[36] Electrophysiological techniques for the study of hormone action in the central nervous system

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[36] E l e c t r o p h y s i o l o g i c a l T e c h n i q u e s for t h e S t u d y o f H o r m o n e A c t i o n in t h e C e n t r a l N e r v o u s S y s t e m B y B. J. HOttER and G. R. SmGINS

For optimum interpretation of results, analysis of the electrophysiological actions of neurohormones and cyclic nucleotides on central nervous elements requires application of these substances in the immediate environment of a given neuron while simultaneously recording extracellular or transmembrane potentials. Microiontophoresis of small quantities of ionized drugs from multibarreled microelectrodes has evolved into the most practical method for testing the electrophysiological actions of putative neurohormones. This chapter will deal with the various technical aspects of microiontophoresis of neurohormones and cyclic nucleotides, with the acquisition of data from such studies, and with some limitations of these techniques. Design of Microiontophoretic Circuitry

If a potential of a given polarity is applied to an ionized solution in a micropipette barrel, ions of the same polarity will be ejected in accordance with Faraday's law, M = n I T / Z F , where M = moles of drug ejected; s

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FIo. 1. A diagrammatic representation of a single neuronal unit in which axosomatic (A), axodendritic (B), and interaxonic (C) junctions are exhibited as possible sites of drug action. The tip sizes of concentric dual pipettes and multibarrel pipettes are drawn to approximate scale.

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Z = equivalents/mole; F = Faraday's constant; I = amperes of ejection current; T = seconds of ejection time; and n = transport constant. Conversely, potentials of opposite polarity applied to the same drug barrel may be used as "holding currents" to minimize diffusion of drug. Although election of the drug from the pipette appears to vary linearly with charge in any given pipette, the transport constant varies widely from one pipette to the next, making the prediction of absolute amounts of drug release practically impossible. 1,2 These considerations have led to specifications of election currents of drugs in microiontophoretie studies rather than moles of drug released. Since the dc resistance of the drug pipettes may reach 500 megohms, the electronic microiontophoretic devices must be designed to pump constant currents (up to several hundred nanoamperes) through extremely high resistances. Moreover, in order to minimize electrotonic effects of the iontophoretic currents, provision should be made for current neutralization by passage of an equal but opposite current through a barrel filled with sodium chloride (balance barrel; see below). Two such designs are shown in Fig. 2. The circuit~ in Fig. 2A makes use of vacuum phototubes (P1, P2) whose current output may be varied by changing the illumination (L). The output will be largely independent of the inserted series resistance (R). The circuit 4 shown in Fig. 2B makes use of an operational amplifier wired in a "Howland current pump" configuration. Both these circuits use large output voltages ( > 100 V) to permit high currents through large external resistances. It is generally advantageous to use low noise power sources, such as batteries or highly filtered and shielded power supplies, to avoid electrical interference with the recorded bioelectric signal.

Electrodes for Microiontophoresis Since the placement near neurons of drug and recording electrodes cannot be performed under direct microscopic visualization in the central nervous system, all micropipettes must have their tips fixed in close proximity prior to insertion in the tissue, in order to ensure drug application at the site of neuronal recording. For extracellular recording, the 5-barreled pipette is most widely used. It is constructed by fusing 5 pieces of Pyrex tubing 3 mm in diameter so that 4 of the pieces are radially placed around the fifth. The tubes are then fused and initially drawn IB. 2 K. 3 G. H.

J. Hoffer, N. H. Neff, and G. R. Siggins, J. Neuropharmacol. 10, 175 (1971). Krnjevic, R. Laverty, and D. Sharman, Brit. J. Pharmacol. 30, 491 (1964). Salmoiraghi and F. Weight, Anesthesiology 28, 54 (1967). Geller and D. Woodward, EEG C[in. Neurophysiol. 33, 430 (1972).

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Fla. 2. (A) Schematic showing the principle of the electrophoresis circuit. (B) Schematic circuit for one of three identical drug channels. The input operational amplifier (top) sums three possible control voltage signals, pumping and holding currents, and external inputs; the outputs of this operational amplifier go to ttle input of the pumping circuit, to a chart recorder and to one input of the balance channel (bottom). Control voltages from the three drug-injection channels are summed, and the resulting voltage controls the balance current source. to a blunt tip by hand. The tubes are then redrawn in a vertical micropipette puller so that the resulting tip is 1 ~m or less (Fig. 3). After these pipettes are pulled, they can be filled to the fine tip by boiling in distilled water for about 30 minutes and can be stored in this condition for up to 2 weeks. Just prior to use, the distilled water is removed to the "shoulder" of each pipette tip by aspiration with P E 10 tubing, and the appropriate drug and salt solutions are placed in the 5

432

NEURAL TISSUE A

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R

II

FIG. 3. Steps in the preparation of multibarrel glass mivropipette electrodes. The outer glass capillaries are fused to a central barrel (A) and partially drawn out by hand (B). In the final step, the electrode is further drawn out to tip sizes less than 0.5 ~m by commercially available electrode pullers (C). When filled and ready for use, the overall tip diameter is increased to 3-6 ~m by gently tapping the assembly. barrels. The central barrel, used for recording neuronal activity, is filled with 5 M NaC1, and one of the peripheral barrels, if used for current neutralization, is filled with 3 M NaCl. The remaining 3 peripheral barrels are filled with drug solution. Diffusion of the solutions into the pipette tip is facilitated by centrifugation at 2000 rpm for 30 minutes. For this purpose, we have used a specially designed, plastic device for holding the pipettes firmly in place in standard centrifuge cups, with the tips facing centrifugally. Finally, the electrode tip is manually bumped under microscopic control to a total outer diameter of 3-6 ~m. This allows a recording barrel size small enough (2-4 megohms resistance) to isolate action potentials from single neurons while keeping drug barrel resistances low enough for constant current ejection. The pH of the drug solution is usually adjusted to produce maximal ionization and solubilization of the agent. For example, when cyclic A M P is dissolved as the free acid, it is dissolved in 0.25 N sodium hydroxide to yield a 0.5 M solution with a pH of 6-8. Several neurohormones may be used as either cations or anions, depending on the pH. In such instances, use as a cation is preferable since interaction with the zeta potential of the glass pipette tip promotes ejection. Microiontophoresis of angiotensin, for example, is effective only if cationic currents are usedS; no responses are seen if anodal currents are applied to the pipette. In general, holding currents range from 10 to 40 nA and ejection cur°R. Nicoll and J. Barker, Nature (London) 233, 172 (1972).

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~'IG. 4. Completed electrode.

rents may be as high as 200 nA. If cells are particularly sensitive to a particular substance, significant responses may result simply by removing the retaining current and allowing the drug to diffuse from the pipette tip. Electrodes for recording transmelnbranc potentials during extracellular drug ejection can be constructed by cementing a fine single-barrel recording pipette (tip diameter <0.5 ~m) parallel with a standard multibarreled drug micropipette. 6 Best results are usually obtained if the tip of the single-barreled pipette is 20-40 ~m in front of the multibarreled pipette (Fig. 4). It is of utmost importance that the tip of the drug electrode rests firmly against the shaft of the recording electrode. If there is any space between them, they will often separate as they pass through nervous tissue, considerably lessening the chance of obtaining drug responses from the recorded neuron. 6A. P. Oliver, EEG Cli~. Ne~rophysioL 31, 284 (1971).

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Recording Techniques Extracellular Recording The electrophysiological effects of putative neurohormones on neurons, evaluated by extracellular recording of action potentials, can usually be causally related to changes in spontaneous, or drug or stimulusinduced discharge rate. It must be emphasized, however, that increases or decreases in discharge rate do not, by themselves, unequivocally indicate excitation or inhibition at the membrane level (see below). In order to simplify data collection and analysis of spontaneous activity, the extracellular action potentials under study are usually displayed on an oscilloscope and simultaneously fed into a "window" discriminator to separate them from base-line noise and action potentials of neighboring neurons. The discriminator output is then converted to fixed amplitude pulses, which are subsequently integrated over a preselected interval (usually 1 second). The fixed amplitude pulses may also be displayed on a second channel of the oscilloscope for comparison with the original action potentials. The output of the integration is displayed on one channel of an ink writer, to provide a continuous analog record of the discharge rate. A comparison of this display with the original action potential record is shown in Fig. 5. In many cases, neurons show very low spontaneous discharge rates. It is often advantageous, in such cases, to induce a steady and regular background discharge to facilitate testing excitatory and inhibitory agents. Applications of small amounts of excitatory amino acids, such as glutamate or DL-homocysteate, from another barrel of the.pipette is often used to induce such discharge. 7 Alternatively, a digital computerbased signal averaging paradigm can be used with regularly repeated drug pulses as the "stimulus." This latter technique has the advantage of eliminating the potential artifacts induced by direct interaction of ~he specific agonist with the excitatory amino acids. The effects of locally applied hormones on driven as well as spontaneous activity may also be studied. Antidromic spikes and orthodromic excitation and inhibition s may all be used. Such studies help to determine whether drugs act on postsynaptic membrane in general, at postsynaptic receptors, or a~ the presynaptic level. Both pre- and postsynaptic action of many putative transmitters and drugs have been demonstrated s in this fashion. Differentiating pre- and postjunctional action of drugs is also facili7j. Godfraind, K. Krnjevic, and R. Pumain, Nature (London) 228, 675 (,1970). s A. Tebecis and A. DiMara, Exp. Brain Res. 14, 480 (1972).

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Fie. 5. Effects of microiontophoresis of norepinephrine (NE) on spontaneous Purkinje cell discharge rate. (A) and (B) illustrate two different Purkinje cells. Left half of figure shows integrated polygraph record. Right half of figure shows action potentials themselves. Numbers after drug indicate ejection currents in nanoamperes, and brackets indicate duration of drug ejection. rated by combining microiontophoresis with either surgical or chemical lesions of afferent pathways. The recent discovery of drugs which destroy specific classes of fibers, such as 6-hydroxydopamine for adrenergic terminals, 9 m a y permit even more detailed analysis of presynaptic drug actions.

Intracellular Recording Most intracellular studies of t r a n s m i t t e r actions on neurons emphasize effects on membrane potentials, with a depolarizing response indicative of an excitatory action and a hyperpolarization, inhibitory action. H o w ever, underlying these potential changes, and perhaps responsible for their generation, m a y be. changes in m e m b r a n e conductance or permeability to one or more ion species. In this section, techniques for measurement of t r a n s m e m b r a n e potentials and conductance during microiontophoresis of neurohormones and cyclic nucleotides will be presented. Since only one pipette can be inserted into most m a m m a l i a n neurons, the most common procedure for determining m e m b r a n e ionic conductance utilizes a Wheatstone bridge circuit for passage of current pulses through ~'F. E. Bloom, B. J. Hoffer, and G. R. Siggins, Biol. Psychiat. 4, 157 (1972).

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the same recording pipette used to measure membrane potential. A good example of such a circuit is the one designed by Araki and Otani. 1° The high resistance leg of the bridge (108 to 109 ohms) is often provided in many commercially available cathode followers. In brief, the usual procedure for recording membrane potential and determining input resistance (inversely proportional to the sum of all ionic conductances) is as follows: (1) Continually pass constant current pulses of about 20-100 msec duration and 10-s to 10-29 amperes intensity (these parameters will depend upon the electrical properties of the specific neuronal membrane and should be empirically determined) through the recording pipettes. (2) With the pipette in the brain but still extracellular, constantly adjust the variable bridge resistor until the bridge is balanced (i.e., the current between the leg with microelectrode and that with the variable resistor is zero). Ideally, the dc potential will then be flat except for the fast "on" and "off" transients of the pulse. (3) With small movements of the micromanipulator, a sudden negative deflection of the dc potential may indicate penetration of a neuron. This can be verified by the size of the potential (--30 to --70 mV), by the presence of large (40-150 mV) positive-going action potentials, and a sudden unbalancing of the bridge. The magnitude of the unbalanced current pulse should now reflect the input resistance of the membrane, provided that the on/off phases of the pulse do not rise so abruptly as to appear discontinuous (a slowly rising or falling potential reflects membrane capacitance). The input resistance is calculated by Ohm's law, dividing the voltage deflection of the pulse by the pulse current. (4) After verifying that the membrane potential is large (--40 to --70 mV) and steady (indicating relatively little injury), determine the effect of drug applications, with a suitable control period after the drug effects have worn off. (5) Note the effect on input resistance of artificial (current-induced) changes in membrane potential (see below). (6) Withdraw the pipette from the cell, note the zero potential, and verify that the Wheatstone bridge is again null-balanced. It may be helpful to first determine whether it is possible to obtain uninjured intracellular records from a given neuron type with single-barrel pipettes. The attachment of additional barrels to the recording pipette, for microiontophoresis of drugs, often greatly reduces the chances of obtaining good records. In our laboratory, it has been determined that two extracellular barrels are optimal for extracellular application of drugs during intracellular recording of transmembrane potentials in cerebellar Purkinje neurons. The double extracellular barrels are heated together, ~oT. Araki and T. Otani, J. Neurophysiol. 18, 472 (1955).

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t w i s t e d once, a n d t h e n p u l l e d to a fine t i p 2 T h e y are t h e n b u m p e d , glued to t h e r e c o r d i n g p i p e t t e , a n d filled2 One e x t r a c e l l u l a r b a r r e l u s u a l l y cont a i n s a d r u g solution, while t h e o t h e r h a s 3 M NaC1 for use as a " b a l a n c e " b a r r e l to n e u t r a l i z e e l e c t r o t o n i c effects. T h e i n t r a c e l l u l a r r e c o r d i n g elect r o d e is u s u a l l y filled w i t h 3 M KC1, K.,SO~, or p o t a s s i u m citrate. W h i l e KC1 is m o s t often used, K c i t r a t e should be t e s t e d if it is s u s p e c t e d t h a t l e a k a g e of C1 or S04 into t h e n e u r o n m i g h t a l t e r the response u n d e r investigation. Such e l e c t r o d e a s s e m b l i e s m a k e it possible to m e a s u r e the effects of n e u r o t r a n s m i t t e r s a n d cyclic n u c l e o t i d e s on r e s t i n g m e m b r a n e p o t e n t i a l , m e m b r a n e c o n d u c t a n c e , a n d p o s t s y n a p t i c p o t e n t i a l s (Fig. 6). T h e s e a n a l y s e s are n e c e s s a r y to d e t e r m i n e t h e e l e c t r o p h y s i o l o g i c a l m e c h a n i s m of

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FIG. 6. Intracellular recordings from rat cerebellar Purkinje cells. (A) Schematic representation of a three-barrel micropipette with a Purkinje cell. The intracellular electrode protrudes beyond the orifices of the two extracellular microelectrophoresis barrels. (B) Multispiked spontaneous climbing fiber discharge obtained during intracellular recording from a Purkinje cell. Number in parentheses is resting potential in millivolts (mV); calibration bars are 20 msec and 25 mV. (C) Changes in membrane potential and membrane resistance of four different Purkinje cells in response to ~-aminobutyrate (GABA), norepinephrine (NE), dibutyryl cyclic AMP (DB cyclic AMP), and cyclic AMP. All specimens in each horizontal row of records are from the same cell. Solid bar above each record indicates the extracellular electrophoresis of the indicated drug (100-150 nA). Number in parentheses below each recording is resting potential in millivolts; calibration bar under membrane potential records is 10 seconds and 20 mV for NE, DB cyclic AMP, and cyclic AMP, and is 5 seconds and 10 mV for GABA. The effective input resistance was judged by the size of pulses resulting from the passage across the membrane of a brief constant current (1 nA) pulse before, during, and after electrophoresis of the respective drugs (1 mV = 1 megohm). Discontinuities in the fast transients of the pulses result from the loss of high frequencies (>10 kHz) and from the chopped nature of the frequency-modulaled magnetic tape recording used. All "pulse" records were graphically normalized to the same base-line level. Calibration bar on right indicates 80 msec and 15 mV for all pulse records.

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drug action and, hopefully, to eventually correlate the electrophysiological and biochemical effects of these substances. Problems with Use of the Iontophoretic Technique

Quantitating Release The great variation in transport number, from one pipette to another, has already been discussed. Because of this problem, it is virtually impossible to predict the amount of drug released by a given ejection current. Moreover, the determination of the effective concentration of the drug at the membrane receptors is still further removed, since the exact distance of the electrode from the neuron is unknown, and the assumption of free diffusion through a homogeneous medium is certainly untenable to the central nervous system.

Artifactual Responses The major artifacts associated with extracellular recording and iontophoretic administration of neurohormones involve pH effects, local anesthetic action, and electrotonic effects of the iontophoretic current. As noted above, the pH of drug solutions is often manipulated to promote ionization and ejection. Although some studies 11,12 have suggested that solutions of biogenic amines with pH of less than 4.0 may elicit artifactual excitation, recent work from several laboratories have provided convincing evidence against this simplistic view. Cortical neurons have been shown to possess identical responses to norepinephrine at pH 3.0 and pH 5.0. As a further control, some drugs may be eiected as cations from low pH solutions and as anions from high pH solutions. An identity of action would argue against pH effects. Many neuroactive drugs possess powerful local anesthetic side effects and can thereby reduce discharge rates by inactivating the membrane spike generating mechanism rather than by producing physiological hyperpolarization and conductance changes. Several types of controls are useful in distinguishing local anesthetic action from inhibition. First, local anesthetic action usually elicits a marked and progressive diminution of the amplitude of the action potentials prior to their disappearance, whereas inhibition usually does not. In the case of norepinephrine effects of cerebellar Purkinje cells, the inhibition may actually be associated with an increase in action potential size. Second, during a true, nonn R. C. Frederickson, L. M. Jordan, and J. W. Phillis, Brain Res. 35, 556 (1971). 12L. M. Jordan, R. C. Frederickson, J. W. Phillis, and N. Lake, Brain Res. 40, 552 (1972).

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anesthetic inhibition, neurons can usually be excited by microiontophoretic administration of a depolarizing ionic curren~ or an amino acid, such as glutamate or DL-homocysteate, from another barrel of the same pipette. Cells which have been locally anesthetized do not often respond to such amino acid or cathode applications (Fig. 7). Electrotonic effects of the iontophoretic currents are another potential source of artifact. These currents can be neutralized by the use of a "balance current," that is, automatic passage, through the sodium chloride barrel, of a current equal in magnitude but opposite in direction to the sum of the currents passing through the other three barrels. When functioning properly, balance currents tend to eliminate any potential difference between the pipette tip and ground. However, if there is still a question of influence of current on responses of a particular neuron, currents equivalent to the magnitude and polarity of the drug current may be passed through the sodium chloride barrel. If the cell then manifests a response to a sodium or chloride ejection similar to that after drug ejection, an electrotonic artifact is a strong possibility. There are many problems and artifacts associated with single neuron A J'~

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FIG. 7. Effects of microelectrophoretic application of adenine nucleotides on spontaneous Purkinje cell discharge. (A) and (B) illustrate continuous records from the same cell. Duration of drug application is indicated by arrows, and numbers after each drug indicates ejection current in nanoamperes. The black bar in (A) represents application of a cathodal current through the micropipette. The restoration of spontaneous discharge by cathodal current indicates that the slowing seen with cyclic A M P is not secondary to local anesthetic action or hyperdepolarization (cathodal block).

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recording from brain and these multiply when mieroiontophoresis of substances is also employed. One of the most difficult problems is movement, whether of Cell or mieroelectrode. Use of the most stable mieromanipulator and stereotaxie apparatus is mandatory. Slight pulsations of nervous tissue, such as arise from respiration and blood pressure, are often troublesome. They may be diminished by pneumothorax and rapid shallow artifieial respiration, drainage of the cerebrospinal fluid, and/or application of a "pressor foot" to the exposed brain surface. 1'~ When unbalanced iontophoretic current is used to apply drugs extracellularly while recording intracellularly, resistive electrical coupling between recording and drug barrels is a maior source of artifact, often producing large changes (5-30 mV) in membrane potential. As a result, proper controls must be performed with the recording electrode outside the cell to determine the voltage-producing effect of iontophoretic currents. These voltage changes must then be algebraicly subtracted from the potentials produced during cell penetration. It is also important to note whether any changes in bridge balance occur with the recording electrode outside the cell during drug ejection. Use of a NaCl-filled extracellular balance barrel usually reduces resistive coupling to less than 1 mV, and thus greatly minimizes this artifactual problem. Interpretation

One final reservation is in order, relating to the limitations of microiontophoresis. By proper regard for each of the necessary experimental controls, it is possible to get reproducible effects of many neurohormones on the electrophysiological activities of neurons. However, such data alone do not indicate that these responses are a refection of an underlying hormonal or cyclic nueleotide-chemical input to the cell under study. Critical evidence to corroborate this inference involves selective activation of endogeneous afferent fibers or hormonal inputs to an identifiable cell type ~4 and histochemical demonstration of synapses containing the neurohormone under study. 15 With regard to circulating hormones, it should be determined that the endogenous hormone actually reaches the neuron under study. When used in such an interdisciplinary fashion, microiontophoresis may become a powerful analytical tool for evaluating the electrophysiological responses to putative humoral substances in the central nervous system. 13B. J. Hoffer,G. R. Siggins, and F. E. Bloom,Brain Res. 25, 523 (1971). 14G. R. Siggins, B. J. Hoffer, A. P. Oliver, and F. E. Bloom, Nature (London) 233, 481 (1971). 1~F. E. Bloom, S. Algeri, A. Groppetti, A. t~evuelta, and E. Costa, Science 166, 1284 (1969).