Electrophysiological Effects of Three Groups of Glutamate Metabotropic Receptors in Rat Piriform Cortex

Transcription

1 Cellular and Molecular Neurobiology, Vol. 26, Nos. 4 6, July/August 2006 ( C 2006) DOI: /s Electrophysiological Effects of Three Groups of Glutamate Metabotropic Receptors in Rat Piriform Cortex Y. Tan, 1 N. Hori, 1 and D. O. Carpenter 1,2 Received August 10, 2005; accepted September 9, 2005; Published online: August 2, 2006 SUMMARY 1. The effects of three metabotropic glutamate receptor (mglur) agonists were tested in two pathways of rat piriform cortex. The group I, II and III mglur agonists used were RS-3,5-dihydroxyphenenylglycine (DHPG) ( µm), (2S,1 S,2 S)-2- Carboxycyclopropyl (L-CCG) ( µm) and L( + )-2-amino-4-phosphonobutyric acid (L-AP4) (5 500 µm), respectively. 2. The effects of the three groups of agonists on synaptic transmission in the two piriform cortex pathways also were examined. All three agonists reduced the amplitude of the monosynaptic EPSPs generated by stimulation of the lateral olfactory tract (LOT) or of the association fiber pathway (ASSN). This was always accompanied by an increase in paired pulse facilitation. 3. Group I and II mglur agonists had similar synaptic effects on the two pathways, while the group III mglur agonist suppressed the LOT pathway more than the association pathway. 4. The group II and III mglur agonists had no effect on passive membrane properties of pyramidal neurons. Group I agonists depolarized the pyramidal neuron membrane potential, and enhanced both membrane resistance and noise. 5. Our data suggest that all three types of mglurs modulate synaptic transmission in both of these pathways in piriform cortex. Only group I agonists alter post-synaptic membrane properties, while all three types of receptor regulate synaptic transmission. Groups I and II are equally potent in the LOT and association fiber pathways, while group III receptors are more potent in the LOT than the association fiber pathways. KEY WORDS: fepsp; mglurs; piriform cortex; synaptic depression. INTRODUCTION L-glutamate is the predominant fast excitatory neurotransmitter in the mammalian central nervous system, but also acts through mglurs to exert more long-lasting actions (Conn and Pin, 1997). The mglur family consists of eight known subtypes that are further categorized into three groups by their pharmacological properties, homology of their amino acid sequences, coupled second messengers and effectors. Group I (mglur1 and 5) receptors are positively coupled via G q /G 11 -like 1 Department of Environmental Health Sciences, School of Public Health, and Institute for Health and the Environment, University at Albany, Five University Place, A 217, Rensselaer, NY 12144, USA. 2 To whom correspondence should be addressed at carpent@uamail.albany.edu /06/ /1 C 2006 Springer Science+Business Media, Inc.

2 916 Tan, Hori, and Carpenter G-proteins to inositol phosphate hydrolysis, while group II (mglur2 and 3) and III (mglur4, 6, 7, and 8) receptors are negatively coupled via G i /G o -like G-proteins to adenylyl cyclase. The piriform cortex is the primary olfactory cortical region in the mammalian brain. It has a more structured anatomical organization than neocortical regions with the pyramidal neurons in a distinct layer (Haberly, 1990). There is evidence from pharmacological (Sugitani et al., 2004) and immunohistochemical studies (Benitez et al., 2000; Wada et al., 1998) for the existence and localization of some types of mglurs in piriform cortex. However, the details on what subtypes of mglurs are present and what their physiologic roles are is unknown for both the lateral olfactory tract (LOT) and association tract pathways. Metabotropic glutamate receptors are known to exist on both presynaptic terminals and postsynaptic sites, and have important roles as modulators of various calcium and potassium ionic currents (Anwyl, 1999). These receptors play important roles in synaptic plasticity (Riedel and Reymann, 1996; Bortolotto et al., 1999). Much of the information available on the effects of metabotropic glutamate receptors comes from hippocampus. Gereau and Conn (1995) reported the two distinct presynaptic receptors (group I and group III) at the Schaffer collateral- CA1 synapse, both of which reduced excitatory synaptic transmission. Davis and Laroache (1996) reported that metabotropic receptors have differential actions when comparing CA1 and dentate areas of hippocampus, which is consistent with the demonstration of differential immunohistochemical localization of immunoreactivity for the three types of receptors in the three primary hippocampal pathways (perforant pathway, mossy fiber pathway and Schaffer collateral) (Shigemoto et al., 1997). They reported group II and III receptors are primarily presynaptic, whereas group I receptors are postsynaptic. To make things even more complex Mannaioni et al. (2001) found that metabotropic receptors 1 and 5, both group I receptors, differentially regulate CA1 pyramidal cell function. We have employed electrophysiological techniques to investigate the effects of the three groups of mglurs on the synaptic transmission in the LOT and association pathways of rat piriform cortex. MATERIALS AND METHODS Young adult male Wistar rats ( g) were euthanized by cervical dislocation. One half of the brain was trimmed in parallel to the lateral olfactory tract (LOT). Piriform cortex slices were cut at 450 µm thickness on a vibratome (OTS 3000, FHC, Inc.) in ice-cold modified Ringer s solution (in mm: sucrose 212.5; KCl 3.5; KH 2 PO 4 1.2; MgSO 4 1.3; CaCl 2 2.4; NaHCO 3 26, glucose 10) saturated with 95% O 2 and 5% CO 2. Slices were then incubated in oxygenated 95% O 2 and 5% CO 2 normal Ringer (125 mm NaCl instead of mm sucrose) for 1 8 h at 34 C before recording. After the recovery period slices were transferred one at a time to a submersion chamber for recording as previously described (Hori et al., 1988).

3 Synaptic Depression of mglurs in Piriform Cortex 917 Field excitatory postsynaptic potentials (fepsps) were recorded in the apical dendrites of pyramidal neurons in response to LOT and/or association fiber stimulation by a monopolar stimulating electrode. Extracellular recording electrodes were pulled from thin-wall glass capillaries (OD 1.2 mm, ID 0.9 mm) and filled with normal Ringer s solution (tip 25 µm, with impedance of less than 2 M ). Paired pulses with an interpulse interval of 35 msec were delivered via a stimulus isolator (A360, World Precision Instruments), driven by a PulseMaster A300 at a frequency of Hz. The pulse was na, 50 µsec duration, adjusted so that half of the maximal EPSP was elicited as by first pulse. Intracellular recordings were made from pyramidal cell neurons using thick-wall (1.2 mm o.d., 0.6 mm i.d.) borosilicate glass electrodes filled with 3 M potassium acetate (ph 7.3; impedance M ) using a Neurodata IR183 amplifier. Input resistance was measured by passing hyperpolarizing current pulses through a bridge circuit, as previously described in studies of hippocampal neurons (Tan et al., 2003). Data were collected with the Whole Cell Analysis Program (John Dempster) and/or a Gould 2400 S chart recorder for off-line analysis. Values are reported as mean ± SEM. The Student s T test was used for statistical analysis and a P value of less than 0.01 was accepted as being a significant level. RESULTS AsshowninFig.1, a brief perfusion of three types of mglur agonists induced an acute reduction of fepsp and an increment of PPF ratio. The PPF ratio is known to reflect presynaptic changes (Hess et al., 1987) in terminal calcium concentration (Debanne et al., 1996). The reduction in fepsp could reflect either presynaptic or postsynaptic actions, but the fact that all three groups of metabotropic agonists resulted in increases in the PPF ratio is evidence that there is a presynaptic action of all of them. The effects of group I agonist, DHPG, were irreversible, but the effects of the group II and III could be recovered after washout. In Fig. 2A, 5-min perfusion of ACPD (25 µm) a broad spectrum agonist (for group I and II), induced a reduction in the fepsp (72.2 ± 4.8%) and an increase (120.8 ± 1.8%) in PPF in the LOT pathway. We then applied specific group I and II mglur agonists, DHPG and CCCG, respectively, to examine the effects. In order to compare the effects of the agonists on the two piriform pathways a pair of electrodes were placed on the LOT and ASSN pathways, and stimuli were delivered every 30 s alternatively to the two pathways and the signal from each pathway was collected with two independent computers for offline analysis. We ascertained that the signals recorded from the two pathways are independent by cross-checking the effects of paired stimuli. In half of the slices a cut right below the LOT fiber pathway was made to physically separate the signals from the two pathways. The Group I mglur agonist, DHPG, induced a similar increase of the fepsp (LOT: 77.1 ± 1.2, ASSN: 76.3 ± 2.6) and a comparative decrease of the PPF ratio (LOT: ± 1.4, ASSN: ± 4.9) in both pathways.

4 918 Tan, Hori, and Carpenter Fig. 1. One of three experiments showing the effects of three mglur agonists on synaptic transmission in the piriform LOT pathway. Five minutes perfusion of mglur agonists L-CCG-I (50 µm), APB (50 µm) and DHPG (25 µm) (indicated by the black bars) resulted in a marked decrement in the fepsp and increment in PPF. It is known that group II and III mglurs frequently exist as presynaptic autoreceptors (Conn and Pin, 1997). We employed CCCG and L-AP4 to examine the effects of these receptors. As showed in Fig. 3, CCCG, the specific group II agonist, caused a marked decrease in the fepsp and increase in PPF. The actions were almost exactly equal in both pathways. The dose-response relationship was quite steep, with there being essentially no effect of 20 µm, and maximal action at 50 µm. More interestingly, L-AP4, the specific group III agonist, induced a differential effect in the two pathways. While the fepsp was depressed and PPF increased in both pathways, there is a larger effect on LOT than on ASSN pathways (Fig. 4). Figure 5 shows the dose-dependence of the actions of L-AP4 on both the fepsp (Fig. 5a and c) and PPF ratio (Fig. 5b and d). The dose-response relationship for L-AP-4 is also very steep for both the fepsp and PPF, and is similar for both actions. The effects of L-AP4 were blocked by a group III antagonist, MSOP (Fig. 6). This provides additional support for the specificity of L-AP-4 acting at group III metabotropic receptors. We were not able to obtain satisfactory blockade of the very potent group I and II receptor agonists. In order to determine whether groups I, II or III agonists had post-synaptic actions in addition to presynaptic depression of transmitter release we made intracellular recordings in piriform cortex pyramidal cells. There were no postsynaptic effects of either CCCG or L-AP-4 (data not shown), indicating that group II and III metabotropic receptors in piriform cortex are exclusively presynaptic. However, the group I agonist, DHPG, triggered membrane depolarization and an increased input

5 Synaptic Depression of mglurs in Piriform Cortex 919 Fig. 2. Effects of ACPD and DHPG on the excitatory pathways in piriform cortex. A. ACPD, a non-specific mglur (group I and II) agonist, reduced the fepsp to 72.2 ± 4.8%, and increased the PPF ratio to ± 1.8% of control. B. DHPG, a specific group I agonist, had similar effects on the fepsp (LOT: 77.1 ± 1.2%, ASSN: 76.3 ± 2.6%) and PPF (LOT: ± 1.4, ± 4.9%) in the LOT and ASSN pathways. resistance which was very long lasting (greater than 2 20 min) (Fig. 7). In addition there was a striking increase in the membrane noise, as reflected by the thickened baseline shown in Fig. 7. The increased noise was correlated with the time course of the increased input resistance. DISCUSSION Our observations document the multiple and diverse roles served by glutamate within even a very restricted brain region. The pyramidal neurons of piriform

6 920 Tan, Hori, and Carpenter Fig. 3. Effects of the group II mglur agonist, CCCG, on two pathways in piriform cortex. A. A brief perfusion of CCCG (50 µm for 5 min) caused a decrease in the fepsp (LOT: 77.1 ± 1.2%, ASSN: 76.3 ± 2.6%) and an increase in the PPF ratio (LOT: ± 1.4%, ASSN: ± 4.9%). B. CCCG at concentrations of 20 µm, 50 µm and 100 µm induced a decrease of 98.1 ± 4.7, 78.9 ± 2.5% and 79.9 ± 2.5% in fepsp in the LOT pathway, and an increase in PPF ratio of ± 1.7%, ± 2.0% and ± 4.4%, respectively. The effects of CCCG on two pathways are not significantly different. The dashed line is the control baseline. cortex exhibit all three major types of ionotropic glutamate receptors (NMDA, AMPA and kainite receptors) (ffrench-mullen et al., 1986), and our present results show all three major categories of metabotropic receptors. Furthermore, these various receptors are not randomly distributed, but some are on presynaptic terminals whereas others are on postsynaptic membranes; in addition the pattern of presynaptic receptors is not the same on inputs from different pathways. Ong et al. (1998) have shown that in cortical neurons there is differential

7 Synaptic Depression of mglurs in Piriform Cortex 921 Fig. 4. Differential effects the group III agonist, L-AP4, on two pathways in piriform cortex. A 5-min perfusion of L-AP4 (50 µm) induced a decrease in fepsp (LOT: 39.9 ± 6.3%, ASSN: 73.8 ± 5.8%) and an increase in PPF ratio (LOT: ± 8.4%, ± 8.4%) in the two pathways in piriform cortex. The effects of both agonists on both pathways are statistically significant (p < 0.01) locations of metabotropic subtypes between postsynaptic sites on cell bodies and dendrites. A major purpose of metabotropic glutamate receptors appears to be the regulation of transmitter release, and we find all three types of metabotropic receptors on presynaptic terminals. While the efficacy of groups I and II receptors appear approximately equal on LOT and association fiber terminals, group III receptors are more potent on LOT terminals than on association fibers. Our observations provide physiologic confirmation of some previous immunolocation studies. Kinoshita et al. (1996) reported finding mglur8, a type III metabotropic receptor, concentrated in axon terminals in layer 1a of piriform cortex, which is the termination of the LOT fibers. Kinzie et al. (1997) reported finding mglur7 and Benitez et al. (2000) showed the presence of mglur4a, both also a type III receptor, in both layer 1a, where LOT fibers terminate, and in layer 1b, where the association fibers terminate. Type III metabotropic glutamate receptors in LOT terminals have been shown to mediate the habituation to odors (Best et al., 2005). It is possible that the greater alteration of the EPSP and PPF which we see in the LOT pathway as compared to the association fiber pathway may reflect the lack of mglur8 in layer 1b. In contrast to group II and III, group I receptors appear to be located on both presynaptic terminals and postsynaptic neurons. This is also the case in hippocampal CA1 (Tan et al., 2003). Sugitani et al. (2002) have reported calcium imaging studies on guinea pig piriform cortex that show a late rise in intracellular calcium upon stimulation of the association fiber pathway in the presence of blockers of AMPA and NMDA receptors, which was blocked by ( + )-alpha-methyl-4-

8 922 Tan, Hori, and Carpenter Fig. 5. Dose-dependent effects of L-AP-4 on the fepsp (A, C) and PPF ratio (B, D). The r 2 for C is 96.6% and D 98.0%.

9 Synaptic Depression of mglurs in Piriform Cortex 923 Fig. 6. Antagonism of the effects of L-AP-4 by MSOP (50 µm). carboxyphenylglycine, an group I/II mglur antagonist. Wada et al. (1998) did not find changes in mglur1 or mglur5 levels after lesion of the LOT, which led Sugitani et al. (2002) to suggest that the postsynaptic group I receptors are associated specifically with the association fiber pathway. However, if these receptors directly alter membrane potential and resistance it is likely that all synaptic transmission is affected, even if they are relatively more concentrated in the 1b region. Our observations provide additional evidence for the multiple roles glutamate plays in the mammalian cortex, which are mediated by the multiple ionotropic and metabotropic receptors located at both pre- and post-synaptic sites. Fig. 7. The effects of a brief perfusion of DHPG on membrane potential noise and input resistance. An intracellular recording was made from one pyramidal neuron. A brief perfusion of DHPG (20 µm) causes a membrane depolarization, increase in input resistance as measured by constant current pulses, and noise enhancement. Scale bar: horizontal,10 sec: vertical, 10 mv.

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