In vivo radiotracers for vesicular neurotransmitter transporters

Widespread hemorrhagic manifestations commonly occur in patients with severe heat stroke. The pathogenesis of hemostatic disorders in these patients is not fully understood, although it is believed to be multifactorial in origin. The present investigation was designed to study the changes in blood platelets caused by heat stress in an experimental model of five merino sheep. The experiments were performed in two groups of five merino sheep each. In one group the sheep were subjected to a combination of heat (elevated environmental temperature) and exertional stress, and allowed to proceed throughout the experiment until a state of near collapse was reached (Task A). In the other group (Task B) the animals were heated in the same manner as those in Task A and also subjected to exertional heat; however, when the temperature reached 43.6 +/- 0.2 degrees C, the critical core temperature (CCT), they were subjected to evaporative cooling in a climatic chamber. Serial changes in the platelet counts and platelet functions were measured throughout the duration of the experiments. At the core temperature (CT) of 42.1 degrees C and above there was a significant impairment of adhesion of platelets to glass beads. During the early phases of elevation of CT, platelets showed hyperaggregation in the presence of different agonists (such as, collagen, ADP, ristocetin); this was followed by hypoaggregation when the CCT was raised above 43.6 +/- 0.2 degrees C. However, these impairments of platelet functions occurring at elevated CT and CCT were found to reverse to normal within 24 hours after the animals were cooled to 39 degrees C. It was also found that the hyperaggregation of platelets to different agonists induced by raised CT could be partially prevented by prior in vitro treatment of platelets with apyrase, a known enzyme destroying of ADP. The results of these experiments indicate that heat stress induced by exposing merino sheep to elevated controlled temperature directly activates the platelets. This may be an important contributing factor in causing altered hemostasis in heat stroke activated directly by heat. This mechanism may be operating in altered hemostasis in heat stroke.     

In vivo assessment of neurotransmitter system in cardiovascular diseases. Clinical issues

Cardiac neurotransmitter systems, especially the adrenergic receptor pathway, are impaired in heart diseases. In patients with heart failure, these abnormalities contribute to arrhythmogenesis and to progression of cardiac dysfunction. The use of MIBG with single photon imaging has provided useful information on the mechanisms of ventricular arrhythmias, and on the causes of death in patients with heart failure or hypertrophic cardiomyopathy. It has been suggested as a prognostic indicator in patients with heart failure. Positron Emission Tomography (PET) now allows us to obtain noninvasively the quantitative determination of regional receptor density and affinity in humans as well as innervation integrity and functioning. These measurements are based upon the synthesis of a radioligand, usually either a selective receptor antagonist or a false neurotransmitter labeled with a positron-emitting radioisotope. Mathematical compartmental models are fitted to activity-versus-time curves obtained during saturation or displacement experiments in order to calculate the rate constants and the receptor density in the myocardium. PET has only recently begun to be applied to the study of cardiac physiology and disease. PET and SPECT cardiac neuroimaging techniques are able to demonstrate the physiological regulation of receptors, and to provide the possibility of studying regional abnormalities of cardiac neurotransmission, especially in arrhythmogenic cardiomyopathy. Furthermore these non invasive techniques could be useful in exploring the alteration of neurotransmission in the early stage of heart disease and could allow repeated scintigraphic examinations in order to evaluate the effects of cardiac medications.     

In vivo visualization of neurotransmitter function in the human brain by PET

Moving along a microtubule, kinesin follows a course parallel to the protofilaments; but it is not known whether kinesin binds exclusively on a single protofilament. The presence of zinc during tubulin polymerization induces sheets where neighboring protofilaments are antiparallel. If kinesin could support the motility of these zinc-sheets, then the binding site for a kinesin molecule would be limited to a single protofilament. Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] reported that kinesin moves along zinc-sheets. We found that zinc-sheets grown under their conditions often had a microtubule-like structure along one edge. We confirmed the possibility that the motility observed by Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] is attributed to the microtubule-like structure rather than the zinc-sheet. To resolve the question of whether kinesin can recognize an antiparallel protofilament lattice, we investigated the kinesin-mediated motility of zinc-macrotubes. At higher free zinc concentrations, zinc-sheets roll up as macrotubes, free of edges. In the presence of 10 microM taxol and 100 nM free Zn2+ at pH 6.8, the samples were shown by electron microscopy to contain only macrotubes. Under these buffer conditions, kinesin could bind strongly to axonemal doublets in the presence of AMP-PNP, and generate motility in the presence of ATP, but kinesin did not bind to nor move the macrotubes. This shows that kinesin cannot bind efficiently to nor move on the anti-parallel lattice; it is possible (though not necessary) that the groove between two parallel protofilaments is required for kinesin's motility.     

Effects of nitric oxide synthase gene knockout on neurotransmitter release in vivo

Previous studies suggest that low bone mass is a complication of alcoholic liver disease. Nevertheless, little is known about bone mass and bone metabolism in viral cirrhosis. To evaluate the prevalence and magnitude of hepatic osteopenia in these patients, bone remodeling status, and its relationship with the severity of liver disease and serum levels of insulin-like growth factor I (IGF-I), we studied 32 consecutive patients with viral cirrhosis and no history of alcohol intake. Bone mineral density (BMD) was measured by dual x-ray absorptiometry in the lumbar spine (LS) and femoral neck (FN), and the values were expressed as the z score. Bone metabolism markers and hormone profiles were measured. Patients with viral cirrhosis showed reduced BMD in all sites (LS: -1.27 +/- 1.06, P < .001; FN: -0.48 +/- 0.96; P < .01). Of the 32 patients, 53% met the diagnostic criteria for osteoporosis. In patients, urine deoxypyridinoline (D-Pyr) as a marker of bone resorption and serum bone alkaline phosphatase (b-AP) as a marker of bone formation were significantly higher than in control subjects (P < .001 and P < .01, respectively). Serum IGF-I was lower than in control subjects (P < .001), and significant differences were also found between patients with and without osteoporosis (P < .05). BMD in LS correlated with severity of the disease, with serum levels of IGF-I, and with urine D-Pyr. Our findings show that viral cirrhosis is a major cause of osteoporosis in men, and that low serum IGF-I levels seem to play a role in the bone mass loss in these patients. The biochemical markers of bone remodeling suggest high-turnover osteoporosis in patients with viral cirrhosis.     

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-microseconds time resolution, respectively, for studying neurotransmitter receptors in giant (approximately 20-microns diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.     

Synaptic physiology and ultrastructure in comatose mutants define an in vivo role for NSF in neurotransmitter release

N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thought to play a key role in vesicular transport in all eukaryotic cells. Although NSF was proposed to function in the trafficking of synaptic vesicles responsible for neurotransmitter release, only recently have in vivo experiments begun to reveal a specific function for NSF in this process. Our previous work showed that mutations in a Drosophila NSF gene, dNSF1, are responsible for the temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, we perform electrophysiological and ultrastructural analyses in three different comt alleles to investigate the function of dNSF1 at native synapses in vivo. Electrophysiological analysis of postsynaptic potentials and currents at adult neuromuscular synapses revealed that in the absence of repetitive stimulation, comt synapses exhibit wild-type neurotransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures revealed a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast calcium-triggered fusion. To define dNSF1 function further, we used transmission electron microscopy to examine the distribution of vesicles within synaptic terminals, and observed a marked accumulation of docked vesicles at restrictive temperatures in comt. Together, the results reported here define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-triggered fusion.     

Astrocytic glutamate uptake and prion protein expression

Factors influencing glutamate uptake by astrocytes may indirectly influence neuronal survival. Elevated extracellular glutamate may be excitotoxic or may exacerbate neurodegeneration in various neurological diseases. By using a cell culture model, we have investigated the influence of astrocytic prion protein (PrPc) expression on glutamate uptake. Type 1 astrocytes expressing PrPc have a higher rate of Na+-dependent glutamate uptake than PrPc-deficient type 1 astrocytes. This difference is exacerbated when serum free media is used to culture the astrocytes. Further analysis suggested that a decrease in substrate affinity is responsible for the sensitivity of PrP-deficient astrocytic glutamate uptake to culture conditions. PrPc has been shown to bind copper. Greater sensitivity of cells to copper concentrations may be responsible for the decreased substrate affinity observed. PrPc-deficient cerebellar cells are more sensitive to glutamate toxicity in the presence of copper. These results show that glutamate uptake from astrocytes is dependent on PrPc expression which in turn may be related to copper metabolism.     

G protein alpha subunit G alpha z couples neurotransmitter receptors to ion channels in sympathetic neurons

The functional roles subserved by G(alpha)z, a G protein alpha subunit found predominantly in neuronal tissues, have remained largely undefined. Here, we report that G(alpha)z coupled neurotransmitter receptors to N-type Ca2+ channels when transiently overexpressed in rat sympathetic neurons. The G(alpha)z-mediated inhibition was voltage dependent and PTX insensitive. Recovery from G(alpha)z-mediated inhibition was extremely slow but accelerated by coexpression with RGS proteins. G(alpha)z selectively interacted with a subset of receptors that ordinarily couple to N-type Ca2+ channels via PTX-sensitive Go/i proteins. In addition, G(alpha)z rescued the activation of heterologously expressed GIRK channels in PTX-treated neurons. These results suggest that G(alpha)z is capable of coupling receptors to ion channels and might underlie PTX-insensitive ion channel modulation observed in neurons under physiological and pathological conditions.     

5-HT receptors in mammalian brain: receptor autoradiography and in situ hybridization studies of new ligands and newly identified receptors

In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combined in situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT1A receptors in monkey brain, labelled with the agonist 3H-8-OH-DPAT and the antagonist 3H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT1A receptors in monkey brain. The labelling conditions to visualize 5-HT1F receptors in guinea pig brain, namely 3H-sumatriptan in the presence of 10(-8) M 5-CT to block 5-HT1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed by in situ hybridization. By using 3H-ketanserin and 3H-mesulergine in parallel with in situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT2A and 5-HT2C binding sites in monkey brain. 5-HT4 receptors were localized in the brain of several species including humans by using 125I-SB 207710. In situ hybridization experiments performed in guinea pig confirmed that 5-HT4 receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT7 binding sites were labelled in rat and guinea pig brains by incubating with 3H-5-CT in the presence of 100 microM WAY 100135 and 250 microM GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.     

Dopamine D2 receptors quantified in vivo in human narcolepsy

The correlation of clinical with psychological and social data is an attempt to find predictors of the definite long-term outcome after brain injury. 34 patients were reexamined 3 to 8 years after the accident using a number of psychological tests. Additionally, life quality was defined and evaluated. Only patients with an initial Glasgow Coma Scale-Score of 3-12, an intracranial traumatic lesion on computertomography and age 16-65 years at the time of accident were included in this study. Patients exhibited a uniform pattern of disturbances in psychosocial long-term outcome. These disturbances were compared with initial clinical data: memory, attention and learning were significantly correlated with the duration of coma and the presence of additional extracerebral injuries. From the initial computerized tomography, the findings 'compression of basal cisterns' and 'intracerebral contusion' showed to be predictors of the cerebral function. Late social status and behavior, defined as quality of life, were clearly related with initial clinical findings. In conclusion, there are early clinical predictors of the long term social and psychological outcome after brain injury.     

In vitro and in vivo detection of functional somatostatin receptors in canine insulinomas

Ten dogs with hypoglycemia due to insulinomas were studied to assess the expression of somatostatin receptors (SSTRs) in canine insulinomas and its potential diagnostic value. METHODS: The response of circulating glucose and insulin concentrations to the subcutaneous administration of a somatostatin analog, octreotide, was measured. SSTRs were visualized in vitro by autoradiography. [Iodine-125-Tyr3]-octreotide and [125I-Tyr11]-somatostatin-14 (SRIF-14) were used as radioligands. SPECT was performed 6 hr after the injection of [111In-DTPA-D-Phe1]-octreotide. RESULTS: After subcutaneous injection of 50 micrograms octreotide, plasma glucose concentration rose from 2.3 +/- 0.2 mmol/liter to 3.2 +/- 0.3 mmol/liter at 3.5 hr (p < 0.05) and plasma insulin concentration decreased from 451 +/- 135 pmol/liter to a nadir of 249 +/- 115 pmol/liter at 30 min (p < 0.05). In vitro autoradiography revealed that all primary insulinomas and their metastases had specific SSTRs for both [125I-Tyr3]-octreotide and [126I-Tyr11]-SRIF-14. Scatchard analysis of SSTR binding in the tumor tissue of one dog revealed high-affinity binding sites for [125I-Tyr3]-octreotide (dissociation constant (Kd) 1.7 nM, maximum binding capacity (Bmax) 499 fmol/mg membrane protein). The primary tumor and/or metastases in five of six dogs could be visualized and localized by SPECT with [111In-DTPA-D-Phe1]-octreotide. In the remaining dog, multiple metastases (< 3 mm) were found in the liver at necropsy, apparently too small to be visualized by SPECT. CONCLUSION: The in vitro autoradiography and ligand binding studies indicate that canine insulinomas express one type of SSTR. This is in contrast with findings in humans where, on the basis of ligand binding studies, different subtypes of SSTRs have been identified. The uniformity of SSTRs, their high frequency of expression and the high incidence of metastatic disease make canine insulinomas very suitable for investigation of the value of SRIF analogs in the diagnosis and treatment of metastasized endocrine pancreatic tumors.     

Role of GABAB receptors in the control of hypoglossal motoneurons in vivo

There is little information on GABAB receptor-mediated effects on orofacial motoneurons. We recorded the inspiratory activity from both hypoglossal (XII) nerves in urethane-anesthetized, paralyzed, vagotomized and artificially ventilated rats. A GABAB receptor agonist, baclofen, or antagonist, CGP-35348, was microinjected into one XII nucleus. Baclofen rapidly reduced the XII nerve activity in a dose-dependent manner by over 50%. The antagonist caused a delayed suppression of activity by 40%. We conclude that: (1) GABAB receptors within the XII nucleus may suppress the activity of inspiratory XII motoneurons, but they are not tonically active under the conditions of our experiment; (2) there is a net endogenous excitatory effect in XII motoneurons that is mediated by GABAB receptors located in the reticular formation surrounding the XII nucleus.     

Orchestration of neuronal migration by activity of ion channels, neurotransmitter receptors, and intracellular Ca2+ fluctuations

The real-time observation of cell movement in acute cerebellar slices reveals that granule cells alter their shape concomitantly with changes in the mode and rate of migration as they traverse different cortical layers. Although the origin of local environmental cues responsible for these position-specific changes in migratory behavior remains unclear, several signaling mechanisms involved in controlling granule cell movement have emerged. The onset of one such mechanism is marked by the expression of voltage-gated ion channels and neurotransmitter receptors in postmitotic cells prior to the initiation of their migration. Granule cells start their radial migration after the expression of N-type Ca2+ channels and the N-methyl-D-aspartate subtype of glutamate receptors on the plasmalemmal surface. Blockade of the channel or receptor activity significantly decreases the rate of cell movement, indicating that the activation of these membrane constituents provides an essential signal for the translocation of granule cells. Another signal that controls the rate of cell migration is embedded in the combined amplitude and frequency components of Ca2+ fluctuations in the somata of migrating granule cells. Interestingly, each phase of Ca2+ fluctuation controls a separate phase of saltatory movement in the granule cells: The cells move forward during the phase of transient Ca2+ elevation and remain stationary during the troughs. Consequently, the changes in the amplitude and frequency components of Ca2+ fluctuations directly affect granule cell movement: Reducing the amplitude or frequency of Ca2+ fluctuations slows down the speed of cell movement, while the enhancement of these components accelerates migration. These findings suggest that signaling molecules present in the local cellular milieu encountered on the migratory route control the shape and motility of granule cells by modifying Ca2+ fluctuations in the soma through the activation of specific ion channels and neurotransmitter receptors.     

Astrocytic messenger RNA responses to striatal deafferentation in male rat

This investigation describes the schedule and regional distribution of astrocytic responses in striatum following deafferentation by unilateral frontal cortex ablation. In the ipsilateral deafferented striatum, glial fibrillary acidic protein and clusterin (sulfated glycoprotein-2) messengerRNA showed peak elevations by 10 days postlesioning (Northern blots). Vimentin messengerRNA responded faster, with a transient elevation by three days postlesioning. The messengerRNA for glial fibrillary acidic protein, clusterin and vimentin returned toward control levels by 27 days postlesioning. However, the neuronal marker growth-associated protein messengerRNA, was decreased at all postlesion times. By in situ hybridization, the increased glial fibrillary acidic protein messengerRNA and clusterin messengerRNA signals were localized mainly to the dorsal half of the ipsilateral deafferented striatum and followed the same schedule as found by Northern blots. Glial fibrillary acidic protein messengerRNA was widely diffused in the dorsal striatum and was excluded from fascicles of the internal capsule; a similar distribution was found for glial fibrillary acidic protein-immunopositive astrocytes. While clusterin messengerRNA signal showed a distinct clustering, its immunoreactivity appeared as deposits in the deafferented striatal neuropil; Western blots confirmed the immunocytochemical results. By in situ hybridization, vimentin messengerRNA was mostly localized to the cortical wound cavity dorsal to the deafferented striatum and overlapped the distribution of vimentin-immunopositive cells. These findings suggest a coordination of striatal astrocytic messengerRNA responses with the degeneration of corticostriatal afferents. We also compared these same parameters with those from published reports on the hippocampus after deafferenting lesions. Certain astrocyte molecular responses to deafferentation are detected about five days earlier in the hippocampus than in the striatum. This different schedule in response to decortication may pertain to differences in synaptic remodeling in the hippocampus vs striatum.     

Sulpiride, but not haloperidol, up-regulates gamma-hydroxybutyrate receptors in vivo and in cultured cells

Five days of gamma-hydroxybutyrate (GHB) administration (3 x 500 mg kg(-1) day(-1) i.p.) to rats resulted in a significant decrease in the density of GHB receptors measured in the whole rat brain without modification of their corresponding affinity. Similar administration of (-)-sulpiride (2 X 100 mg kg(-1) day(-1) i.p. for 5 days) induces an up-regulation of GHB receptors without change in their dissociation constants (Kd). Haloperidol (2 X 2 mg day(-1) i.p. for 5 days) showed no effect. Administered chronically via osmotic minipumps directly into the lateral ventricles, (-)-sulpiride (60 microg day(-1) for 7 days) and GHB (600 microg day(-1) for 7 days) up-regulated and down-regulated rat brain GHB receptors, respectively. Finally, in a mouse hybridoma cell line (NCB-20 cells) expressing GHB receptors, the treatment of these cells with 1 mM GHB, 100 microM (-)-sulpiride or 1 mM GABA decreases, increases and induces no change, respectively, in the density of GHB receptors after 3 days of treatments. These results indicate that chronic GHB treatment modifies the expression of its receptor and that sulpiride also induces plastic changes in GHB receptors perhaps via antagonistic properties.     

In vivo roles of receptor tyrosine kinases and cytokine receptors in early thymocyte development

The early phases of T-cell development require both cell-cell interactions and soluble factors provided by stromal cells within the thymic microenvironment. Still, the precise nature of the signals delivered in vivo by cytokines (resulting in survival, proliferation or differentiation) remains unclear. Recent studies using mice deficient in cytokines or in their receptors have helped to identify essential signaling pathways required for the development of intrathymic precursors to mature alpha beta and gamma delta T cells. In addition, cytokine requirements for the development of natural killer cells were revealed in such mutants. The results obtained demonstrate that the development of all classes of lymphocytes (natural killer, gamma delta T cells and alpha beta T cells) is cytokine dependent, but the specific requirements differ for each lineage.     

Expression of platelet-derived growth factor ligands and receptors by rat aortic endothelium in vivo

In situ hybridization and immunostaining were used to study the time course of expression for platelet-derived growth factor (PDGF) ligands and receptors in endothelium of the rat aorta after injury. The PDGF-A and -B chains were expressed in endothelial cells at the wound edge within 4 h after injury, but no expression was detectable in uninjured endothelium. PDGF alpha-receptor was expressed in a pattern similar to the PDGF-A chain, while expression of PDGF beta-receptor was not detected at any time. Expression of the PDGF-B chain remained elevated in endothelial cells at the leading edge even at later measurements when these cells had stopped replicating. Smooth muscle cells (SMCs), which are absent from the intima of the normal aorta and are known to express PDGF beta-receptors, were predominantly found to migrate into the intima near the endothelial leading edge where PDGF-B was expressed. These data suggest a paracrine role for endothelial PDGF in SMC migration.     

Roles of neurotransmitter receptors in behavior: Recovery of function following decreases in muscarinic receptor density induced by cholinesterase inhibition.

Cholinergic neurotransmitter levels were elevated in rat brain by reducing its inactivating enzyme, acetylcholinesterase (AChE), with an anti-AChE agent. Elevated levels result in decreases in cholinergic (muscarinic) receptors. Withdrawal of agent after 10 days of chronic treatment began a gradual return of neurochemical variables toward normal states, yet not fully achieving them within the following 29 days of the experiment. All behavioral and physiological variables measured showed significant effects at the start of the treatment period, developing tolerance at different rates as treatments continued. They also recovered differentially during withdrawal. Results are consistent with a theoretical model in which thresholds for normal functioning of different behavioral and physiological processes are associated with different receptor densities. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Glial heterogeneity and developing neurotransmitter systems

Evidence from the developing serotonergic system provides support for the working hypothesis that an important function of receptors expressed by developing glia may be to mediate the effects of neurotransmitters on growth and differentiation of the neurons that utilize them, by regulating the production of glial-derived neurotrophic factors. Regional heterogeneity of glial expression may reflect the specific roles played by these receptors within different compartments of a developing neurotransmitter system. During the construction of neural pathways, the specificity of these neurotransmitter-mediated neuronal-glial interactions would require appropriate spatiotemporal expression of glial receptors and neurotrophic factors, as well as specificity of these factors for neurons of appropriate neurotransmitter phenotypes.