Negligible In Vitro Recovery of Macromolecules from Microdialysis Using 100 kDa Probes and Dextran in Perfusion Fluid.

Microdialysis is applied in neurointensive care to monitor cerebral glucose metabolism. If recoverable, macromolecules may also serve as biomarkers in brain disease and provide clues to their passage across the blood-brain barrier. Our study aimed to investigate the in vitro recovery of human micro- and macromolecules using microdialysis catheters and perfusion fluids approved for clinical use. In vitro microdialysis of a bulk solution containing physiological or supraphysiological concentrations of glucose, lactate, pyruvate, human IgG, serum albumin, and hemoglobin was performed using two different catheters and perfusion fluids. One had a membrane cut-off of 20 kDa and was used with a standard CNS perfusion fluid, and the other had a membrane cut-off of 100 kDa and was perfused with the same solution supplemented with dextran. The flow rate was 0.3 µl/min. We used both push and push-pull methods. Dialysate samples were collected at 2-h intervals for 6 h and analyzed for relative recovery of each substance. The mean relative recovery of glucose, pyruvate, and lactate was > 90% in all but two sets of experiments. In contrast, the relative recovery of human IgG, serum albumin, and hemoglobin from both bulk solutions was below the lower limit of quantification (LLOQ). Using a push-pull method, recovery of human IgG, serum albumin, and hemoglobin from a bulk solution with supraphysiological concentrations were above LLOQ but with low relative recovery (range 0.9%-1.6%). In summary, exchanging the microdialysis setup from a 20 kDa catheter with a standard perfusion fluid for a 100 kDa catheter with a perfusion solution containing dextran did not affect the relative recovery of glucose and its metabolites. However, it did not result in any useful recovery of the investigated macromolecules at physiological levels, either with or without a push-pull pump system.

Therapeutic potential of metabotropic glutamate receptor modulators.

Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and is a major player in complex brain functions. Glutamatergic transmission is primarily mediated by ionotropic glutamate receptors, which include NMDA, AMPA and kainate receptors. However, glutamate exerts modulatory actions through a family of metabotropic G-protein-coupled glutamate receptors (mGluRs). Dysfunctions of glutamatergic neurotransmission have been implicated in the etiology of several diseases. Therefore, pharmacological modulation of ionotropic glutamate receptors has been widely investigated as a potential therapeutic strategy for the treatment of several disorders associated with glutamatergic dysfunction. However, blockade of ionotropic glutamate receptors might be accompanied by severe side effects due to their vital role in many important physiological functions. A different strategy aimed at pharmacologically interfering with mGluR function has recently gained interest. Many subtype selective agonists and antagonists have been identified and widely used in preclinical studies as an attempt to elucidate the role of specific mGluRs subtypes in glutamatergic transmission. These studies have allowed linkage between specific subtypes and various physiological functions and more importantly to pathological states. This article reviews the currently available knowledge regarding the therapeutic potential of targeting mGluRs in the treatment of several CNS disorders, including schizophrenia, addiction, major depressive disorder and anxiety, Fragile X Syndrome, Parkinson's disease, Alzheimer's disease and pain.

Distinct electrophysiological properties of glutamatergic, cholinergic and GABAergic rat septohippocampal neurons: novel implications for hippocampal rhythmicity.

The medial septum-diagonal band complex (MSDB) contains cholinergic and non-cholinergic neurons known to play key roles in learning and memory processing, and in the generation of hippocampal theta rhythm. Electrophysiologically, several classes of neurons have been described in the MSDB, but their chemical identity remains to be fully established. By combining electrophysiology with single-cell RT-PCR, we have identified four classes of neurons in the MSDB in vitro. The first class displayed slow-firing and little or no Ih, and expressed choline acetyl-transferase mRNA (ChAT). The second class was fast-firing, had a substantial Ih and expressed glutamic acid decarboxylase 67 mRNA (GAD67), sometimes co-localized with ChAT mRNAs. A third class exhibited fast- and burst-firing, had an important Ih and expressed GAD67 mRNA also occasionally co-localized with ChAT mRNAs. The ionic mechanism underlying the bursts involved a low-threshold spike and a prominent Ih current, conductances often associated with pacemaker activity. Interestingly, we identified a fourth class that expressed transcripts solely for one or two of the vesicular glutamate transporters (VGLUT1 and VGLUT2), but not ChAT or GAD. Some putative glutamatergic neurons displayed electrophysiological properties similar to ChAT-positive slow-firing neurons such as the occurrence of a very small Ih, but nearly half of glutamatergic neurons exhibited cluster firing with intrinsically generated voltage-dependent subthreshold membrane oscillations. Neurons belonging to each of the four described classes were found among septohippocampal neurons by retrograde labelling. We provide results suggesting that slow-firing cholinergic, fast-firing and burst-firing GABAergic, and cluster-firing glutamatergic neurons, may each uniquely contribute to hippocampal rhythmicity in vivo.

Endogenous neurotensin down-regulates dopamine efflux in the nucleus accumbens as revealed by SR-142948A, a selective neurotensin receptor antagonist.

SR-142948A belongs to the second generation of potent, selective, non-peptide antagonists of neurotensin receptors. It was used to investigate the role of endogenous neurotensin in the regulation of dopamine efflux in the nucleus accumbens and striatum of anaesthetized and pargyline-treated rats. All the data were obtained using in vivo electrochemistry. Electrically evoked (20 Hz, 10 s) dopamine efflux was monitored by differential pulse amperometry, whereas variations in basal (tonic) dopamine efflux were monitored by differential normal pulse voltammetry. Like the first-generation compound SR-48692, SR-142948A did not affect the tonic and evoked dopamine efflux, but dose-dependently enhanced haloperidol (50 microg/kg, i.p.) induced facilitation of the electrically evoked dopamine release in the nucleus accumbens. In contrast to SR-48692, SR-142948A dose-dependently potentiated haloperidol (50 microg/kg, i.p.) induced increase in the basal dopamine level in the nucleus accumbens. This potentiating effect did not appear in the striatum. When dopaminergic and/or neurotensinergic transmissions were modified by a higher dose of haloperidol (0.5 mg/kg, i.p.), apomorphine, amphetamine or nomifensine, SR-142948A pre-treatment affected only the effect of apomorphine on the basal dopamine level in the nucleus accumbens. These results strengthen the hypothesis that endogenous neurotensin could exert a negative control on mesolimbic dopamine efflux.

Insulin-like growth factor-I and its receptor in the frontal cortex, hippocampus, and cerebellum of normal human and alzheimer disease brains.

Assimilated evidence indicates that the neurotoxic potential of amyloid beta (Abeta) peptide and an alteration in the level of growth factor(s) may possibly be involved in the loss of neurons observed in the brain of patients suffering from Alzheimer disease (AD), the prevalent cause of dementia in the elderly. In the present study, using receptor binding assays and immunocytochemistry, we evaluated the pharmacological profile of insulin-like growth factor-I (IGF-I) receptors and the distribution of IGF-I immunoreactivity in the frontal cortex, hippocampus, and cerebellum of AD and age-matched control brains. In control brains, [(125)I]IGF-I binding was inhibited more potently by IGF-I than by Des(1-3)IGF-I, IGF-II or insulin. The IC(50) values for IGF-I in the frontal cortex, hippocampus, and cerebellum of the normal brain did not differ significantly from the corresponding regions of the AD brain. Additionally, neither K(D) nor B(max) values were found to differ in the hippocampus of AD brains from the controls. At the regional levels, [(125)I]IGF-I binding sites in the AD brain also remained unaltered compared to the controls. As for the peptide itself, IGF-I immunoreactivity, in normal control brains, was evident primarily in a subpopulation of astrocytes in the frontal cortex and hippocampus, and in certain Purkinje cells of the cerebellum. In AD brains, a subset of Abeta-containing neuritic plaques, apart from astrocytes, exhibit IGF-I immunoreactivity. These results, taken together, suggest a role for IGF-I in compensatory plasticity and/or survival of the susceptible neurons in AD brains.

Comparative effects of neurotensin, neurotensin(8-13) and [D-Tyr(11)]neurotensin applied into the ventral tegmental area on extracellular dopamine in the rat prefrontal cortex and nucleus accumbens.

Ejections of 10(-5)-10(-3)M neurotensin into the ventral tegmental area increased dopamine efflux measured by electrochemical approaches in the prefrontal cortex of anaesthetized rats. In the same conditions, the effects evoked on dopamine efflux by 10(-5)M neurotensin(8-13) and [D-Tyr(11)]neurotensin were different from each other and depended on the explored area: the prefrontal cortex and the caudal and rostral nucleus accumbens. In the prefrontal cortex, neurotensin(8-13) was as potent as neurotensin, whereas [D-Tyr(11)]neurotensin was ineffective. In the caudal nucleus accumbens, when considering the initial intensity of the effect, neurotensin(8-13) and neurotensin appeared more potent than [D-Tyr(11)]neurotensin. In contrast, in the rostral nucleus accumbens, neurotensin(8-13) was less potent than [D-Tyr(11)]neurotensin and neurotensin. These results support the differential involvement of two pharmacologically distinct neurotensin receptor entities on ventral tegmental area neurons in the modulation of mesolimbic and mesocortical dopaminergic activity.

Differential effects of neurotensin on dopamine release in the caudal and rostral nucleus accumbens: a combined in vivo electrochemical and electrophysiological study.

The time-course of variations in extracellular dopamine concentration following local pressure ejection of 10(-7) to 10(-3) M neurotensin into the ventral tegmental area of the rat was determined in the minute range in the nucleus accumbens by means of differential normal pulse voltammetry associated with carbon fibre electrodes. The effects of neurotensin ejection into the ventral tegmental area were further investigated on the firing activity of the corresponding dopaminergic neurons. The lowest concentration of neurotensin (10(-7) M) enhanced the extracellular dopamine concentration throughout the nucleus accumbens and stimulated the discharge activity of ventral tegmental area dopaminergic neurons. The two highest concentrations of neurotensin (10(-5) M and 10(-3) M) evoked two patterns of responses on the extracellular dopamine concentration and on the discharge activity of dopaminergic neurons. The extracellular dopamine concentration was increased above basal levels in the caudal part of the nucleus accumbens. In the rostral part, the evoked changes exhibited a multiphasic time-course characterized by a decreasing phase below baseline. The firing rate of dopaminergic neurons was either increased or decreased, depending on the neuron being tested. In fact, neurotensin ejection was always followed by an exacerbation of bursting activity, the resulting effect on the mean firing rate being related to the duration of the interburst intervals. Indeed, short interburst intervals permitted an increase in mean firing rate whereas long interburst intervals, indicative of excessive depolarization, led to a decrease in mean firing rate. These results suggest that variations in extracellular dopamine concentration evoked by neurotensin administration into the ventral tegmental area are the result of neurotensin-evoked changes in dopaminergic activity. Moreover, the differential effects evoked by high concentrations of neurotensin could be attributable to two subpopulations of ventral tegmental area dopaminergic neurons which could project differentially to the caudal and the rostral parts of the nucleus accumbens.

On the involvement of a tonic dopamine D2-autoinhibition in the regulation of pulse-to-pulse-evoked dopamine release in the rat striatum in vivo.

The dopamine overflow evoked by trains of electrical stimulation pulses applied to the ascending dopaminergic pathway was measured with continuous amperometry in the striatum of anesthetised rats. As previously observed in in vitro studies, a pulse by pulse analysis showed a fall in dopamine overflow evoked by pulses 2 to 6, compared to the response evoked by pulse 1. However, in contrast with in vitro findings, the present in vivo data showed that the dopamine receptor antagonist haloperidol i) completely reverses the fall in dopamine overflow between pulse 1 and subsequent pulses, ii) enhances the dopamine overflow elicited by pulse 1. These results suggest that in vivo, both basal and pulse-evoked dopamine overflow results in stimulation of dopamine D2-type autoreceptors and therefore in regulation of dopamine release.

Latent inhibition in conditioned emotional response: c-fos immunolabelling evidence for brain areas involved in the rat.

Latent inhibition refers to the fact that the formation of a conditioned association between a conditioned and an unconditioned stimulus is delayed by prior exposure to the conditioned stimulus. Latent inhibition is often investigated in the context of the conditioned emotional response, in which a tone serves as the conditioned and a footshock as the unconditioned stimulus. Such a paradigm was used for the present experiments in which some rats had been pre-exposed to the tone. Two hours after a subsequent exposure to the tone, c-fos immunocytochemistry was used to map activated brain areas. The density of immunoreactive neurones was measured in brain areas involved in audition, fear, stress and memory. For the basic conditioning group, pre-exposure to the tone decreased the density of labelled cells in the auditory system, areas involved in fear and stress and a number of limbic areas, namely the amygdala, the Ammon's horn of the hippocampus and the entorhinal cortex. In contrast, the density increased in three limbic areas: the dentate gyrus, the subiculum and the nucleus accumbens. Taken together, these data suggest that latent inhibition corresponds to alterations of sensory processing which renders difficult to state about the alteration of the transfers of the sensory information to structures involved in the control of emotional responses. As some brain areas show a specific increase of activity in cases of latent inhibition, further studies will investigate how the latter brain areas contribute to the other cell density alterations reported in this study and to the latent inhibition phenomenon itself.