Michael R. Palmer

Improved ceramic-based multisite microelectrode for rapid measurements of L-glutamate in the CNS.

This paper describes improvements and further characterization of a ceramic-based multisite microelectrode for in vivo measurements of L-glutamate. Improvements include increased recording area, insulation deposition using photolithography for more uniform recording sites and forming the microelectrodes using a diamond saw providing smoother microelectrode edges. The new microelectrodes are triangular in shape, 1 cm in length and taper from 1 mm to a 2-5 microm tip. Details on performing in vivo measurements are given, including microelectrode preparation, pitfalls of the recording method and approaches to enhance reproducibility of the technique. The detection limit for L-glutamate was lowered to approximately 0.5 microM and a self-referencing recording technique was utilized to remove interferents as well as decrease noise. Applications of the microelectrodes to study L-glutamate uptake and release in rat prefrontal cortex, cortex, cerebellum and striatum are included.

Glial cell line-derived neurotrophic factor improves survival of ventral mesencephalic grafts to the 6-hydroxydopamine lesioned striatum

Abstract One approach to replace lost dopaminergic neurons in Parkinson’s disease is to transplant fetal mesencephalic tissue into the striatum. In an attempt to expand the developmental window useful for grafting of mesencephalic tissue and increase the fiber outgrowth from grafted dopaminergic neurons, we have pretreated fetal mesencephalic tissue with the dopaminotrophic factor glial cell line-derived neurotrophic factor (GDNF). Mesencephalic tissue pieces from embryonic day 18–19 Fischer 344 rats were preincubated for 20 min with GDNF (1 μg/μl) or vehicle. Two tissue pieces were then transplanted into the striatum of rats that had been unilaterally lesioned by medial forebrain bundle injections of 6-hydroxydopamine. The animals were tested for apomorphine-induced rotations prior to intracranial grafting. Host rats received intrastriatal injections of 10 μg GDNF or control solution at 10 days and 4 weeks postgrafting. The animals were tested in the rotometer twice monthly following transplantation. Despite the fact that these transplants were from a suboptimal donor stage, the rotations were significantly decreased in both transplanted groups. Immunohistochemical evaluation of the host brains revealed that the overall size of transplanted mesencephalic tissue was significantly increased in the GDNF-treated animals, and that the average size of transplanted tyrosine hydroxylase (TH)-positive neurons was also increased. Furthermore, we found that the innervation density of surrounding host striatal tissue was significantly increased in the GDNF-treated group, as compared with controls. Taken together, these results suggest that treatment of intrastriatal ventral mesencephalon grafts with GDNF can optimize the conditions for intracranial grafting and thus improve the chances for functional recovery following the intrastriatal grafting procedure.

Characterization of the techniques of pressure ejection and microiontophoresis using in vivo electrochemistry.

Catecholamine levels in frontal cortex were determined in vivo by electrochemical detection after the local application of dopamine (DA) from multibarrel micropipettes by pressure ejection or microiontophoresis. Tissue DA levels were linearly related to microapplication doses with either technique and reached steady state with longer application times. Furthermore, the plateau DA tissue concentrations were clearly related to ejection pressure or iontophoretic current. Using either microapplication technique, the tissue DA levels decreased as distance between the recording electrode and the tip of the drug pipette increased. However, pressure ejected and iontophoretically applied drug differed in their concentration versus time dynamics. Thus, although similar tissue concentrations of drug can be generated by the two techniques, the time dynamics of the drug effects may not be comparable. The quantitative use of these drug application techniques requires a minimal amount of variance in release between pipettes in order to effectively measure small sensitivity differences. Although the 10-fold variance with microiontophoresis does not appear resolvable at present, improved pipette construction techniques permit the variability in dosage to be limited to a maximum of 3-fold with pressure ejection. In addition, the present data also suggest that this variance can be further minimized by holding either ejection duration or ejection pressure constant when establishing dose-response relationships.

Multiple single-unit recordings in the striatum of freely moving animals: effects of apomorphine and D-amphetamine in normal and unilateral 6-hydroxydopamine-lesioned rats.

Ensembles of striatal neurons were recorded in freely moving normal and unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats using chronically implanted electrode arrays. Animals received bilateral striatal implants of two 16-microwire arrays 1 week before recordings. Identified striatal neurons were categorized as medium spiny-like and large aspiny-like based on a combination of their activity autocorrelations and firing rates. Baseline firing rates of medium spiny-like neurons in the 6-OHDA-lesioned striata were significantly faster than were firing rates of the same neurons in the intact hemispheres of 6-OHDA-lesioned rats or normal animals. However, firing rates of large aspiny-like neurons were faster in both hemispheres of the 6-OHDA-lesioned rats as compared to normal animals. Interestingly, firing rates of neurons in all groups decreased by fivefold or greater under urethane anesthesia, although the relative firing rates between hemispheres were unchanged. d-Amphetamine (5.0 mg/kg, s.c.) increased the firing rates of both types of striatal neurons by twofold or greater in normal rats and in the intact hemispheres of 6-OHDA-lesioned animals. By contrast, this treatment did not alter neuron firing in the 6-OHDA-lesioned striata. Apomorphine (0.05 mg/kg, s.c.) did not affect neuronal firing rates either in normal rat striatum or in the unlesioned hemispheres of 6-OHDA-lesioned animals. However, it did significantly increase the firing rate of the medium spiny-like neurons in 6-OHDA-lesioned striata. These results demonstrate that the dopaminergic innervation of the striatum differentially influences two electrophysiologically distinct sets of striatal neurons in freely moving rats.

Hippocampal noradrenergic responses in vivo and in vitro

SummaryPressure ejection of l-norepinephrine (NE) in the in vivo rat hippocampus generally produced depression of pyramidal cell spontaneous activity. In addition, both excitation and biphasic responses were observed. NE-induced inhibition of firing rate was effectively antagonized by concurrent administration of the alpha antagonist phentolamine, but was largely unaltered by the beta antagonist timolol. On the other hand, NE-induced elevation in spontaneous firing rate was effectively blocked by timolol, and largely unaffected by phentolamine. Another beta antagonist, sotalol, did not selectively antagonize either NE-induced inhibition or NE-induced excitation. The beta agonist 2-fluoro-NE produced increases in pyramidal cell firing rates in most cells studied, while the alpha agonist 6-fluoro-NE inhibited the majority of cells examined. The effects of sotalol were also examined on alpha and beta receptor-mediated field responses in the in vitro hippocampal slice. Sotalol was shown to be a selective beta antagonist in this system, blocking excitation evoked by the beta agonist isoproterenol while having no effect on inhibition elicited by the alpha agonist clonidine; however, the potency of sotalol (Ki=3.5μM) was considerably less than that of timolol (Ki=50 nM). Taken together, these results suggest that NE-induced depression and elevation in hippocampal pyramidal cell spontaneous discharge in vivo are mediated via alpha and beta adrenoceptors, respectively.

Leukotriene C elicits a prolonged excitation of cerebellar Purkinje neurons

Leukotriene C-1 (LTC-1), a recently characterized potent agonist in bioassays for the slow reacting substance of anaphylaxis (SRS-A), was tested for electrophysiological effects on central neurons. LTC-1 consistently elicited a unique prolonged excitatory action on cerebellar Purkinje (P) cells when administered locally by pressure ejection from a multibarreled micropipette. This excitation could be reversed by subsequent pressure ejection of FPL 55712 (FPL), a SRS-A end-organ antagonist. Prior application of FPL prevented the LTC-1-induced responses altogether. Heat-inactivated LTC-1 had no effect on P cell discharge. These data suggest that LTC-1, as has been recently found with many other peripherally acting neurohormones, may also function to regulate excitability of central neurons.

ETHANOL POTENTIATION OF GABA-INDUCED ELECTROPHYSIOLOGICAL RESPONSES IN CEREBELLUM : REQUIREMENT FOR CATECHOLAMINE MODULATION

In this study, we confirmed that microiontophoretically applied norepinephrine (NE) and isoproterenol potentiate the depressant effects of locally-applied gamma-aminobutyric acid (GABA) on cerebellar Purkinje neurons of anesthetized rats. Although ethanol (EtOH) does not reliably or efficaciously potentiate GABA-induced depressions of neuronal activity, we found that systemic or locally-applied EtOH does markedly potentiate GABA-induced inhibitions of Purkinje neuron firing rate if that response is concomitantly modulated by NE or isoproterenol. This study suggests that the EtOH sensitivity of the GABA mechanism of electrophysiological responses in the cerebellar cortex is regulated by the neuromodulatory effect of beta-adrenergic receptor activation.

Electrophysiological responses to adenosine analogs in rat hippocampus and cerebellum: Evidence for mediation by adenosine receptors of the A1 subtype

Adenosine has profound depressant effects upon the electrophysiological activity of the brain, but the adenosine receptor subtypes which mediate these responses are uncertain. In order to resolve this question, we have characterized the effects of two adenosine analogs which differ in their relative potencies at adenosine A1 and A2 receptors. The effects of these adenosine analogs were examined on spontaneous firing rate of Purkinje neurons in the rat cerebellum in situ, in cerebellar brain slices in vitro, and on synaptic transmission in the rat hippocampus in vitro. Although the A2 agonist appeared to be more potent with local drug application techniques in situ, our in vitro results suggest that the A1 receptor subtype is involved in the electrophysiological actions of these drugs in both rat cerebellum and hippocampus. Furthermore, these data indicate that the physical properties of some adenosine analogs may reduce apparent drug potencies when they are studied with local application techniques.

Differential electrophysiological and behavioral responses to optically active derivatives of phencyclidine

SummaryDextro-and levorotatory isomers of 1-(1-phenylcyclohexyl)-3-methylpiperidine (PCMP) were synthesized. Both isomers inhibited spontaneous cerebellar Purkinje neuron firing when applied locally by pressure ejection. This effect was dose-dependent, with the (+)-isomer about 5–7 times more potent than the (−)-isomer. Both isomers also depressed rotarod performance in mice. Again, the (+)-isomer was about 5 times more potent than the (−)-isomer. Both rotarod performance and Purkinje cell discharge were depressed maximally 10–15 min after i.p. injection of drug. Our results suggest a correlation between behavioral performance and central neuron electrophysiological activity and suggest that the central actions of PCP or its derivatives are probably mediated at one locus, by a stereospecific mechanism.

Chapter 7 Grafts, growth factors and grafts that make growth factors

Publisher Summary This chapter presents an in vivo screening system based upon the intraocular grafting of fetal brain tissue, which can be used to monitor temporal and regional specificity of growth factor effects. It focuses on the most well-known nerve growth stimulating factor, nerve growth factor (NGF), and describes its presence in the central nervous system (CNS) as well as the fate of intraparenchymally injected NGF. The fate of NGF injected directly into brain parenchyma as monitored by immunohistochemical techniques is described in the chapter. Genetically engineered fibroblasts secreting large amounts of NGF that have been grafted to the CNS explain the way such cells can support surrounding cholinergic systems and rescue axotomized cholinergic neurons. The functions of NGF in the CNS might extend beyond the current set of cholinergic neurons. Thus, it is conceivable that NGF sensitivity is more widespread during development.