA-induced depolarization is excitatory or inhibitory would be determined by the reversal potential of the GABA-evoked current, and the temporal relationship between GABA-evoked depolarizations and other excitatory events. 6. We conclude that the reversal potential of the GABA-evoked current shifts negatively from depolarizing to hyperpolarizing in developing hypothalamus. Consequently, GABA neurotransmission may serve both excitatory and inhibitory roles during early development.

1. The developmental changes in gamma-aminobutyrate (GABA)-, glutamate-, and glycine-mediated currents in cultured embryonic neurons (n = 134) from rat hypothalamus were studied with the use of whole cell voltage-clamp recording. 2. GABA-evoked currents were detected in neurons cultured from 15-day embryos (E15) a few hours after plating. Every neuron studied from the time of plating at E15 to 2 wk later responded to GABA (30 microM). The peak and steady-state currents evoked by GABA increased by four- to fivefold within 2 wk in culture. The time constants of the desensitization of GABA currents did not change during this period. The properties of the responses to GABA were not altered by different culture densities or substrates. 3. Glycine activated receptors that were pharmacologically distinct from GABA receptors on hypothalamic neurons. The glycine responses increased by > 50-fold within 2 wk in culture. The percentage of cells responding to glycine (500 microM) was 20% at 0 days in vitro (DIV), and increased to 100% at 6 DIV. Astrocytes increased both the amplitude of glycine-mediated currents and the percentage of cells responding to glycine. 4. Glutamate-mediated currents developed later than GABA-mediated currents. The percentage of cells responding to glutamate (500 microM) increased within the 1st wk, from 20% on the day of plating to 100% after 6 DIV. Both the peak currents and the steady-state currents mediated by glutamate increased by 20-fold during the 2 wk in culture. Both the amplitude of the responses to glutamate and the percentage of cells responding to glutamate were increased by growing neurons either on an astrocyte substrate or in high-density cultures. 5. The currents and conductance changes elicited by GABA were greater than those generated by glutamate or glycine throughout the period examined. This difference was particularly evident in younger cells. After 3 days in vitro, GABA (30 microM) elicited a mean current of 1,648 pA, whereas glutamate (500 microM) only elicited a 266-pA current, and glycine (500 microM) elicited a 278-pA current from neurons growing on an astrocyte layer. 6. The expression of amino acid receptors was heterogeneous among hypothalamic neurons in younger cultures. Whereas all neurons expressed GABA receptors, some developing neurons did not express detectable glutamate receptors or glycine receptors. 7. Each of the three amino acid-evoked currents increased from E15 (1 DIV) to E20 (1 DIV), indicating an intrinsic development in the expression of the amino acid receptors in vivo. The GABA, glutamate, and glycine currents at E15, 10 DIV were similar to the currents at E20, 5 DIV (both 25 days after conception), suggesting parallel developmental patterns for amino acid receptor expression in vitro and in vivo. 8. Together, these data suggest that GABA may play a major role in early development because hypothalamic neurons are more sensitive to GABA than to either glutamate or glycine. However, glutamate and glycine receptors appear more sensitive to regulation by the local environment than GABA receptors because culture density and the astrocyte substrate have greater inductive effects on glutamate and glycine receptors than on GABA receptors.

Glutamate can play a number of roles in the developing brain, including modulation of gene expression, cell motility, neurite growth and neuronal survival, all critical for the final organization and function of the mature brain. These functions are dependent on the early expression of glutamate receptors and on glutamate release in developing neurons. This subject has received little attention in the hypothalamus, despite glutamate's critical role as an excitatory transmitter in hypothalamic control of circadian rhythms, endocrine secretion, temperature regulation, and autonomic control. A total of 10,922 rat hypothalamic neurons were studied with digital Ca2+ imaging with the ratiometric dye fura-2 to examine their responses to glutamate receptor agonists and antagonists during embryonic development and maturation in vitro. Functional glutamate receptors were found very early in development (embryonic day 15-E15) with both Ca2+ imaging and with patch clamp recording. This is a time when the hypothalamus is beginning to undergo neurogenesis. Ca2+ responses from N-methyl-D-aspartate receptors developed later than those from non-N-methyl-D-aspartate ionotropic receptors that responded to kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate. The responses of immature E15 cells after one day in vitro were compared with more mature cells after six days in vitro to examine the response to repeated 3 min applications of 100 microM kainate (n = 108). Immature cells showed similar Ca2+ rises (+232nM Ca2+) with each kainate stimulation. In contrast, more mature cells showed an initial Ca2+ rise of 307 nM, with the second rise only to 147 nM above the initial baseline. Immature cells more quickly returned to their pre-kainate baseline than did older cells. The expression of metabotropic glutamate receptors was studied with the selective agonist trans-1-amino-cyclopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five days in vitro. E16 astrocytes showed a greater response than did neurons to conditions that would activate the metabotropic glutamate receptor. A dramatic increase in the percentage of cells that responded to N-methyl-D-aspartate was found after only a few days in culture. Only a small number of E15 cells studied on the day of culture (4% of 694 cells) showed a response to 100 microM N-methyl-D-aspartate. Thirty-eight percent of 120 E18 cells cultured for one day in vitro showed an N-methyl-D-aspartate response.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Synaptic transmission between olfactory receptor neurons and mitral/tufted cells was examined using a whole-cell recording technique in a hemisected preparation of the turtle olfactory bulb. To determine the olfactory receptor neuron transmitter, we isolated components of the synaptic response of mitral/tufted cells to olfactory nerve stimulation using postsynaptic receptor antagonists. 2. Low-intensity stimulation of the olfactory nerve evoked monosynaptic excitatory postsynaptic potentials in mitral/tufted cells that consisted of a rapid and prolonged depolarization with little contribution from other bulb neurons. The exogenous application of glutamate mimicked the response of mitral/tufted cells to olfactory nerve stimulation. 3. Olfactory nerve stimulation evoked in mitral/tufted cells a two component response that was reversibly blocked by glutamate receptor antagonists. The first, a rapid depolarization of short duration, was sensitive to the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX); the second, a depolarization of slower onset but longer duration, was sensitive to the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5). When DNQX and AP5 were both present the postsynaptic response was completely abolished. These results strongly support the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral/tufted cell synapse.

1. It is generally presumed that gamma-aminobutyric acid (GABA) mediates inhibition in the mammalian brain, whereas glycine is restricted to the brain stem and spinal cord. Recent immunocytochemical and molecular biological studies have demonstrated, however, a widespread distribution of glycine receptors through-out the CNS, including the olfactory bulb. To explore the functional significance of glycine receptors in the olfactory bulb we have used primary culture and whole-cell voltage-clamp recording techniques to test the hypothesis that glycine, as well as GABA, exerts inhibitory actions on olfactory bulb neurons. 2. Cultures of olfactory bulb neurons contain two primary populations of morphologically distinct neurons, mitral/tufted cells and interneurons (granule and periglomerular cells). In all mitral/tufted cells and interneurons examined, both glycine and GABA evoked concentration-dependent desensitizing currents mediated by chloride, similar to those seen in mammalian neurons elsewhere in the brain and spinal cord. 3. The median effective concentration (EC50) for glycine was 125 microM, with a Hill coefficient of 1.7, whereas the EC50 and Hill coefficient for GABA were 52 microM and 1.8, respectively. These values are similar to values previously reported for other central neurons. 4. At moderate concentrations (> 1 microM) strychnine nonselectively antagonized both glycine- and GABA-evoked currents. At low concentrations (< or = 1 microM) strychnine blocked glycine-mediated currents but had little effect on GABA-mediated currents. Similarly, picrotoxin was a nonselective antagonist for glycine- and GABA-mediated currents at high concentrations (100 microM), but was selective for GABA at low concentrations (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Noradrenergic modulation of the glutamatergic-GABAergic synapses between mitral/tufted (M/T) and granule cells has been implicated in some forms of olfactory learning (5), but the mechanism of action is unknown. Intracellular stimulation of M/T cells in primary culture, evoked glutamate-mediated excitatory postsynaptic potentials (EPSPs) in granule cells that were reversibly inhibited by approximately 50% during application of norepinephrine (NE). NE had no effect, however, on the membrane current evoked by the application of glutamate, indicating a presynaptic site of action. The effect of NE on EPSPs was mimicked by the alpha receptor agonist clonidine, but not by the beta receptor agonist isoproteronol. NE also inhibited spontaneous GABAergic inhibitory postsynaptic potentials recorded in M/T cells, by a presynaptic alpha-adrenergic mediated mechanism. NE and clonidine also inhibited high threshold calcium currents. The effects of NE on calcium currents were irreversible in the presence of internal GTP gamma S and prevented by pertussis toxin, suggesting a G protein-coupled mechanism. Pertussis toxin also prevented the effects of NE on synaptic transmission. These results support previous results suggesting a disinhibitory role for NE in the olfactory bulb. This action is, at least in part, due to a reduction in mitral cell mediated granule cell excitation through inhibition of presynaptic calcium influx.

Recent work in molecular biology and synaptic physiology has significantly increased our understanding of inhibitory and excitatory mechanisms in the olfactory bulb. Multiple subtypes of amino acid receptors with different functional and neuromodulatory properties are likely to play key roles in processing odor information transduced and relayed to the olfactory bulb by the olfactory sensory neurons, and in modulating that information during olfactory learning.

1. Olfactory bulb neurons were dissociated from neonatal rats and plated at low density on a confluent layer of olfactory bulb astrocytes. Intracellular stimulation of presumptive mitral/tufted (M/T) cells evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in adjacent neurons. Whole-cell recording techniques and a flow-pipe drug delivery system were used to compare EPSPs with voltage-clamp recordings of currents evoked by excitatory amino acids (EAA) including N-acetylaspartylglutamate (NAAG), a putative mitral cell transmitter. 2. Cultured olfactory bulb neurons were morphologically and physiologically distinct. Large pyramidal-shaped neurons were present, which were NAAG immunoreactive; stimulation of these neurons invariably evoked EPSPs, suggesting that they were M/T cells. The majority of small bipolar neurons were glutamic acid decarboxylase (GAD) immunoreactive consistent with granule or periglomerular gamma-aminobutyric acid (GABA)ergic interneurons. 3. Monosynaptic EPSPs between M/T cells could be separated into fast and slow components by the use of EAA receptor antagonists. A fast component with a time-to-peak of 7.7 +/- 1.0 (SE) ms and half-width of 31.8 +/- 7.4 ms was blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX, 2.5 microM). The slow component (time-to-peak = 41.4 +/- 7.2 ms; half-width = 218.9 +/- 40.4 ms) was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5, 100 microM). 4. Under voltage clamp, flow-pipe applications of NAAG (10-1,000 microM) evoked inward currents at a holding potential of -60 mV in Mg-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Because norepinephrine (NE) reuptake inhibitors have been reported to block 6-hydroxydopamine (6-OHDA) induced NE depletion, we wondered whether a reuptake inhibitor would block 6-OHDA's effects on visual cortical plasticity. We found, however, that desmethylimipramine (DMI) did not reduce 6-OHDA-induced NE depletion at 6-OHDA doses sufficient to prevent the effects of monocular deprivation. We also found that DMI itself induced transient NE depletion. We used this last result to further examine the NE hypothesis of depletion. In contrast to 6-OHDA-induced NE depletion, DMI-induced NE depletion was not accompanied by changes in visual cortical plasticity.

Under some circumstances intraventricular administration of 6-OHDA decreases visual cortical plasticity of kittens; the mechanism for this change is not known, but depletion of norepinephrine (NE) is not the entire explanation. We have examined the effects of 6-OHDA treatment on beta adrenergic receptor binding in kitten visual cortex. Subjects were given vehicle solution alone, a low dose of 6-OHDA which depleted cortical NE without affecting visuocortical plasticity, or a higher dose of 6-OHDA which depleted cortical NE and decreased visuocortical plasticity. Drugs were administered in single daily injections via intraventricular cannulas. Saturation assays were performed on homogenates of visual cortical tissue using 125I-pindolol (30-400 pM) along w/ isoproterenol (237 microM) as a cold competitor. We measured radioactivity bound to tissue and retained on filters and analyzed the data using the EBDA computer program (McPherson 1983, 1985); we determined the affinity constant (Kd) and receptor density (Bmax) in multiple assays for each animal. Despite 75-90% NE depletion in both experimental groups, only the group receiving the lower dose of 6-OHDA showed any evidence of supersensitivity. The Kds did not differ among the groups. The data suggest that the effects of 6-OHDA on visuocortical plasticity are not secondary to beta adrenergic supersensitivity.

We recorded from the visual cortex of 4 groups of monocularly deprived kittens. Three groups were treated with intraventricular 6-hydroxydopamine (6-OHDA) at different times relative to monocular deprivation (MD). One group received only vehicle solution and MD. 6-OHDA caused the greatest decrease in plasticity in the kittens receiving 6-OHDA throughout the deprivation period; that is, these kittens were the least affected by MD. 6-OHDA caused a smaller decrease in plasticity in kittens receiving 6-OHDA just prior to eyelid suture and a still smaller decrease in kittens waiting a week between 6-OHDA treatment and eyelid suture. The kittens in all groups receiving 6-OHDA were equally depleted of norepinephrine (NE). We conclude that 6-OHDA decreases plasticity in the visual cortex; however, the time course of this decrease is better related to the time course of the 6-OHDA treatment than to the time course of NE depletion.