Nandor Ludvig

Simultaneous single-cell recording and microdialysis within the same brain site in freely behaving rats: a novel neurobiological method

We present a method for performing intracerebral microdialysis in freely behaving rats while recording the firing of neurons within the dialysis site. Studying hippocampal theta cells and complex-spike cells with this technique, it has been found that: (1) when the microdialysis fluid contained only artificial cerebrospinal fluid, both types of neurons displayed normal electrical activity, (2) the simultaneous single-cell recording/microdialysis procedure could be readily performed for as long as 3 days, and (3) inclusion of drugs into the microdialysis fluid, at appropriate concentrations, caused clear changes in firing pattern. For example, microdialysis with 1% lidocaine completely abolished, whereas that with 50 mM K+ markedly increased, the neuronal electrical activity. These cellular changes developed without apparent EEG or behavioral manifestations and were reversible. In some of the experiments, the extracellular concentrations of glutamate and aspartate in the recording/dialysis site were also measured. The described method allows the extracellular environment of recorded brain cells to be manipulated by drugs delivered through the microdialysis probe and simultaneously allows determination of the neurochemical composition of that environment over a remarkably long period of time and in intact, physiologically functioning, neural network. Such studies will provide new insights into the molecular basis of neuronal activity in the brain in the context of behavior, including learning.

Single-cell recording from the brain of freely moving monkeys.

Single-cell recording from the brain of non-human primates has traditionally been performed in monkeys seated in a primate chair. However, this arrangement makes long-term recordings difficult, causes stress that may confound the data, and prevents the manifestation of natural behaviors. Extending our previous neurophysiological studies in non-human primates (Ludvig et al. Brain Res. Protocols 2000;5:75-85), we have developed a method for recording the electrical activity of single hippocampal neurons in freely moving squirrel monkeys (Saimiri sciureus). The recording sessions lasted for up to 6 h, during which the monkeys moved freely around on the walls and the floor of a large test chamber and collected food pellets. Stable action potential waveforms were readily kept throughout the sessions. The following factors proved to be critical in this study: (a) selecting squirrel monkeys for the experiments, (b) using a driveable bundle of microwires for the recordings, (c) using a special recording cable, (d) implanting the microwires into the brain without causing neurological deficits, and (e) running the recording sessions in a special test chamber. The described method allows long-term extracellular recordings from the brain of non-human primates, without the stress of chairing, during a wide range of natural behaviors. Using this model, new insights can be obtained into the unique firing repertoire of the neurons of the primate brain.

Place Cells Can Flexibly Terminate and Develop Their Spatial Firing. A New Theory for Their Function

In this study, hippocampal place cells were recorded in a behavioral paradigm previously not employed in place-cell research. Rats were exposed to the same fixed environment for as long as 8-24 h without interruption, while the firing of CA1 and CA3 place cells was monitored continuously. The first finding was that all place cells that were detected at the beginning of the recording sessions ceased to produce location-specific firing in their original firing fields within 2-12 h. This was observed despite the fact that the animals kept visiting the original firing fields, the hippocampal EEG was virtually unchanged, and the discriminated action potentials of the cells could be clearly recorded. The second finding was that some complex-spike cells that produced no spatially selective firing pattern at the beginning of the recording sessions developed location-specific discharges within 3-12 h. Thus, place cells can flexibly terminate and develop their spatial firing. even in a fixed environment and during similar behaviors, if that environment is explored continuously for a prolonged period. To explain this phenomenon, a new place-cell theory is outlined. Accordingly, the high-frequency discharges of these neurons may serve to create, under multiple extrahippocampal control and within limited periods, stable engrams for specific spatial sites in the association cortex where the cognitive map probably resides. After the creation of a stable engram, or in the absence of favorable extrahippocampal inputs, place cells may suspend their location-specific firing in the original field, and initiate the processing of another spatial site.

Manipulation of pyramidal cell firing in the hippocampus of freely behaving rats by local application of K+ via microdialysis.

In this study, microdialysis was performed in the hippocampus of freely behaving rats, and the firing of pyramidal cells, including place cells, was recorded at the site of the microdialysis probe. For 10‐min periods, the artificial cerebrospinal fluid (ACSF) in the microdialysis system was replaced with ACSF containing 50 mM K+(high K+ solution). Complementary in vitro tests determined that microdialysis with such high K+ solution produced an outflow of 5% of the perfused K+ from the microdialysis probe. Application of K+ with this method into the CA1 region significantly increased the firing of the local pyramidal cells, including place cells, during both movement and sleep. On average, K+ exposures increased the firing rate of the neurons to 306% and 448% of the control firing rate during movement and sleep, respectively. After the termination of the K+ outflow, the cells continued to discharge for 5–30 min with a significantly higher frequency than before the K+ challenge. This phenomenon also occurred in both behavioral states. During the period of enhanced firing, the out‐of‐field firing rate of the recorded place cells was dramatically increased. It was also found that during the K+ applications, otherwise silent pyramidal cells often became electrically active. The K+ ‐induced firing modifications were usually not accompanied by behavioral or EEG changes. The data raise the possibility that transient elevations in the extracellular K+ concentration contribute to the ionic/molecular processes which are responsible for plastic firing pattern modifications in hippocampus. Pharmacological manipulation of place cells with the described method offers a new strategy to understand the molecular bases of spatial memory.

The Suppressant Effect of Ethanol, Delivered via Intrahippocampal Microdialysis, on the Firing of Local Pyramidal Cells in Freely Behaving Rats

Intrahippocampal microdialysis was performed on 14 freely behaving rats, and the firing of pyramidal cells within the dialysis area was recorded. In one group of rats, the microdialysis was conducted only with artificial cerebrospinal fluid (ACSF) for 2-4 h. In this control group, the recorded neurons displayed normal firing patterns. In another group, ACSF was replaced for 30-60 min with various concentrations of ethanol to deliver this drug via the microdialysis probe into the cell recording area. Ethanol at the concentration of 5% (w/v) significantly and reversibly suppressed the firing of the recorded neurons. The marked firing rate alterations were not accompanied with apparent changes in the hippocampal EEG activity or the behavior of the rats, indicating localized drug actions. These data demonstrate for the first time that in the physiologically functioning brain, ethanol exerts principally a suppressant effect on the electrical activity of hippocampal pyramidal cells.

Delivering drugs, via microdialysis, into the environment of extracellularly recorded hippocampal neurons in behaving primates.

Hippocampal neurons in primates have been extensively studied with electrophysiological and neuroanatomical methods. Much less effort has been devoted to examining these cells with contemporary pharmacological techniques. Therefore, we modified a recently developed integrative technique (N. Ludvig, P.E. Potter, S.E. Fox, Simultaneous single-cell recording and microdialysis within the same brain site in freely behaving rats: a novel neurobiological method, J. Neurosci. Methods 55 (1994) 31-40 [9] ) for cellular neuropharmacological studies in behaving monkeys. A driveable microelectrode-microdialysis probe guide assembly was implanted stereotaxically into the left hippocampus of squirrel monkeys (Saimiri sciureus) under isoflurane anesthesia. The assembly was covered with a protective cap. After 3 weeks of postsurgical recovery and behavioral training, the experimental subject was seated in a primate chair. For 4-5 h, single-cell recording and microdialysis were simultaneously performed in the hippocampal implantation site. The technique allowed the recording of both complex-spike cells and fast-firing neurons without the use of head restraint. The control microdialysis solution, artificial cerebrospinal fluid (ACSF), was replaced with either 1 M ethanol or 500 microM N-methyl-D-aspartate (NMDA) for 10-30 min intervals. The ethanol perfusions principally suppressed the firing of the neurons in the dialysis area. The NMDA perfusions initially increased the firing of local neurons, then caused electrical silence. These drug delivery/cell recording sessions were performed with 1-4 day intersession intervals over a 1-month period. The described method provides a tool to elaborate the pharmacology of primate hippocampal neurons during behavior and without the confounding effects of systemic drug administrations.

Cellular electrophysiological changes in the hippocampus of freely behaving rats during local microdialysis with epileptogenic concentration of N-methyl-D-aspartate.

N-methyl-D-aspartate (NMDA) receptor dysfunctions are thought to be involved in the pathophysiology of seizures of hippocampal origin. While the cellular effects of excessive NMDA receptor stimulation have been widely studied in vitro, no data are available on the sequence of cellular electrophysiological events that follow the overstimulation of hippocampal NMDA receptors in awake, behaving subjects. Therefore, the present study addressed this problem. Intrahippocampal microdialysis with 500 microM NMDA was performed in freely behaving rats, and the electrical activity of single neurons in the dialysis area were monitored. In all recorded neurons (n = 9), regardless of their type, NMDA induced a long-lasting electrical silence preceded in most cells by a brief but robust firing rate increase. During these firing rate increases, place cells lost the spatial selectivity of their discharges, and a gradual reduction in the amplitude of the action potentials was also observed. Remarkably, electroencephalographic (EEG) seizures developed exclusively after the appearance of cellular electrical silence in the recording/dialysis site. The NMDA-induced electrophysiological changes were reversible. This study demonstrates that the combined single-cell recording-intracerebral microdialysis technique can be readily used for inducing focal epileptiform events in the hippocampus and monitoring the induced cellular electrophysiological events in behaving animals.

Evidence for the ability of hippocampal neurons to develop acute tolerance to ethanol in behaving rats

BACKGROUND The cellular mechanisms underlying acute tolerance to alcohol are unclear. This study aimed to determine whether hippocampal neurons have the ability to develop acute tolerance to alcohol in behaving rats. METHODS Intrahippocampal microdialysis was performed in freely behaving rats, and the firing of single neurons in the dialysis area was recorded. The control microdialysis fluid, artificial cerebrospinal fluid (ACSF), was replaced with 1 M ethanol in ACSF for a 30 min period. One hour later, the ethanol perfusion was repeated. To test the functional integrity of the microdialysis probe in situ, each microdialysis session was completed with recording the effect of a 10-20 min perfusion of 500 microM N-methyl-D-aspartate (NMDA). The extracellular concentration profile of ethanol during intrahippocampal microdialysis with 1 M ethanol was estimated in a separate study in anesthetized rats. The ethanol content was measured in tissue slices surrounding the probe with gas chromatography (GC), and the generated data were analyzed with a mathematical model for microdialysis to estimate the concentration of ethanol at the recording site. RESULTS The predominant effect of the first intrahippocampal microdialysis with ethanol was a decrease in firing rate in both pyramidal cells and interneurons. In contrast, such firing rate decrease did not develop during the second ethanol perfusion. Subsequent NMDA perfusion still induced robust changes in the electrical activity of the neurons. The estimated extracellular ethanol concentration at the recording site was 45-70 mM. CONCLUSION This study revealed that hippocampal neurons have the ability to develop acute tolerance to a single exposure of clinically relevant concentrations of ethanol in behaving rats, without influences from the rest of the body.

Microdialysis-coupled place cell detection in the hippocampus: A new strategy for the search for cognition enhancer drugs

1. The MPCD method in freely moving rats is a new neuroscience technique. It is able to detect the location-specific firing of hippocampal place cells, and to deliver, via microdialysis, various drug solutions into the extracellular environment of the detected neurons. Place cells are critical elements of the neural system in brain which governs cognitive processes. It is emphasized in this article that effective cognition enhancer drugs must selectively and significantly affect the firing of these cells. 2. By using MPCD, it is possible to recognize drug combinations which can increase the location-specific firing of place cells to an optimal level. This paper proposes that such pharmacological action facilitates engram-creation in extrahippocampal cortical areas, improving cognitive functions. Thus, an MPCD-based research strategy may lead to the rational development of a new generation of cognition enhancer drugs for the treatment of learning and memory disorders, including Alzheimers disease (AD).