c‘omp. Bio~hcm. Ph.w;o/. Vol 8OC, No. 2, pp. 361-370. 1985 Printed in Great Britain

0306-4492,‘85 53.00 + 0.00 ,_ 1985 Perganon Press Ltd

THE EFFECT OF SOME DRUGS ON THE MITRAL CELL ODOR-EVOKED RESPONSES IN

THE GECKO OLFACTORY BULB

KEIICHI TONOSAKI* and TATSUAKI SHIBUYA~

Zoological Institute, Faculty of Science, Tokyo Kyoiku University, 3-29-1, Otsuka, Bunkyo-ku, Tokyo, Japan

(Receiced 9 August 1984)

Abstract-l. The activity of odor-evoked olfactory mitral cell response of the gecko was recorded extracellularly by glass microelectrodes.

2. The activities of the mitral cell observed during the presentation of the odor (n-amyl acetate) could be described as excitation, suppression or zero.

3. The present experiments were undertaken to study the neural activities of the mitral cell in the olfactory bulb by perfusion application of some drugs (cobalt chloride, carnosine, norepinephrine, GABA and u-t_-homocysteate) on the olfactory bulb surface or iontophoretic application of some drugs (carnosine, norepinephrine, GABA and u-L-homocysteate) to the glomerulus and the external plexiform layer to change the physiological environment.

4. The effect of the drugs suggested that the synaptic neurons on the mitral cell have different chemical characteristics

INTRODUCTION

The axon of the olfactory receptor cell synapses first with the primary dendrite of the mitral cell in the glomerulus layer. Periglomerular cells also exist in the glomeruli, they connect with the primary dendrite of a mitral cell and the axon of the olfactory receptor cell. Moreover, each periglomerular cell seems to relate to several glomeruli. The secondary dendrite of the mitral cell has synapses with the dendrite of the granule cell in the external plexiform layer. In the deeper region of the olfactory bulb, there are many granule cells and they do not have any axons. These morphological studies were made with light and electron microscopes by various investigators (Allison, 1953; Gasser, 1956; Clark, 1957; Andres, 1965, 1970; Wenzel and Sieck, 1966; Price and Powell, 1970a,b,c). The main synaptic regions in the olfactory bulb are roughly restricted to two layers, these are the glomerulus layer and the external plexi- form layer. The gecko (G&o gecko) used in this experiment possesses distinctive morphological and spatial differences between these layers. From these facts, it can be considered that odor information from the olfactory receptor cell may be modified mainly in the glomerulus and external plexiform layers. Then the neural pathway, propagating the odor informa- tion in the olfactory bulb, may be revealed by the response to odor in the olfactory bulb affected by the application of some drugs to each layer to change the physiological environment.

*Present address: Department of Oral Physiology, Gifu College of Dentistry, Takano, Hozumi, Motosu, Gifu N-02, Japan.

tPresent address: Institute of Bioiogicat Science, The Uni- versity of Tsukuba, Sakuramura, Ibaraki 300-3 1, Japan.

There are some descriptions of the mitral cell activity in reptiles in response to odor stimulation (Mathews, 1972; Shibuya et al., 1977; Tonosaki and Shibuya, 1979; Tonosaki, 1982). In general, the effect of odor stimulation on the mitral cell activity could be described as excitation, suppression or zero. How- ever, the effects of drugs on the odor-evoked electrical response of the mitral cell have been reported in a few papers (Shibuya ef al., 1977; Tonosaki and Shibuya, 1979).

The present investigation was undertaken to study the neuronal activities and mechanisms at the second- ary neuron level with synapses in the olfactory bulb by applying some drugs to the synaptic layers.

MATERIALS AND METHODS

I. Preparation qf the material

The gecko (Gekko gecko) (body wt, 5&80 g) was used in this experiment. The animal was anesthetized by i.p. injec- tion of 50% ethyl urethane (0.2 ml/100 g body wt), and then placed in the dark room for about 30min for sufficient anesthetization. After the head was fixed on the plate with a holder, a part of the skin on the head was removed so as to expose the olfactory bulb and olfactory tract by taking off a part of the skull with a dental drill. The dura on the olfactory bulb was removed with care so that the glass microelectrode could be inserted.

2. Dissection for drug application

A small depression was made on the olfactory bulb of the gecko for convenience of perfusing the solution. Another small depression was similarly made on the lateral olfactory tract. These depressions were separated from each other by a wall of bone about 0.5 mm in thickness. The wall was coated with a paraffin or bone wax, so that the physiological solution did not ooze out to other portions. The position of the tip of the recording and iontophoretic electrodes could be recognized from the electrical observation of the field potenttats (Shepherd, 1963a,b; Phillips et al., 1963).

361

362 KEIICHI TONOSAKI and TATSUAKI SHIBUYA

3. Method of upplication of drugs

The drugs used in this experiment were cobalt chloride, camosine, norepinephrine, 7-amino butylic acid (GABA) and o-L-homocysteate. It is known that carnosine (Mar- golis, 1974; Neidle and Kandera, 1974; Ferriero and Mar- golis, 1975), and norepinephrine (Lorenzo, 1963; Shan- taveerappa and Bourne, 1965; Lichtensteiger, 1966; Brenells, 1974) are found substantially in the olfactory bulb. GABA and D-L-homocysteate are effective drugs on the central nervous system.

3.i. The method of perfusion on application of drugs. Perfusion application of a drug solution was carried out as follows. The syringe was filled with drug solution. The vinyl pipes (2 mm in diameter) connected with six syringes filled with the respective drug solutions were led to the olfactory bulb surface through a Teflon tube with a special joint. The solution flowed constantly at a rate of about O.O4ml/sec. One of the six syringes was filled with the physiological solution. All drug solutions were diluted at a concentration of lo-’ M with the physiolo~cai solution.

The response of the mitral ceil to odor stimuli was first recorded under control conditions using the physiological solution. Then the drug solution was allowed to flow into the depression on the olfactory bulb. After 3 min, the odor stimulation was applied and the response was recorded. The drug solution was then replaced by the physiological solu- tion. After the spontaneous impulse activity recovered to the control level, the procedure of the next experiment was started.

3% The method qf j~nto~~oretic a~~~jcation of drugs. The method of iontophoretic application of drugs has been described by various investigators (Curtis et al., 1951; Globus, 1973; Nicoll, 1969; McLennan, 1971). The ionto- phoretic technique is based on the movement of ions by an electric current. The drugs used in this experiment were carnosine (0.25 M), norepinephrine (1 M), GABA (0.5 M) and D-L-homocysteate (0.2 M). These were used at the same concentration and at a suitable pH as described by McLennan (1971).

An iontophoretic electrode was made similarly to the recording electrode mentioned below. The tip of the elec- trode was about 1 /lrn in diameter. To avoid artifacts caused by applied electrical current on the electrical record, a bridge circuit was used with automatic current balance. The polar- izing current could be varied and its intensity was monitored with an ammeter. In the experiment, a current of 20 nA was passed through the electrode for 25 sec. The odor stimu- lation was begun 15 set after switching on the iontophoretic current. The backing current was at lOOnA to avoid an outflow of the drug from the iontophoretic electrode.

The glomerulus layer of the gecko forms a sheet at about 200gm from the surface of the olfactory bulb and the thickness of the layer was about 100 pm. The thickness of the external plexiform layer was about 20&3OO~m below the glomerulus layer. The tip of the iontophoretic electrode could be placed within the layer by measuring the scale of the manipulator. Two iontophoretic electrodes were mounted in a holder with tips separated vertically by about 200pm. These electrodes were inserted obliquely into the olfactory bulb at an angle of about 45” to the horizontal plane. The tip of the upper electrode was positioned in the glomerulus layer. That of the lower one was in the external plexiform layer. The position of the tips of these electrodes could be detected by the marking method or field potential method (Phillips et nl., 1963).

The iontophoretic experiments were performed as fol- lows. The response of the mitral cell to odor stimuli was recorded as a control. Then a drug was applied to the glomerulus layer or the external plexiform layer by the iontophoretic method, and odor stimulation was applied to the olfactory cavity. and the responses were recorded. The impulse activity completely recovered to control level after

several minutes and the procedure of the next experiment was started.

4. Odor stimulation

n-Amy1 acetate vapor was applied to the olfactory epi~elium as the stimulant. This odor has been employed for olfaction experiments (Adrian, 1953).

An olfactometer was used for the odor stimulation in the present experiments (Tucker, 1963a). Air flowed through silica gel and charcoal in washed glass bottles from an air compressor and was bubbled into liquid n-amyl acetate in a glass bottle, and saturated n-amyl acetate vapor was obtained as the odor (concentration: 10’). On the other hand, the air passing through silica gel, charcoal and deodorized distilled water was used as pure air. The saturated vapor of the odor and the pure air were mixed through howmeters and the experimental stimulation was made at various concentrations of odor vapor from 10” to 10m4. In this study the concentration of n-amyl acetate odor was adjusted to lo-*. The odor concentration is referred to as per unit vapor saturation ((partial vapor pressure)/(saturated vapor pressure)). Deodorized air (pure air) was used as the carrier gas of the odorants.

The flow rate was adjusted to 3 l/min in all experiments. The duration of application of odor stimuli was usually for about 3 sec. The pure air flowed ~roughout the experiment except for the duration of odor stimulation at the same flow rate mentioned above. The mixed gas of odor and pure air could be obtained by switching the channel of a three-way valve. The Teflon tube nozzle (2 mm in diameter) was led to the nares. The time interval between odor stimulations was at least for 1 min, so as to abolish a possible after effect of the previous stimulation.

5. Recording techniques

The proximal end of the microelectrode was dipped into 2 M KC1 solution or 6% aqueous solution of procion brown, and the solution went to the tip by capillary action. The microelectrode was placed in a desiccator bottle at reduced pressure for a few minutes. The microelectrode was completely filled with the solution on return to atmospheric pressure. The tip diameter of the microelectrode was supposedly less than 0.1 pm and the resistance was lo@250 MR. The microelectrode was set in a holder and slowly advanced into the preparation vertically with the oil manipulator.

Impulses recorded as the electrical discharges with equal height and similar duration in the olfactory bulb in response to odor could be obtained at a depth of 450-550/1m below the upper surface. These impulses were led to ac. main amphfiers via a pre-amplifier. The signals were led to an oscilloscope. The vertical movement of the spot on the oscilloscope screen was photographed. An audio monitor and a monitor oscilloscope were additionally used.

These experiments were performed at room temperature (15~20°C).

RESULTS

I. E.xtraceliular recordings of the mitral cell responses

When the microelectrode gradually penetrated into the superficial layer of the olfactory bulb, spontane- ous impulses were observed at a depth of 450-550 Hrn below the surface. The depth coincided with a mitral cell body layer as determined by the pattern of antidromic summed potentials and the correlations with histological sections, according to the method of Phillips et al. (1963).

One hundred and fifty-five mitral cell responses were recorded extracelluiarly in 25 experiments.

Pharmacology of gecko mitral cells 363

Impulses of the mitrai cell recorded extracellularly in response to odors were always initially positive and were monophasic or diphasic in shape. The height of the impulse was l-5 mV and the duration was l-3 msec. Regular bursting of impulses was not ob- served either in the presence or absence of odor stimuli. The impulse activity continued even if the olfactory bulb was isolated from the rest of the brain or the olfactory epitheiium was destroyed.

In general, the activities of the mitral cell observed during the presentation of the odor could be placed into one of two categories, excitation and suppression types.

2. Effect of some drugs

2.i. Effect of drugs on odor responses of the mitral cell by perfusion application. Figure 1 shows an example of the effect of cobatt chloride. When cobalt chloride was perfused on the olfactory bulb surface, neither excitation nor the suppression responses to n-amyl acetate were observed (Fig. IA-C,P and lB-C,P). Only the “spontaneous” impulse activity was seen.

Records shown in Fig. 2 are an example of the action of carnosine at 10.* odor concentration. In perfusion application of carnosine to the olfactory bulb surface the mitral cell, which indicated the excitation type of response under the control condi- tions, showed an increase in the spontaneous activity rate; however, the number of impulses during the odor stimulation was slightly decreased (Fig. ZA-C,P). When carnosine was applied to the olfac- tory bulb surface the mitral cell, which indicated the suppression type of response, increased spontaneous

A

c

P

B

C

P

COBALT CHLORIDE

activity rate and number of impulses during the odor stimulation (Fig. 2B-C,P).

Figure 3 is an example of perfusion application of norepinephrine on the olfactory bulb surface at lo-’ odor concentration. When the response was of the excitation type, the spontaneous activity decreased its rate a small amount (Fig. 3A-C,P). The number of impulses during the odor stimulation also decreased (Fig. 3A-P). When norepinephrine was applied to the suppression type of response under the control condi- tions, the spontaneous activity decreased slightly, though the suppression phenomenon on odor stimu- lation was maintained (Fig. 3B-C,P).

Figure 4 shows an example of the effect of GABA. When GABA was perfused on the olfactory bulb surface, the excitation type of response at 10d2 odor concentration showed a decrease in the number of spontaneous impulses and impulses caused in re- sponse to the odor (Fig. 4A-C,P). The impulses in response to the odor were ciearly observed (Fig. 4A-P). When the odor response showed a suppression type of response under control conditions at 10w2, the spontaneous activity rate slightly decreased and the suppression of stimulation by the odor was main- tained (Fig. 4B-C,P).

The records shown in Fig. 5 are examples of the effect of D-L-homocysteate on the response to 10e2 odor. The excitation type of response increased the spontaneous activity rate and the number of impulses during odor stimulation (Fig. SA-C,P). When the suppression type of response to odor was shown in control conditions, o-L-homocysteate increased the spontaneous activity rate and the number of impulses during the stimulation (Fig. SB-C,P).

Fig. 1. The effects of perfusion application of cobalt chloride (IO-’ M) on the olfactory bulb surface. A: The excitation type of response to n-amyl acetate (10m2: concentration). B: The suppression type of response to n-amyl acetate (iOm2: concentration). C: The response in control conditions of perfusion application of the physiological solution. P: The response caused by perfusion appl~~tion of the drug solution. The horizontal lines below each record show the odor stimulation. Calibration: I mV, 1 sec.

364

A

C

P

B

C

P

KEIICHE TONO~AKI and TATSUAKI SHIBUYA

CARNOSINE

Fig. 2. The effect of perfusion application of carnosine (IO-?M) on the olfactory bulb surface. The symbols are as in Fig. 1.

NOREPINEPHRINE

A

C

Fig. 3. The effect of perfusion application of norepinephrine (10m3 M) on the olfactory bulb surface. The symbols are as in Fig. 1.

A

c

P

0

C

P

Pharmacology of gecko mitral cells

GABA

Fig. 4. The effect of perfusion application of GABA (10m3 M) on the olfactory bulb surface. The symbols are as in Fig. 1.

0-L-HOMOCY ST E ATE

365

Fig. 5. The effect of perfusion application of o-L-homocysteate (IO-’ M) on the olfactory bulb surface. The symbols are as in Fig. 1.

366 KEIICHI TONOSAKI and TATSUAKI SHIBUYA

CARNOSINE

Fig. 6. The effect of iontophoretic application of carnosine (0.25 M) to the glomerulus and external plexiform layers. A: The excitation type of response to n-amyl acetate (IO-*: concentration). B: The suppression type of response to n-amyl acetate (10.‘: concentration). C: The response in control conditions. G: The response caused by the iontophoretic application of the drug to the glomerulus layer. E: The response caused by the iontophoretic application of the drug to the external plexiform layer. The

horizontal lines below each record show the odor stimulation. Calibration: 1 mV, 1 sec.

2.ii. EfSect of drugs on odor responses of the mitral cell by iontophoretic application. The records shown in Fig. 6 are examples of the effect of carnosine on the response to 10m2 odor concentration. When the mitral cell showed an excitation type of response in control conditions (Fig. 6A-C), carnosine was applied iontophoretically near to the region connected to the dendrites of the mitral cells in the glomerulus layer.

The spontaneous activity rate and the number of impulses decreased during stimulation (Fig. 6A-C,G). The discharge burst of the excitation observed in Fig. 6A-C disappeared. When carnosine was applied to a part of the external plexiform layer, the spontaneous activity increased and the number of impulses caused by the odor enhanced (Fig. 6A-C,E). In the sup- pression type of response, the spontaneous activity decreased with carnosine, but the suppression was

maintained at the glomerulus layer (Fig. 6%C,G). When carnosine was applied to the external plexiform layer, the spontaneous activity was relatively in- creased and a few impulses during stimulation were observed (Fig. 6B-C,E).

Figure 7 shows an example of the effect of nor- epinephrine on the response at 10w2. In the excitation type of response of the mitral cell to odor, there was a decrease in the spontaneous activity and the num- ber of impulses during stimulation on iontophoretic application of norepinephrine to the glomerulus layer (Fig. 7A-C,G). A decrease was always observed. When the suppression type of response appeared in control conditions, the spontaneous activity was slightly decreased by the application of norepine- phrine to the glomerulus layer (Fig. “IS-C,G). When norepinephrine was applied to the external plexiform layer, the excitation type of response also showed a

Pha~acology of gecko mitral cells

NOREPWJEPHRINE

367

E

Fig. 7. The effect of iontophoretic application of norepinephrine (1 M) to the glomerulus and external plexiform layers. The symbols are as in Fig. 6.

slight decrease in spontaneous activity and the num- ber of impulses during stimulation (Fig. 7A-C,E). In the suppression type of response to odor, the sponta- neous activity decreased and the suppression was maintained (Fig. 7B-C,E).

The records shown in Fig. 8 are examples of the effect of GABA on the response at lo-*. When GABA was applied iontophoretically to the glomeru- lus layer, it was hard to tell whether the spontaneous activity and the number of impulses to odor changed or not (Fig. 8A-C,G). This was observed when the response in control conditions showed an excitation or suppression type of response to odor (Fig. 8A-G and 8B-G). When GABA was applied iontophoreti- tally to the external plexiform layer, a decrease in the spontaneous activity and the number of impulses during stimulation was always observed on the exci- tation type of response (Fig. 8A-C,E). In the case of the suppression type of response in control con- ditions, the spontaneous activity was apt to decrease and the suppression to odor be maintained when GABA was applied iontophoreticalIy to the external plexiform layer (Fig. 8B-C,E).

Figure 9 shows an example of the effect of D-L-homocysteate on the response at lo-*. When the

drug was applied iontophoretically to the glomerulus layer, the spontaneous activity and the number of impulses during stimulation in the case of the ex- citation type of response in control conditions had a small increase (Fig. 9A-C,G). In the suppression type of response, there was also an increase in the sponta- neous activity, and the suppression to odor was maintained (Fig. 9B-C.G). In the case of the ex- citation type of response, when the drug was applied iontophoretically to the external plexiform layer, the spontaneous activity and the number of impulses during stimulation increased slightly (Fig. 9A-C,E). In the case of the suppression type of response, when the drug was applied iontophoretically to the external plexiform layer the spontaneous activity increased and the suppressive response to odor was maintained (Fig. OB-C,E).

DISCUSSION

In these experiments, many kinds of drugs which are known to show physiological actions on the central neurons were used to test the odor responses of the mitral cells. Carnosine, norepinephrine, GABA and D-L-homocysteate had some effects on the olfac-

368 KEIICHI TONOSAKI and TATSUAKI SHIBUYA

GABA

B

C

G

E

Fig. 8. The effect of iontophoretic application of GABA (0.5 M) to the glomerulus and external plexiform layers. The symbols are as in Fig. 6.

tory nervous elements in the olfactory bulb. It may be suggested that the experimental results reflected mainly the action of the drugs upon the function of the nervous elements which compose the neural path- way of olfaction.

The fast green or procion brown solution which was perfused on the olfactory bulb surface for about 3 min permeated to the mitral cell layer by diffusion. The effect of perfusion application of cobalt chloride, carnosine, norepinephrine, GABA and D-V_-homo- cysteate showed at least additional effects composed to iontophoretic application of these drugs on the glomerulus and the external plexiform layers.

When cobalt chloride (known as a blocker of chemical synaptic transmission) was perfused on the olfactory bulb surface, both the excitation and sup- pression responses to odor disappeared. The sponta- neous impulse activity continued even if the olfactory bulb was isolated from the rest of the brain or the olfactory epithelium was destroyed. It is suggested that the spontaneous impulse was produced by the intrinsic activity of the mitral cell. It can be consid-

ered that the neural connections to the mitral cell modify the odor response of the mitral cell.

When carnosine (known as an excitatory substance in the central nervous system (Himwich and Agrawal, 1969; Margolis, 1974; Neidle and Kandera, 1974)) was applied iontophoretically to the glomerulus layer, the number of impulses during stimulation decreased. When carnosine was iontophoretically ap- plied to the external plexiform layer, the spontaneous impulses and the number of impulses during stimu- lation increased. Carnosine applied to the glomerulus layer should probably affect the interneuron between the olfactory receptor cell axon and the primary dendrite of the mitral cell to suppress the activity of the mitral cell. A similar response was considered to be the effect of norepinephrine on the glomerulus layer. From morphological studies (Andres, 1970; Price and Powell, 197Oc), it was reported that there were periglomerular cells in the glomerulus layer and that they play the role of an inhibitory interneuron to the primary dendrite of the mitral cell. This might explain the decreased number of impulses when there

Pharmacology of gecko mitral cells 369

D-L-HOMOCYST E ATE

A

Bc- GG EE

Fig. 9. The effect of iontophoretic application of D-L-homocysteate (0.2 M) to the glomerulus and external plexiform layers. The symbols are as in Fig. 6.

is a high concentration of odor stimulation (Shibuya et al., 1977). The action of the mitral cell in response to odor might be controlled at the glomerulus layer and suppressed by the action of periglomerular cells when the olfactory receptor cells were sufficiently excited by high odor concentration.

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