Localization of immunoreactivity for deleted in colorectal cancer (DCC), the receptor for the guidance factor netrin-1, in ventral tier dopamine projection pathways in adult rodents

DCC (deleted in colorectal cancer)-the receptor of the netrin-1 neuronal guidance factor-is expressed and is active in the central nervous system (CNS) during development, but is down-regulated during maturation. The substantia nigra contains the highest level of netrin-1 mRNA in the adult rodent brain, and corresponding mRNA for DCC has also been detected in this region but has not been localized to any particular neuron type. In this study, an antibody raised against DCC was used to determine if the protein was expressed by adult dopamine neurons, and identify their distribution and projections. Significant DCC-immunoreactivity was detected in midbrain, where it was localized to ventrally displaced A9 dopamine neurons in the substantia nigra, and ventromedial A10 dopamine neurons predominantly situated in and around the interfascicular nucleus. Strong immunoreactivity was not detected in dopamine neurons found elsewhere, or in non-dopamine-containing neurons in the midbrain. Terminal fields selectively labeled with DCC antibody corresponded to known nigrostriatal projections to the dorsolateral striatal patches and dorsomedial shell of the accumbens, and were also detected in prefrontal cortex, septum, lateral habenular and ventral pallidum. The unique distribution of DCC-immunoreactivity in adult ventral midbrain dopamine neurons suggests that netrin-1/DCC signaling could function in plasticity and remodeling previously identified in dopamine projection pathways. In particular, a recent report that DCC is regulated through the ubiquitin-proteosome system via Siah/Sina proteins, is consistent with a potential involvement in genetic and sporadic forms of Parkinson's disease. (c) 2005 IBRO. Published by Elsevier Ltd. All rights reserved.

A method for drug infusion into the lateral median eminence and arcuate nucleus of sheep

The role of catecholamines in the control of the GnRH pulse generator is unclear as studies have relied on the use of peripheral or intracerebroventricular injections, which lack specificity in relation to the anatomical site of action. Direct brain site infusions have been used, however, these are limited by the ability to accurately target small brain regions. One such area of interest in the control of GnRH is the median eminence and arcuate nucleus within the medial basal hypothalamus. Here we describe a method of stereotaxically targeting this area in a large animal (sheep) and an infusion system to deliver drugs into unrestrained conscious animals. To test our technique we infused the dopamine agonist, quinpirole or vehicle into the medial basal hypothalamus of ovariectomised ewes. Quinpirole significantly suppressed LH pulsatility only in animals with injectors located close to the lateral median eminence. This in vivo result supports the hypothesis that dopamine inhibits GnRH secretion by presynaptic inhibition in the lateral median eminence. Also infusion of quinpirole into the medial basal hypothalamus suppressed prolactin secretion providing in vivo evidence that is consistent with the hypothesis that there are stimulatory autoreceptors on tubero-infundibular dopamine neurons. (C) 1997 Elsevier Science B.V.

Event-related potential correlates of impaired visuospatial working memory in schizophrenia

Previous studies have reported that patients with schizophrenia demonstrate impaired performance during working memory (WM) tasks. The current study aimed to determine whether WM impairments in schizophrenia are accompanied by reduced slow wave (SW) activity during on-line maintenance of mnemonic information. Event-related potentials were obtained from patients with schizophrenia and well controls as they performed a visuospatial delayed response task. On 50% of trials, a distractor stimulus was introduced during the delay. Compared with controls, patients with schizophrenia produced less SW memory negativity, particularly over the right hemisphere, together with reduced frontal enhancement of SW memory negativity in response to distraction. The results indicate that patients with schizophrenia generate less maintenance phase neuronal activity during WM performance, especially under conditions of distraction.

Modulating the rate and rhythmicity of perceptual rivalry alternations with the mixed 5-HT2A and 5-HT1A agonist psilocybin

Binocular rivalry occurs when different images are presented simultaneously to corresponding points within the left and right eyes. Under these conditions, the observer's perception will alternate between the two perceptual alternatives. Motivated by the reported link between the rate of perceptual alternations, symptoms of psychosis and an incidental observation that the rhythmicity of perceptual alternations during binocular rivalry was greatly increased 10 h after the consumption of LSD, this study aimed to investigate the pharmacology underlying binocular rivalry and to explore the connection between the timing of perceptual switching and psychosis. Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine, PY) was chosen for the study because, like LSD, it is known to act as an agonist at serotonin (5-HT)(1A) and 5-HT2A receptors and to produce an altered state sometimes marked by psychosis-like symptoms. A total of 12 healthy human volunteers were tested under placebo, low-dose ( 115 mg/kg) and high-dose ( 250 mg/kg) PY conditions. In line with predictions, under both low- and high-dose conditions, the results show that at 90 min postadministration ( the peak of drug action), rate and rhythmicity of perceptual alternations were significantly reduced from placebo levels. Following the 90 min testing period, the perceptual switch rate successively increased, with some individuals showing increases well beyond pretest levels at the final testing, 360 min postadministration. However, as some subjects had still not returned to pretest levels by this time, the mean phase duration at 360 min was not found to differ significantly from placebo. Reflecting the drug-induced changes in rivalry phase durations, subjects showed clear changes in psychological state as indexed by the 5D-ASC ( altered states of consciousness) rating scales. This study suggests the involvement of serotonergic pathways in binocular rivalry and supports the previously proposed role of a brainstem oscillator in perceptual rivalry alternations and symptoms of psychosis.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

PACAP stimulates dopamine neuronal activity in the medial basal hypothalamus and inhibits prolactin

In sheep intracerebroventricular injection of PACAP (10 nmol) significantly (P < 0.01) stimulated the levels of the dopamine metabolite DOPAC within the medial basal hypothalamus las measured by in vivo microdialysis) and this effect was temporally correlated with a significant (P < 0.05) suppression in peripheral prolactin concentrations. This result is in accord with the hypothesis that PACAP suppresses prolactin secretion from the anterior pituitary gland by stimulating dopamine release from tuberoinfundibular dopaminergic neurons. (C) 1998 Elsevier Science B.V.

Differential involvement of rat medial prefrontal cortex dopamine receptors in modulation of hypothalamic-pituitary-adrenal axis responses to different stressors

Recent investigations have implicated the medial prefrontal cortex (mPFC) in modulation of subcortical pathways that contribute to the generation of behavioural, autonomic and endocrine responses to stress. However, little is known of the mechanisms involved. One of the key neurotransmitters involved in mPFC function is dopamine, and we therefore aimed, in this investigation, to examine the role of mPFC dopamine in response to stress in Wistar rats. In this regard, we infused dopamine antagonists SCH23390 or sulpiride into the mPFC via retrodialysis. We then examined changes in numbers of cells expressing the c-fos immediate-early gene protein product, Fos, in subcortical neuronal populations associated with regulation of hypothalamic-pituitary-adrenal (HPA) axis stress responses in response to either of two stressors; systemic injection of interleukin-1beta, or air puff. The D-1 antagonist, SCH23390, and the D-2 antagonist, sulpiride, both attenuated expression of Fos in the medial parvocellular hypothalamic paraventricular nucleus (mpPVN) corticotropin-releasing factor cells at the apex of the HPA axis, as well as in most extra-hypothalamic brain regions examined in response to interleukin-1beta. By contrast, SCH23390 failed to affect Fos expression in response to air puff in any brain region examined, while sulpiride resulted in an attenuation of the air puff-induced response in only the mpPVN and the bed nucleus of the stria terminalis. These results indicate that the mPFC differentially processes the response to different stressors and that the two types of dopamine receptor may have different roles.

Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30-75 nS. The dose response curve for calcium exhibited an EC50 of about 26 mu M. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between -80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P-Na/P-Cl of 0.034. The halide permeability sequence was P-Cl > P-F > P-I > P-Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN-, acetate(-), and gluconate(-), with the permeability sequence P-Cl > P-SCN > gluconaie. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 Angstrom.

Hypothalamic dopamine D1 receptors are involved in the stimulation of prolactin secretion by high environmental temperature in the female sheep

Recent evidence suggests that dopamine, acting via its D1 receptors, may function as a neurotransmitter in intrahypothalamic pathways involved in the stimulation of prolactin secretion. Functional dopamine D1 receptors are present in the ventromedial hypothalamic nucleus (VMH) and we hypothesized that they might be part of a prolactin-stimulatory pathway activated by stress. We tested this hypothesis in a series of experiments on sheep involving two different forms of stressors, audiovisual (barking dog) and high environmental temperature. We attempted to block the stimulation of prolactin secretion by infusion into the VMH of an antagonist specific for the D1 receptor. Ovariectomised, oestradiol-implanted merino ewes were surgically implanted with bilateral guide tubes directed at the VMH. After a 180 min pretreatment period, the ewes either were or were not exposed to a stressor (30 min of barking dog or 120 min at 35 degrees C, 65% relative humidity). D1 receptor antagonist, SCH23390 or vehicle (0.9% saline) was infused into the VMH (1.7 mu l/h, 120 nmol/h) for 60 min prior to and during the stressor period. Blood was sampled every 15 min via jugular cannulae and the plasma was assayed for prolactin, cortisol and growth hormone (GH). Both stressors significantly increased prolactin concentrations over control levels. SCH23390 infusion significantly attenuated the prolactin response to high environmental temperature, but had no effect on the prolactin response to audiovisual stress. Cortisol concentrations were significantly increased by audiovisual stress only and were not affected by SCH23390, GH concentrations were not changed by either stressor or infusion. Drug infusion alone did not affect the concentration of the hormones. The data suggest that the VMH D1 receptors are involved in a prolactin stimulatory pathway in response to high environmental temperature. The inability of the D1 antagonist to affect the response to the barking dog indicates that this pathway is stress-specific, implying that there is more than one mechanism or pathway involved in the prolactin response to different stressors.

New techniques for biopsy and culture of human olfactory epithelial neurons

Objective: To improve the success of culturing olfactory neurons from human nasal mucosa by investigating the intranasal distribution of the olfactory epithelium and devising new techniques for growing human olfactory epithelium in vitro. Design: Ninety-seven biopsy specimens were obtained from 33 individuals, aged 21 to 74 years, collected from 6 regions of the nasal cavity. Each biopsy specimen was bisected, and 1 piece was processed for immunohistochemistry or electron microscopy while the other piece was dissected further for explant culture. Four culture techniques were performed, including whole explants and explanted biopsy slices. Five days after plating, neuronal differentiation was induced by means of a medium that contained basic fibroblast growth factor. After another 5 days, cultures were processed for immunocytochemical analysis. Results: The probability of finding olfactory epithelium in a biopsy specimen ranged from 30% to 76%, depending on its location. The dorsoposterior regions of the nasal septum and the superior turbinate provided the highest probability, but, surprisingly, olfactory epithelium was also found anteriorly and ventrally on both septum and turbinates. A new method of culturing the olfactory epithelium was devised. This slice culture technique improved the success rate for generating olfactory neurons from 10% to 90%. Conclusions: This study explains and overcomes most of the variability in the success in observing neurogenesis in cultures of adult human olfactory epithelium. The techniques presented here make the human olfactory epithelium a useful model for clinical research into certain olfactory dysfunctions and a model for the causes of neurodevelopmental and neurodegenerative diseases.

Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-D-aspartic acid) receptors, The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density.

Photolytic manipulation of [Ca2+](i) reveals slow kinetics of potassium channels underlying the afterhyperpolarization in hipppocampal pyramidal neurons

The identity of the potassium channel underlying the slow, apamin-insensitive component of the afterhyperpolarization current (sl(AHP)) remains unknown. We studied sl(AHP) in CA1 pyramidal neurons using simultaneous whole-cell recording, calcium fluorescence imaging, and flash photolysis of caged compounds. Intracellular calcium concentration ([Ca2+](i)) peaked earlier and decayed more rapidly than sl(AHP). Loading cells with low concentrations of the calcium chelator EGTA slowed the activation and decay of sl(AHP). In the presence of EGTA, intracellular calcium decayed with two time constants. When [Ca2+](i) was increased rapidly after photolysis of DM-Nitrophen, both apamin-sensitive and apamin-insensitive outward currents were activated. The apamin-sensitive current activated rapidly (<20 msec), whereas the apamin-insensitive current activated more slowly (180 msec). The apamin-insensitive current was reduced by application of serotonin and carbachol, confirming that it was caused by sl(AHP) channels. When [Ca2+](i) was decreased rapidly via photolysis of diazo-2, the decay of sl(AHP) was similar to control (1.7 sec). All results could be reproduced by a model potassium channel gated by calcium, suggesting that the channels underlying sl(AHP) have intrinsically slow kinetics because of their high affinity for calcium.

Neurons in the hilus region of the rat hippocampus are depleted in number by exposure to alcohol during early postnatal life

We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life resulted in a deficit in spatial learning ability. This ability is controlled, at least in part, by the hippocampal formation. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life affected the number of specific neurons in the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol between postnatal days 10 and 15 by placing them for 3 h each day in a chamber containing ethanol vapor. The blood ethanol concentration was about 430 mg/dl at the end of the exposure period. Groups of ethanol-treated (ET) rats, separation controls (SC), and mother-reared controls (MRC) were anesthetized and killed at 16 days of age by perfusion with phosphate-buffered glutaraldehyde (2.5%). The Cavalieri principle was used to determine the volume of various subdivisions of the hippocampal formation (CA1, CA2+CA3, hilus, and granule cell layer), and the physical disector method was used to estimate the numerical densities of neurons within each subdivision. The total number of neurons was calculated by multiplying estimates of the numerical density with the volume. There were, on average, about 441,000 granule cells in the granule cell layer and 153,000 to 177,000 pyramidal cells in both the CA1 and CA2+CA3 regions in all three treatment groups. In the hilus region, ET rats had about 27,000 neuronal cells. This was significantly fewer than the average of 38,000 such neurons estimated to be present in both MRC and SC animals. Thus, neurons in the hilus region may be particularly vulnerable to the effects of a high dose of ethanol exposure during early postnatal life. (C) 2000 Wiley-Liss, Inc.

Central projections of Drosophila sensory neurons in the transition from embryo to larva

Sensory axons of different sensory modalities project into typical domains within insect ganglia. Tactile and gustatory axons project into a ventral layer of neuropil and proprioceptive afferents, including chordotonal axone, into an intermediate or dorsal layer. Here, we describe the central projections of sensory neurons in the first instar Drosophila larva, relating them to the projection of the same sensory afferents in the embryo and to sensory afferents of similar type in other insects. Several neurons show marked morphologic changes in their axon terminals in the transition between the embryo and larva. During a short morphogenetic period late in embryogenesis, the axon terminals of the dorsal bipolar dendrite stretch receptor change their shape and their distribution within the neuromere. In the larva, external sense organ neurons (es) project their axons into a ventral layer of neuropil. Chordotonal sensory neurons (ch) project into a slightly more dorsal region that is comparable to their projection in adults. The multiple dendrite (md) neurons show two distinctive classes of projection. One group of md neurons projects into the ventral-most neuropil region, the same region into which es neurons project. Members of this group are related by lineage to es neurons or share a requirement for expression of the same proneural gene during development. Other md neurons project into a more dorsal region. Sensory receptors projecting into dorsal neuropil possibly provide proprioceptive feedback from the periphery to central motorneurons and are candidates for future genetic and cellular analysis of simple neural circuitry. J. Comp. Neurol. 425:34-44, 2000. (C) 2000 Wiley-Liss, Inc.

Calcium-activated potassium currents in mammalian neurons

1. Influx of calcium via voltage-dependent calcium channels during the action potential lends to increases in cytosolic calcium that can initiate a number of physiological processes. One of these is the activation of potassium currents on the plasmalemma. These calcium-activated potassium currents contribute to action potential repolarization and are largely responsible for the phenomenon of spike frequency adaptation. This refers to the progressive slowing of the frequency of discharge of action potentials during sustained injection of depolarizing current. In some cell types, this adaptation is so marked that despite the presence of depolarizing current, only a single spike (or a few spikes) is initiated, Following cessation of current injection, slow deactivation of calcium-activated potassium currents is also responsible for the prolonged hyperpolarization that often follows, 2. A number of macroscopic calcium-activated potassium currents that can be separated on the basis of kinetic and pharmacological criteria have been described in mammalian neurons. At the single channel level, several types of calcium-activated potassium channels also have been characterized. While for some macroscopic currents the underlying:single channels have been unambiguously defined, for other currents the identity of the underlying channels is not clear. 3. In the present review we describe the properties of the known types of calcium-activated potassium currents in mammalian neurons and indicate the relationship between macroscopic currents and particular single channels.