Introduction

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Recent studies directed at understanding the influence of network events on neuronal membrane properties in the central nervous system have, in large part, been carried out using in vitro preparations. Although these isolated preparations are useful for studying the synaptic pharmacology and membrane biophysics of neurochemically and/or visually identified neurons, extrapolation of observations made in vitro to the intact adult system is often problematic. In addition to the potential caveats related to the impact of the specific physical-chemical conditions used in the in vitro preparation on the viability or membrane biophysics of neurons, the disconnection of the neuron from its extrinsic inputs can have a significant impact on the steady-state properties of the neuronal membrane. For example, spiny projection neurons recorded in vivo in the cortex or striatal complex often exhibit characteristic shifts in membrane potential consisting of "up" (depolarized plateau potential between 65 and 48 mV) and "down" (resting potential between 88 and 75 mV) states (Steriade et al., 1993a,b; Wilson, 1993; O'Donnell and Grace, 1995; Wilson and Kawaguchi, 1996; Paré et al., 1998a,b; Onn and Grace, 1999, 2000; West and Grace, 2002). In both the cerebral cortex and the striatum, the up state is driven by glutamatergic inputs (Mahon et al., 2001). In the striatum, neocortical (dorsal striatum; Wilson, 1993; Mahon et al., 2001) and allocortical (ventral striatum; O'Donnell and Grace, 1995) and/or thalamic (Wilson, 1993) afferents seem to interact to produce the up state. In the cortex the up state is primarily dependent upon synchronous activation of cortico-cortical synaptic connections (Steriade et al., 1993b; Cowan and Wilson, 1994; Amzica and Steriade, 1995; Silberstein, 1995). Consistent with these findings, striatal and cortical neurons recorded in vitro in brain slices do not exhibit bistable membrane activity (Nicola et al., 2000; Lavin and Grace, 2001). Paré and colleagues have further shown that, in addition to being necessary for spontaneous action potential discharge, the tonic activity of synaptic inputs to cortical pyramidal neurons in vivo is sufficient to maintain a membrane input resistance significantly lower than that observed in vitro, consequently altering other "passive" membrane properties such as excitability (Paré et al., 1998b). Together, these studies indicate that the synaptic activity occurring throughout the dendritic tree of the spiny neurons of the cortex and striatum not only sets the natural firing pattern of these neurons but also has a significant impact on the response of the neuronal membrane to the activation of ligand-gated ion channels. At present, however, little is known about the influence of local neurotransmitters or synaptic activity on the steady-state membrane properties of spiny neurons in vivo.

Given the above-mentioned information, it is likely that the influence of a specific local receptor population on neuronal excitability and spontaneous activity will depend on the ongoing activity within circuits that provide synaptic inputs to the neuron. Thus, studies investigating the influence of local neurotransmitter interactions on neuronal activity in intact systems are critical for understanding the influence of network events on the membrane properties of spiny neurons, as well as the modulation of their activity by local receptor stimulation. Toward this end, the current study was undertaken to determine the viability of the use of local microdialysis for temporally and spatially controlled delivery of pharmacological agents during intracellular recordings in vivo. The potential impact of the microdialysis procedure on the membrane properties and synaptic responses of striatal and cortical neurons was assessed by comparing recordings made in control animals (no probe) to recordings from neurons located within 500 µm of a microdialysis probe continuously perfused with artificial cerebral spinal fluid (aCSF). To test the validity of combining the techniques of intracellular recording and microdialysis in vivo, the influence of local pharmacological manipulations of glutamatergic and GABA-ergic receptors on the membrane properties of cortical or striatal neurons was examined. The effect of eliminating the majority of synaptic input via local perfusion of tetrodotoxin (TTX) on the spontaneous activity and membrane properties of cortical neurons was also assessed.

	Experimental Procedures

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Materials. Dulbecco's phosphate-buffered saline, N-methyl-D-aspartate (NMDA), glutamic acid, and tetrodotoxin (TTX) were purchased from Sigma-Aldrich (St. Louis, MO). ()-Bicuculline (BIC) methochloride was purchased from Sigma/RBI (Natick, MA). D-Glucose was purchased from Fisher Scientific (Springfield, NJ). All other reagents were of the highest grade commercially available.

Subjects and Surgery. Male Sprague-Dawley or Fischer 344 rats (Hilltop, Scottdale, PA) weighing 275 to 450 g were used. Before experimentation, animals were housed two per cage under conditions of constant temperature (21-23°C) and maintained on a 12-h light/dark cycle with food and water available ad libitum. All animal procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and adhere to the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Additionally, all efforts were made to minimize the number of animals used and their suffering. Before surgery, animals were deeply anesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a stereotaxic apparatus (Narishige, Tokyo, Japan or David Kopf Instruments, Tujunga, CA). The level of anesthesia was periodically verified via the hind limb compression reflex and maintained using supplemental administration of chloral hydrate (80 mg/ml) via a lateral tail vein (approximately 0.2 ml/0.5 h). Temperature was monitored using a rectal probe and maintained at 36-37°C with a heating pad (Fintronics, Orange, CT).

View larger version (58K): [in this window] [in a new window]   Fig. 1.   In vivo intracellular recordings from identified spiny neurons recorded proximal to the microdialysis probe. a, schematic of the implanted microdialysis probe and recording electrode in the PFC. Electrodes and microdialysis probes were stereotaxically implanted at a rate of 3 to 6 µm/s using a micromanipulator. After implantation, probes were perfused with aCSF (2 µl/min) and allowed to equilibrate in the brain for 2 to 4 h before the initiation of recording. Baseline recordings of stable neurons located proximal to the dialysis probe were made for approximately 10 min during aCSF infusion before the perfusate was switched to a drug-containing solution. b, location of dialysis probe track and biocytin-labeled neuron in the PFC. Top, photomicrograph showing a coronal section of the prefrontal cortex. The dotted outline marks the position of the probe in and up to 500 µm rostral to this section. Damage produced by the probe appears as a tear in the tissue with blood accumulation adjacent to the neuron (arrow) and at the tip of the probe outline. The apical dendrite of a PFC pyramidal neuron extends from right of the arrow toward the midline. Bottom, higher magnification photographic reconstruction of the biocytin-filled PFC pyramidal neuron located near the tip of the arrow in the top panel. Arrows in the top and bottom panels mark the same approximate location in the section. c, physiological recordings from the PFC neuron shown in b. The neuron exhibited spontaneous electrophysiological activity (left, resting membrane potential = 71 mV) and spikes evoked by intracellular injection of depolarizing current pulses (0.6, 1.2 nA) that were characteristic of regular-spiking pyramidal neurons of the cerebral cortex (Connors and Gutnick, 1990). d, schematic representation of the orbital frontal cortical stimulation site (left) and position of the microdialysis probe and recording electrode in the central striatum (right). e, coronal section showing a photomontage of a striatal neuron (solid arrow) labeled after intracellular biocytin injection, magnified in the inset (right). The striatal neuron was located proximal to the active zone of the microdialysis probe (termination point of the probe track indicated by the dashed arrow). D, dorsal; M, medial; cc, corpus callosum. f, electrophysiological activity of the striatal neuron shown in e. The neuron exhibited electrophysiological activity characteristic of medium spiny projection cells under basal conditions (aCSF infusion; resting membrane potential = 82 mV) and responded to pairs of electrical stimuli (1 mA, 200 ms, 0.2 Hz) delivered to the orbital prefrontal cortex. Additionally, the neuron exhibited an enhanced response to the second stimulus and was driven to spike discharge if depolarized with constant positive current (0.8 nA). Asterisks indicate the stimulation artifacts.

Intracellular Recordings. Intracellular electrodes were pulled from 1.0-mm-o.d. borosilicate glass tubing (World Precision Instruments, Sarasota, FL) using a Flaming-Brown P-80/PC electrode puller. Microelectrodes were filled with potassium acetate (2-3 M) solution containing 2% biocytin using a nonmetallic Microfil syringe needle. Intracellular electrodes used for cortical recordings had impedances of 50 to 90 M, whereas electrodes used for striatal recordings ranged from 30 to 100 M as measured in situ. Electrode potentials were amplified via a headstage connected to a Neurodata IR-183 intracellular preamplifier (Cygnus Technology, Delaware Water Gap, PA). Intracellular current was injected via an active bridge circuit integral to the preamplifier. Continuous or event-triggered collection of data of both voltage and current signals from the amplifier were digitized and stored onto a PC via a Microstar data acquisition board interface (Microstar Laboratories, Bellevue, WA) controlled by custom software (Neuroscope; Brian Lowry, Pittsburgh, PA). Output from the amplifier was simultaneously monitored on a Philips PM3337 storage oscilloscope (Fluka, Eindhoven, The Netherlands), digitized (NeuroData NeuroCorder DR 390; Cygnus Technology), and stored on videotape. Cell penetrations were defined as stable when the cells exhibited a resting membrane potential of at least 55 mV; discharged action potentials having amplitudes of at least 45 mV (range 48-82 mV), a positive overshoot; and fired trains of spikes after membrane depolarization. Data were collected for cells defined as stable when these electrophysiological properties were maintained for a minimum period of 5 min. After experimental manipulations, neurons were injected (~10-60 min) with biocytin using a train of depolarizing current injection pulses (~0.5 nA, 300 ms, 2 Hz).

Electrical Stimulation. In striatal experiments, twisted-pair bipolar stimulating electrodes (Plastics One, Roanoke, VA) were implanted into the orbital prefrontal cortex (coordinates: 3.7-4.7 mm anterior to bregma, 0.2-2.3 mm lateral to midline, 2.5-4.0 mm ventral to brain surface) ipsilateral to the recording electrode. Stimulation sites in the medial, ventral, and ventrolateral orbital PFC were selected based on the results of striatal retrograde and anterograde tracing studies (Deniau et al., 1996). Single pulses or pairs of electrical stimuli (100-ms interspike interval) with durations of 200 to 250 µs and intensities between 0.1 and 5.0 mA were generated using an S88 stimulator (Grass Instruments, Quincy, MA) and photoelectric constant current/stimulus isolation unit (PSIU6F; Grass Instruments) and delivered at a frequency of 0.2 Hz.

Procedure for Intracellular Recording during Local Pharmacological Manipulations via Microdialysis. Electrophysiological recordings were initiated approximately 2 to 4 h after probe implantation (Fig. 1). Electrode tips were positioned to enter the brain surface approximately 1 mm lateral or caudal to the probe, and angled at 10° toward the probe. The electrode was then lowered and neurons were impaled at coordinates lying within 500 µm of the active membrane of the probe. After impaling a neuron, the neuron was allowed to stabilize for several minutes until synaptic and/or spike activity reached a steady state. Baseline synaptic activity was then recorded for at least 5 min after which the effects of intracellular injection of hyperpolarizing and depolarizing currents were determined. After steady-state activity and membrane properties were recorded, the aCSF perfused through the probe was switched to an aCSF containing glutamate (500 µM), NMDA (200 µM), BIC (100 µM), or TTX (10 µM) using a zero dead-volume liquid switch (CMA/Microdialysis or Bioanalytical Systems). Due to the recovery of the probes, the concentration of drug in the tissue immediately adjacent to the probe was estimated to be approximately 10% (for 2-mm probes) or 25% (for 4-mm probes) of the concentration in the perfusion fluid, and substantially less at the soma of the neuron being recorded. Time was allowed for the drug to reach the active surface of the probe (dead volume, 6 to 12 µl; time, 3-6 min), after which spontaneous activity and the effects of intracellular current injection were recorded during perfusion of the drug. Effective doses of BIC, TTX, glutamate, and NMDA were derived from previous studies (Karreman and Moghaddam, 1996; West and Galloway, 1997) and were soluble in aCSF.

Data Analysis. Changes in neuronal membrane properties, synaptic activity, and spike activity were analyzed using custom software (Neuroscope; Brian Lowry). Resting membrane properties and spike characteristics (Tables 1 and 2) were determined for control neurons (no probe) and for neurons proximal to the dialysis probes before and after addition of a drug to the perfusion fluid. Baseline resting membrane potential, input resistance, and current threshold were determined from 30- to 60-s epochs taken from traces occurring after at least 5 min of stable recording and not more than 7 min before the onset of drug perfusion. Input resistance of each neuron in the "down" membrane potential state was calculated by injecting a series of hyperpolarizing and depolarizing current pulses intracellularly (150 ms, 0.1-1.5 nA) and plotting the resulting membrane deflections against the amplitude of the current pulse (Figs. 2c and 3c, right). The resulting data points were then fitted to a least-squares regression line and the input resistance was estimated from the slope of the lines. Current threshold was defined as the minimum depolarizing current required to evoke a spike. The spike threshold, spike amplitude, and spike duration were determined from the first spike evoked at each depolarizing current level. The spike threshold was visually identified as the change in slope evident at the transition from the graded depolarization to the onset of the rapid depolarizing phase of the spike. Spike amplitude was defined as the change in voltage between the spike peak and threshold. For cortical cells, spike duration was determined as the duration of the spike at a voltage midway between the spike threshold and peak. For striatal cells, spike duration was measured from the spike threshold to the point where the falling phase of the action potential returned to the membrane potential at spike threshold. To maximize our power to detect differences between neurons in control and perfused animals, individual Student's t tests were conducted on each dependent variable. Potential effects of the microdialysis procedure on the proportion of spontaneously active neurons were assessed via comparisons between control and probe groups using the Fisher's exact test.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Prefrontal cortical neurons recorded in vivo proximal to the dialysis probe exhibit identical membrane properties as neurons recorded in the nonimplanted (control) animal. a, the majority of PFC neurons recorded proximal to the dialysis probe fired spontaneously and exhibited a bistable pattern of membrane activity characterized by rapid and spontaneous transitions from a hyperpolarized state to a depolarized plateau. Inset, time-frequency histogram of the membrane potential (1-mV bins) recorded during a 30-s sampling period. Note that during the majority of the sampling time, the membrane potential fluctuated between a hyperpolarized and depolarized state. b, in the same cell, intracellular injection of 150-ms duration constant current pulses (bottom traces) induced deflections in the membrane potential (top traces). c, plot of the steady-state voltage deflections against the current pulse amplitude results in a regression line which was used to calculate the input resistance of the recorded neuron (44 M).

Histology. After experimentation, animals were deeply anesthetized and perfused transcardially with ice-cold saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Brains were postfixed in 4% paraformaldehyde in PB for 1 to 7 days then cryoprotected with 25% sucrose in PB. Brains were then sectioned into 60- to 80-µm coronal slices and processed to reveal biocytin using a standard, commercially available avidin/biotin procedure kit (Vector Laboratories, Burlingame, CA). Specifically, sections were washed 2 × 5 min in PB, 10 min in 0.2% hydrogen peroxide in PB, 2 × 5 min in PB, and 1 × 5 min in 0.2% Triton X in 0.01 phosphate-buffered saline (PBS). After these rinses sections were incubated for 1 to 10 h in the avidin/biotinylated horseradish peroxidase solution (VECTASTAIN Elite ABC at 1 drop each of reagent A and B per 5 ml of 0.2% Triton in PBS). Sections were then rinsed 2 × 5 min in PBS and 3 × 5 min in 0.05 M Tris-buffered saline (TBS, pH 7.6) and incubated in 0.4% diaminobenzidine-4 HCl/3% nickel ammonium sulfate in TBS for 10 min alone and 10 additional minutes with hydrogen peroxide added (0.05-0.1%). The reaction was quenched with TBS (3 × 5 min). Sections were mounted from water onto gelatin-coated glass slides. After drying, sections were counterstained with a mixture of neutral red/cresyl violet (8:1), dehydrated, and coverslipped. Neurons were identified as striatal medium spiny or cortical pyramidal on the basis of the morphology of the dendrites and were confirmed to lie within 500 µm of the probe track. In brains in which neurons were not filled or recovered, the recording location relative to the probe track was estimated from the 3,3-diaminobenzidine tetrachloride product that had reacted with the small amounts of blood adjacent to the electrode and microdialysis probe tracks.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

In vivo intracellular recordings were made from 29 striatal neurons in 26 rats and 27 cortical neurons in 25 rats (total = 51 rats).

Electrode and Microdialysis Probe Placement. For striatal recordings, all stimulating electrode tips implanted into the cortex were confirmed to lie in the PFC between 3.2 and 4.7 mm anterior to bregma, 0.4 and 2.2 mm lateral to the midline, and 2.8 and 4.3 mm ventral to the dural surface (Paxinos and Watson, 1986). All dialysis probe tips were confirmed to lie within the dorsal striatum between 0.1 mm posterior and 1.7 mm anterior to bregma, 2.0 and 4.5 mm lateral to the midline, and 5.0 and 7.7 mm ventral to the dural surface (Paxinos and Watson, 1986). The majority of recording electrode tracks was observed to pass and terminate within 500 µm of the observed probe track (Fig. 1d). Three biocytin-filled neurons were identified in the central and dorsolateral striatum proximal to the dialysis probe track (Fig. 1e). A fourth biocytin-filled neuron was recovered in a control animal. Three of these neurons were unequivocally identified as medium-sized spiny neurons; the remaining neuron exhibited morphological and physiological characteristics consistent with medium spiny cells.

Electrophysiological Properties and Spontaneous Activity of Striatal and Cortical Neurons Recorded in Tissue Perfused by a Microdialysis Probe. For all recordings, qualitative factors that had the greatest impact on the viability of the neurons were the rate at which the microdialysis probe had been implanted, the duration of the equilibration period, and the overall health of the animal while under anesthesia. Lowering the probe at a rate greater than 500 µm/min markedly decreased the probability of finding a healthy neuron proximal to the probe. The minimum equilibration period seemed to be approximately 2 h, with most recordings occurring more than 3 h after probe insertion. In addition, recording stability was particularly susceptible to the deleterious effects of increases in body temperature or difficulties in respiration. These problems were minimized with a combination of injections of saline through the tail vein, hydrating the air around the snout, and holding the body temperature at 36°C.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Striatal neurons recorded in vivo proximal to the dialysis probe exhibit identical membrane properties as neurons recorded in the intact (control) animal. a, most of the striatal neurons recorded proximal to the dialysis probe did not fire spontaneously but did exhibit a bistable pattern of membrane activity characterized by rapid and spontaneous transitions from a hyperpolarized state to a depolarized plateau. b, time-frequency histogram of the membrane potential (1-mV bins) recorded during a 30-s sampling period from the cell shown in a. Note that during the majority of the sampling time, the membrane potential fluctuated between a hyperpolarized and depolarized state. c, left, in the same cell, intracellular injection of 150-ms duration constant current pulses (bottom traces) induced deflections in the membrane potential (top traces). Right, plot of the steady-state voltage deflections against the current pulse amplitude results in a regression line that was used to calculate the input resistance of the recorded neuron (39 M).

Synaptically Evoked Activity in Striatal Neurons. In striatal neurons recorded in both control and probe groups, postsynaptic potentials and, in some cases, spikes could be evoked by single pulses or pairs of electrical stimuli delivered to the PFC (Fig. 4). To compare the effects of PFC stimulation on cells from control and probe groups, a series of single pulses (0.2 Hz) of electrical stimuli were delivered at gradually increasing stimulus intensities (0.1-3.0 mA). For all cells, the first excitatory postsynaptic potential (EPSP) elicited by PFC stimulation having an amplitude of at least 10 mV was analyzed. Statistical analyses of recordings from cells in control and probe groups revealed no significant differences in the average membrane potential before electrical stimulation, EPSP onset latency, amplitude, duration, or current intensity required to evoke an EPSP greater than 10 mV in amplitude (Table 3).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Synaptic responses evoked in striatal neurons recorded proximal to the microdialysis probe. a, single pulses of electrical stimulation delivered to the PFC evoked single EPSPs in a striatal neuron in a manner that was dependent on the stimulus intensity. At higher stimulus intensities (1 mA), the same striatal neuron fired an action potential. B, paired pulses of electrical stimuli (0.2 Hz, 250 µs, 1 mA) delivered to the PFC evoked EPSPs and sometimes spikes in another striatal neuron. Note that a spike is evoked by the first stimulus only when the membrane potential is in the depolarized up state.

Pharmacological Manipulations. Perfusion of TTX into the PFC increased membrane input resistance from an average of 27 ± 12 to 71 ± 10 m (mean ± S.E.M.), abolished spike activity, and markedly reduced spontaneous subthreshold depolarizations, resulting in an average membrane potential similar to that of the down state (n = 2; Fig. 5). In contrast, seven of seven PFC neurons showed a depolarization of the membrane and increase in spontaneous or current-evoked spike activity during perfusion of glutamate or NMDA via the probe (Fig. 6). In some cases, when cells could be held for a sufficient period (n = 2), the excitatory effects of glutamate or NMDA were observed to wash out after reperfusion with aCSF (Fig. 6b). Interestingly, NMDA had variable effects on input resistance (IR with aCSF = 34.8 ± 11.2 m, IR with NMDA = 26.6 ± 7.5 m; mean ± S.E.M.), seeming to have less of an effect on neurons with high (> 50) input resistances. Nonetheless, NMDA significantly decreased current threshold and increased the number of spikes evoked per unit of depolarizing current injected intracellularly (Fig. 6).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of local perfusion of TTX on PFC neuron physiology. a, left, spontaneous activity exhibited by the neuron during aCSF perfusion. This intrinsically bursting neuron displayed characteristic up and down states with action potentials that discharged during the up state. Inset, at a faster time base, it can be seen that the burst-firing pattern is characterized by spikes with gradually decreasing amplitudes, similar to that reported previously by Steriade et al. (1993a). Right, proportion of time (30-s epoch) that the neuron spent at a given membrane potential. The bimodal distribution indicates that the membrane potential was distributed primarily between two modes, one at approximately 70 mV, the other at 61 mV. b, left, membrane activity during reverse dialysis of TTX (5 min). After the addition of TTX (10 µM) to the aCSF, the bistable membrane potential pattern was depressed and action potentials were eliminated. Instead, the neuron rested in a hyperpolarized state. Right, time-membrane potential histogram shows that in the presence of TTX, the membrane potential is shifted to the left (hyperpolarized) and is distributed around a single mode at approximately 81 mV. c, left, membrane activity after 10 min of reverse dialysis of TTX. The membrane potential hyperpolarized further and depolarized plateau potentials were nearly absent. Right, time-membrane potential histogram shows that after 10 min of TTX infusion, the membrane potential had become more hyperpolarized and unimodal.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Effects of reverse dialysis of glutamate or NMDA on membrane potential and excitability of PFC neurons. a, membrane activity of a PFC neuron during perfusion (2 µl/min) of normal aCSF (left) and 3 min after the onset of reverse dialysis of glutamate (500 µM, right). b, example of changes in membrane activity in a prelimbic neuron during perfusion of glutamate (GLU; 200 µM) through the microdialysis probe and after washout. Left, examples of traces of spontaneous membrane activity at baseline (aCSF), 4 and 8 min after the addition of GLU to the perfusion fluid, and 16 min after the switch back to GLU-free aCSF. Action potentials are truncated. Right, effects of GLU perfusion (8 min) and subsequent washout period (16 min) on resting membrane potential (MP; top) and frequency of up states (bottom). Up states were defined as depolarizing shifts in membrane potential exceeding 7 mV, with a duration of at least 100 ms. The membrane potential depolarized and frequency of up states increased progressively during the 8-min perfusion with GLU. After a subsequent perfusion with drug-free aCSF, the frequency of up states decreased and the neuron repolarized to levels at or below baseline. c, left, average resting membrane potential during perfusion of aCSF and 5 to 10 min after addition of NMDA (200 µM) to the perfusion fluid. NMDA significantly depolarized PFC neurons (n = 5, paired t test, p < 0.05). Middle, current threshold during perfusion of aCSF and during NMDA perfusion. NMDA significantly decreased current threshold (n = 5, paired t test, p < 0.05). Right, example of the responses of a PFC neuron to intracellular injection of 0.8 nA of depolarizing current during perfusion of aCSF (top) or coperfusion with NMDA (bottom). During coperfusion of NMDA, the neuron fires a greater number of spikes in response to depolarizing current injection. Initial membrane potential for both traces was 79 mV.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   Intrastriatal infusion of BIC (100 µM) depolarizes striatal neurons recorded in vivo. a, during aCSF infusion this striatal neuron exhibits rapid spontaneous shifts in the steady-state membrane potential but is not spontaneously active. During local BIC infusion, the membrane potential of this same cell began to depolarize. Approximately 1 to 2 min into BIC infusion, the membrane potential depolarized further and the cell fired multiple action potentials. b, comparisons of time-frequency histograms of the membrane potential (1-mV bins) recorded from the same cell during intrastriatal aCSF infusion (pre-BIC) and 5 to 6 min after local BIC infusion revealed that the GABAA antagonist induced a rightward shift in the membrane potential distribution (recorded over a 60-s sampling period). c, intrastriatal BIC infusion (5-15 min) depolarized the mean ± S.E.M. membrane potential of striatal neurons (n = 4) from approximately 77.2 ± 3.3 (during aCSF infusion) to 71.0 ± 2.6 mV (*, p < 0.05, paired t test).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In this study, cortical and striatal neurons located in proximity to a microdialysis probe were found to exhibit passive membrane properties and intracellularly and synaptically evoked responses indistinguishable from neurons recorded without microdialysis. In most cases, the neurons were estimated to be located less than 500 µm from the dialysis probe. The responsiveness of the spiny neurons to local pharmacological manipulations suggested that the dendritic fields and/or somata of the neurons were within the perfusion volume of the microdialysis probe. Additionally, because the majority of neurons labeled in this study exhibited large dendritic fields that extended into the neuropil for hundreds of micrometers, it is likely that the above-estimated distance that a dialyzed drug would need to diffuse to influence a given neuron is a conservative one.

Influence of Local Microdialysis on Spontaneous and Evoked Activity. Implementation of this technology also revealed for the first time essential information regarding the state of neurons and neural tissue proximal to the microdialysis probe. Thus, cortical and striatal neurons recorded in control subjects versus animals undergoing the microdialysis procedure described herein demonstrate no significant differences in multiple measures of passive membrane properties, spontaneous activity, or intracellular current and synaptically evoked responses. These observations are of significant interest for the interpretation of microdialysis studies monitoring extracellular neurotransmitters levels, particularly when considered in the light of recent concerns raised regarding the functional state of the tissue surrounding the dialysis probe. Specifically, it has been suggested that local microdialysis traumatizes the tissue and creates a functional dead space around the probe (Lu et al., 1998), which may extend for several hundred micrometers. Although previous studies have explored the impact of microdialysis procedures on the regulation of neurotransmitter efflux (DeBeor and Abercrombie, 1996; cf. Moore et al., 1996) and used reverse microdialysis or micropipette infusions to deliver drugs locally near an intracellular recording electrode (Paré et al., 1998a,b; Castro-Alamancos, 2000), the present study is the first to examine the impact of microdialysis on the membrane activity of neurons in the vicinity of the probe. If the probe implantation or dialysis procedure had severely disrupted synaptic activity either mechanically or by altering the diffusion of locally released neurotransmitters, it is likely that significant differences in natural membrane activity, input resistance, and spike characteristics of neurons recorded proximal to the probe would have been observed compared with intact controls (Wilson, 1993; O'Donnell and Grace, 1995). Moreover, the presence of the bistable membrane potential pattern in cortical and striatal neurons, as well as their responses to afferent stimulation and TTX, showed that synaptic inputs to these neurons remained intact in the presence of the dialysis probe. Given that stable neurons recorded in animals undergoing microdialysis in the current study (some of which were located less than 50 µm from the dialysis probe) exhibited electrophysiological properties similar to those recorded in intact controls, it is likely that the effects of local microdialysis on ongoing synaptic activity and neuronal excitability are minimal. Additionally, the finding that reverse dialysis of the GABA_A receptor antagonist BIC depolarized striatal neurons indicates that the neuron is receiving tonic GABA-ergic stimulation and, therefore, the dialysis procedure does not significantly deplete via washout endogenous neurotransmitter surrounding the recorded neuron. This observation is also supported by our previous studies demonstrating that intrastriatal infusion of dopamine D₁ and D₂ receptor antagonists decrease and increase, respectively, the excitability of striatal neurons recorded proximal to the dialysis probe (West and Grace, 2002). Moreover, the effects of DA antagonists were observed in both within- and between-subject experiments, indicating that the duration of microdialysis and other time effects did not influence the responsiveness of the neuron to local drug infusion (West and Grace, 2002). Our previous studies showing that striatal microdialysis does not alter the striatonigral efferent regulation of midbrain dopamine cells (West and Grace, 2000) also suggests that the activity of striatal output neurons is not significantly affected by the microdialysis procedure. These data demonstrate that, provided probe implantation is done with sufficient care, the neuronal environment proximal to the probe is not severely disrupted.

Responsiveness of Frontal Cortical and Striatal Spiny Neurons to Local Pharmacological Manipulations. In the present study, local pharmacological manipulations executed using reverse dialysis showed that in vivo, the "resting" membrane characteristics and spontaneous patterns of membrane activity are determined in large part by afferent activity. The effects of local perfusion of TTX in the PFC observed in the present study are similar to the findings of Paré et al. (1998b), demonstrating that cortical neurons recorded in vivo in the presence of TTX showed an increase in input resistance and hyperpolarization of the membrane. This is also consistent with previous studies showing that neurons recorded in slice preparations have significantly higher input resistances and more hyperpolarized resting membrane potentials than those recorded in vivo (Paré et al., 1998b). These studies indicate that excitatory afferent activity in the cortex normally provides a tonic level of membrane conductance in the pyramidal neuron; functionally maintaining a relatively low input resistance and depolarized membrane potential. Moreover, loss of the spontaneous shifts in membrane potential states after TTX demonstrates conclusively that synaptic input is necessary for the expression of bistable membrane potential properties of spiny neurons, a finding consistent with the effects of lesioning specific afferents in vivo (Steriade et al., 1993b; Wilson, 1993; Amzica and Steriade, 1995; O'Donnell and Grace, 1995) or eliminating extrinsic afferents as with an in vitro preparation (Paré et al., 1998; Lavin and Grace, 2001). Furthermore, the increased excitability produced by NMDA indicates that even in anesthetized subjects, in most cortical neurons there is sufficient excitatory afferent activity to permit the voltage-dependent activation of NMDA receptors. However, in a subset of neurons receiving less excitatory input (i.e., those with higher input resistance), the effects of NMDA activation are revealed only upon intracellular injection of depolarizing current.