- Email: [email protected]
Physiological, pharmacological, and immunohistochemical characterisation of juxtacellularly labelled neurones in rat nucleus tractus solitarius
Autonomic Neuroscience: Basic and Clinical 98 (2002) 12 – 16 www.elsevier.com/locate/autneu
Physiological, pharmacological, and immunohistochemical characterisation of juxtacellularly labelled neurones in rat nucleus tractus solitarius G.A. Jones a,*, I.J. Llewellyn-Smith b, D. Jordan a a
Department of Physiology, Royal Free and University College Medical School, Royal Free Campus, University College London, Rowland Hill Street, London NW3 3PF, UK b Cardiovascular Neuroscience Group, Cardiovascular Medicine and Centre for Neuroscience, Flinders University, Bedford Park, Adelaide South Australia 5042, Australia
Abstract The pharmacology and anatomy of neurones in the nucleus tractus solitarius (NTS) have proved to be difficult to study in vivo because of their generally small size and high packing density. To overcome these problems, we have developed an approach that combines drug application through multibarrelled electrodes with juxtacellular labelling via an attached single-barrelled electrode followed by immunohistochemical processing. This approach has allowed us to assess the responses of individual NTS neurones in vivo to ionotropic glutamate receptor agonists and antagonists and then, to determine whether the neurones expressed the glutamate receptor subunits, GLUR2,3 and NMDAR2a,b. It should also be possible to extend these techniques further and correlate morphology with these features and to examine pharmacologically characterised, dye-filled neurones at the ultrastructural level. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Amino acid receptors; Blood pressure; Ionophoresis; Juxtacellular labelling; Medulla
The nucleus of the solitary tract (NTS) is the primary site of termination for sensory afferents from a variety of cardiovascular and respiratory receptors, including arterial baroreceptors. Information from these receptors is then integrated and processed within the NTS before transmission to the various motoneuronal pools controlling autonomic outflow (Jordan and Spyer, 1986). In these sensory afferent pathways, glutamate is thought to be the main excitatory neurotransmitter, both at the primary level and at subsequent synapses (Zhang and Mifflin, 1998). However, the relative contribution of the different glutamate receptor subtypes at the different postsynaptic sites is unknown. It has been difficult to apply conventional anatomical and pharmacological approaches to the in vivo study of NTS neurones due to the small size and close packing of the neurones in this region. We have combined the use of compound electrodes with the juxtacellular labelling method of Pinault (1996) to perform stable extracellular recordings of NTS neurones that respond to vagal stimulation in vivo, under a
*
Corresponding author. Tel.: +44-171-830-2771; fax: +44-171-4331921. E-mail address: [email protected] (G.A. Jones).
variety of ionophoretic conditions. We then processed the tissue for morphological or immunohistochemical studies on the filled neurones so that we could correlate the anatomical and electrophysiological characteristics of the juxtacellularly labelled neurones. Other studies of the physiology and anatomy of the NTS have used in situ preparations of the brainstem, which retains some of its reflexes (e.g. Deuchars et al., 2000). Some of the data presented here have appeared in abstract form (Jones and Jordan, 2001). Male Sprague – Dawley rats (300 – 360 g) were anaesthetized (pentobarbital sodium, 60 mg kg 1, i.p.), neuromuscularly blocked (gallamine, 8 mg kg 1 or a-bungarotoxin, 150 Ag kg 1, i.v.), and artificially ventilated. Anaesthesia and neuromuscular block were monitored and maintained. All the experiments were carried out under a UK Home Office Licence, in accordance with the regulations of the UK Animals (Scientific Procedures) Act, 1986. Single-unit activities of NTS neurones were recorded using a glass recording electrode attached to a seven-barrelled ionophoretic electrode. The recording electrode was filled with 1.5% Neurobiotin in 0.5 M NaCl (resistance 15– 30 MV), and the other barrels were filled with AMPA (20 mM), NMDA (20 mM), DNQX (20 mM), and AP-5 (20 mM). All drug solutions were made according to manufac-
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 2 2 - X
G.A. Jones et al. / Autonomic Neuroscience: Basic and Clinical 98 (2002) 12–16
turer’s guidelines, diluted in 0.9% saline, and adjusted to pH 8 –8.5 for ionophoresis. Extracellular signals were amplified using an Axoclamp 2A (Axon instruments) and filtered (0.1 –3 kHz). Neurones that responded to electrical stimulation of the vagus nerve (1 ms, 50– 500 AA, 0.6 –1 Hz) were tested before, during, and after application of AP-5 or DNQX (20 – 120 nA), antagonists of NMDA, and non-NMDA receptors, respectively. AMPA and NMDA (0 –100 nA) were applied before, during, and after application of the antagonists to establish effective blockade of the appropriate receptors. Second-order neurones and presumed polysynaptic neurones could be differentiated on the basis of the variability in the latency of their response to the electrical stimulation of the vagus nerve (Se´voz-Couche et al., 2000). Differences between second-order and polysynaptic neurones were not examined in this study. Following drug application, vagally evoked neurones were subjected to positive current pulses (200 ms, 50% duty cycle, 1 – 15 min) delivered through the recording electrode at sufficient amplitude to entrain the cell to fire with the positive current (0.1 – 10 nA). Current amplitude was continuously adjusted to maintain entrainment but limit possible damage to the neurone. Recordings were often maintained subsequent to juxtacellular labelling with no discernible difference in the physiology of the neurone. Subsequent to juxtacellular labelling, neurones were left for at least 2 h, after which, animals were perfused via the ascending aorta with 200 ml heparinised physiological saline followed by at least 500 ml of fixative (4% formaldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were post-fixed in the same fixative for at least 2 days at 4 jC. To determine the morphology of juxtacellularly labelled neurones, 50-Am slices of the brainstem were cut on a Vibratome and incubated for 2 –3 days in ExtrAvidin conjugated to horseradish peroxidase (Sigma) diluted, 1:1000, in immunobuffer (10 mM Tris, 10 mM phosphate buffer, 0.9% saline, 0.05% merthiolate, and 0.03% Triton, pH 7.4). Neurones were then visualised using a glucose oxidase Ni-DAB method (Llewellyn-Smith et al., 1993). To detect immunoreactivity for glutamate receptor subunits in labelled neurones, brainstems were dehydrated rapidly in ethanol and then infiltrated with and embedded in polyethylene glycol (Murphy et al., 1998). Serial sections, 10 Am thick, were cut on a rotary microtome into phosphate buffer and then washed in immunobuffer. After a blocking step in 10% normal horse serum in Tris-phosphate buffered saline containing 0.3% Triton X-100 (TPBS-Triton), the sections were incubated for 24 h at room temperature in rabbit anti-GLUR2,3 (1:250; Chemicon, Temecula, CA) or rabbit anti-NMDAR2a,b (1:500; Chemicon) antiserum made up in the same diluent, which also contained Alexa Fluor 488-streptavidin (Molecular Probes, Eugene, OR). After several washes, the sections were exposed overnight to Cy3anti-rabbit immunoglobulin (Jackson ImmunoResearch, West Grove, PA) in TPBS-Triton containing 1% serum,
13
washed, and mounted serially on untreated slides in buffered glycerol. Sections were viewed with an Olympus widefield AX70 fluorescence microscope and images were collected with a Hamamatsu Orca cooled CCD camera (Hamamatsu City, Japan). Using a seven-barrelled compound glass electrode (Fig. 1), it was possible to apply glutamate receptor agonists and antagonists to vagal-evoked NTS neurones and test their responses to these drugs (Fig. 2). A total of 25 NTS neurones was recorded. Nineteen responded to stimulation of the vagus nerve. Of the 21 neurones tested, all were excited by AMPA, as were all 15 tested with NMDA (Fig. 2). DNQX blocked the response to AMPA in the 10 neurones tested (Fig. 2) and produced inhibition of vagus nerve-evoked activity in 6/6 neurones (not shown). AP-5 blocked the NMDA response in all four neurones tested (Fig. 2), but did not attenuate the vagus nerve-evoked response in 2/2 neurones (not shown). Following completion of the pharmacological studies, neurones were filled juxtacellularly with Neurobiotin. As previously described by Pinault (1996), entrainment of neuronal firing with the positive current pulse was an essential prerequisite for successful filling (Fig. 3A). Over 80% of the neurones (17/21) were recovered after entrainment, filling, and visualisation with Ni-0DAB. Some neurones were successfully retrieved after entrainment for as little as 1 min. However, detection of neuronal morphology improved with increasing entrainment time so that neurones entrained for 2 min or more showed excellent filling of cell bodies, axons,
Fig. 1. Diagram of a seven-barrelled compound electrode used for extracellular recording, juxtacellular labelling, and ionophoresis. Electrodes were fabricated by bending the tip of a seven-barrelled glass electrode and gluing it to a single-barrelled recording electrode. This was reinforced with epoxy resin and metal bars. Tips were separated by 0 – 10 Am. The multibarrelled electrodes were broken back to between 7 and 15 Am and the single-barrelled to around 1 Am. Scale bar, 10 Am.
14
G.A. Jones et al. / Autonomic Neuroscience: Basic and Clinical 98 (2002) 12–16
Fig. 2. Effects of ionophoresis of glutamate receptor agonist and antagonists on vagal-evoked NTS neurones. Panels show continuous ratemeter records (1s bins) illustrating the excitatory effects of AMPA and NMDA on a vagal-evoked NTS neurone before (top left), during (top right), and following (bottom left) the application of AP-5. The bottom right panel shows the ionophoresis of AMPA and NMDA during the ionophoresis of DNQX. The time between panels is not continuous; antagonists were applied for 1 – 2 min. The bars represent drug ejections and the currents are specified.
Fig. 3. Entrainment and juxtacellular labelling of vagal-evoked NTS neurones. (A) Three discontinuous raw traces showing the activity of a neurone during a successful entrainment process. Top panel, the current intensity is not sufficient to entrain the neurone and activity is not related to current ejection. Middle panel, the current amplitude is great enough to entrain the cellular activity to the positive-going phase of current ejection. Note the increase in noise during the transition to entrainment indicated by the arrow. Lower panel, the cell has been successfully entrained and the current intensity lowered so as to avoid damage to the neurone. (B) Photomicrograph of a single, vagally evoked NTS neurone that was recovered after entrainment, juxtacellular labelling, and processing for morphological characterisation. The neurone was filled with Neurobiotin and visualised with ExtrAvidin-HRP and a Ni-DAB reaction. Scale bar, 20 Am. (C) Camera lucida reconstruction of the neurone shown in B. The reconstruction was done using a 40 objective. In both B and C, the axon is indicated by the arrow.
G.A. Jones et al. / Autonomic Neuroscience: Basic and Clinical 98 (2002) 12–16
15
Fig. 4. Immunohistochemistry for the NMDA receptor subunits 2a,b in a vagally evoked, juxtacellularly labelled NTS neuron. (A) Visualisation of Neurobiotin in the vagal-evoked neuron. The soma and a dendrite have been well filled. The nucleus of the neuron is marked with a star. (B) Immunoreactivity for NMDAR2a,b in the neuron. Staining appears as a fluorescent network around the neuronal nucleus (star).
and dendrites (Fig 3B). Indeed, camera lucida reconstructions of neurones filled for 5 min or more have axons that can be traced for several hundred microns (Fig 3C). Juxtacellularly labelled neurones were not as readily detected with Alexa Fluor 488-streptavidin as with ExtrAvidin-HRP. Recovery was consistently obtained with the fluorescent tag only when neurones had been entrained for at least 3 min. Combining detection of the juxtacellular label with Alexa Fluor 488-streptavidin (Fig. 4A) and immunohistochemistry for the glutamate receptor subunits, GLUR2,3 and NMDAR 2a,b (Fig. 4B) allowed us to determine the receptor phenotypes of vagally evoked NTS neurones whose excitatory amino acid pharmacology had been characterised. The GLUR2,3 immunoreactivity filled the neurones (not shown), whereas immunoreactivity for NMDAR2a,b appeared as an anastomosing network around neuronal nuclei (Fig. 4B), probably due to concentration of the antigen in the Golgi apparatus. The critical aspects of the immunohistochemical method were the use of 10-Am sections and a Cy3-labelled secondary antibody so that visualisation of immunoreactivity for glutamate receptor subunits was optimised. The results of this study prove that it is possible to (1) functionally identify a neurone in vivo on the basis of its response to nerve stimulation, (2) define its receptor profile pharmacologically in vivo, (3) juxtacellularly label the neurone, and then (4) either demonstrate its somatodendritic architecture with avidin – peroxidase or its expression of amino acid receptor subunits with fluorescence immunohistochemistry. In addition to nerve stimulation, responses to other physiological manipulations, such as administration of phenyl biguanide into the right atrium or intravenous injection of phenylephrine, can be measured before juxtacellular labelling (Jones and Jordan, unpublished). Compared to intracellular recording, the juxtacellular labelling method is fast, reliable, and noninvasive. Excellent anatomical results can be obtained provided the activity of the targeted neurone is entrained with the positive current pulses from the electrode and entrainment lasts long enough to adequately fill the cell (in our hands, about 2 min for detection with ExtrAvidin-
HRP and 3 min for detection with Alexa Fluor 488-streptavidin). Furthermore, combining a multibarrelled pipette with a pipette filled with Neurobiotin allows the neuronal response to a variety of agonists and antagonists to be tested. Juxtacellular labelling has already proved to be very useful for studying central autonomic neurones (for example, see Schreihofer and Guyenet, 1997; Verberne et al., 1999; Kirouac and Pittman, 1999; Llewellyn-Smith et al., 2001; Aicher et al., 2001). Our addition of pharmacology and complementary detection of receptor subunits to the technique should make it even more powerful.
Acknowledgements This work was supported by the Wellcome Trust (UK), the National Health and Medical Research Council of Australia, and the National Heart Foundation of Australia. We thank Carolyn Martin and Lee Travis for the expert technical assistance and Chris Guillebaud for doing the neuronal reconstruction.
References Aicher, S.A., Schreihofer, A.M., Kraus, J.A., Sharma, S., Milner, T.A., Guyenet, P.G., 2001. Mu-opioid receptors are present in functionally identified sympathoexcitatory neurons in the rat rostral ventrolateral medulla. J. Comp. Neurol. 433, 34 – 47. Deuchars, J., Li, Y.-W., Kasparov, S., Paton, J.F.R., 2000. Morphological and electrophysiological properties of neurones in the dorsal vagal complex of the rat activated by arterial baroreceptors. J. Comp. Neurol. 417, 233 – 249. Jones, G.A., Jordan, D., 2001. Single unit recordings, ionophoresis and histological labelling of nucleus tractus solitarii neurones in vivo: a new development in juxtacellular labelling. J. Physiol. 533, 83 – 84. Jordan, D., Spyer, K.M., 1986. Brainstem integration of cardiovascular and pulmonary afferent activity. Prog. Brain Res. 67, 295 – 314. Kirouac, G.J., Pittman, Q.J., 1999. Identification of barosensitive neurons in the mediobasal forebrain using juxtacellular labeling. Am. J. Physiol. 276, R1766 – R1771. Llewellyn-Smith, I.J., Pilowsky, P., Minson, J.B., 1993. The tungstate-sta-
16
G.A. Jones et al. / Autonomic Neuroscience: Basic and Clinical 98 (2002) 12–16
bilized tetramethylbenzidine reaction for light and electron microscopic immunocytochemistry and for revealing biocytin-filled neurons. J. Neurosci. Methods 46, 27 – 40. Llewellyn-Smith, I.J., Schreihofer, A.M., Guyenet, P.G., 2001. Distribution and amino acid content of enkephalin-immunoreactive inputs onto juxtacellularly-labeled bulbospinal barosensitive neurons in rat rostral ventrolateral medulla. Neuroscience 108, 307 – 322. Murphy, S.M., Matthew, S.E., Rodgers, H.F., Lituri, D.T., Gibbins, I.L., 1998. Synaptic organisation of neuropeptide-containing preganglionic boutons in lumbar sympathetic ganglia of guinea pigs. J. Comp. Neurol. 398, 551 – 567. Pinault, D., 1996. A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin. J. Neurosci. Methods 65, 113 – 136.
Schreihofer, A.M., Guyenet, P.G., 1997. Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo. J. Comp. Neurol. 387, 524 – 536. Se´voz-Couche, C., Wang, Y., Ramage, A.G., Spyer, K.M., Jordan, D., 2000. In vivo modulation of nucleus tractus solitarius (NTS) neurones by activation of 5-hydroxytryptamine(2) receptors in rats. Neuropharmacology 39, 2006 – 2016. Verberne, A.J.M., Stornetta, R.L., Guyenet, P.G., 1999. Properties of C1 and other ventrolateral medullary neurones with hypothalamic projections in the rat. J. Physiol. 517, 477 – 494. Zhang, J., Mifflin, S.W., 1998. Differential roles for NMDA and nonNMDA receptor subtypes in baroreceptor afferent integration in the nucleus of the solitary tract of the rat. J. Physiol. 511, 733 – 745.
Physiological, pharmacological, and immunohistochemical characterisation of juxtacellularly labelled neurones in rat nucleus tractus solitarius G.A. Jones a,*, I.J. Llewellyn-Smith b, D. Jordan a a
Department of Physiology, Royal Free and University College Medical School, Royal Free Campus, University College London, Rowland Hill Street, London NW3 3PF, UK b Cardiovascular Neuroscience Group, Cardiovascular Medicine and Centre for Neuroscience, Flinders University, Bedford Park, Adelaide South Australia 5042, Australia
Abstract The pharmacology and anatomy of neurones in the nucleus tractus solitarius (NTS) have proved to be difficult to study in vivo because of their generally small size and high packing density. To overcome these problems, we have developed an approach that combines drug application through multibarrelled electrodes with juxtacellular labelling via an attached single-barrelled electrode followed by immunohistochemical processing. This approach has allowed us to assess the responses of individual NTS neurones in vivo to ionotropic glutamate receptor agonists and antagonists and then, to determine whether the neurones expressed the glutamate receptor subunits, GLUR2,3 and NMDAR2a,b. It should also be possible to extend these techniques further and correlate morphology with these features and to examine pharmacologically characterised, dye-filled neurones at the ultrastructural level. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Amino acid receptors; Blood pressure; Ionophoresis; Juxtacellular labelling; Medulla
The nucleus of the solitary tract (NTS) is the primary site of termination for sensory afferents from a variety of cardiovascular and respiratory receptors, including arterial baroreceptors. Information from these receptors is then integrated and processed within the NTS before transmission to the various motoneuronal pools controlling autonomic outflow (Jordan and Spyer, 1986). In these sensory afferent pathways, glutamate is thought to be the main excitatory neurotransmitter, both at the primary level and at subsequent synapses (Zhang and Mifflin, 1998). However, the relative contribution of the different glutamate receptor subtypes at the different postsynaptic sites is unknown. It has been difficult to apply conventional anatomical and pharmacological approaches to the in vivo study of NTS neurones due to the small size and close packing of the neurones in this region. We have combined the use of compound electrodes with the juxtacellular labelling method of Pinault (1996) to perform stable extracellular recordings of NTS neurones that respond to vagal stimulation in vivo, under a
*
Corresponding author. Tel.: +44-171-830-2771; fax: +44-171-4331921. E-mail address: [email protected] (G.A. Jones).
variety of ionophoretic conditions. We then processed the tissue for morphological or immunohistochemical studies on the filled neurones so that we could correlate the anatomical and electrophysiological characteristics of the juxtacellularly labelled neurones. Other studies of the physiology and anatomy of the NTS have used in situ preparations of the brainstem, which retains some of its reflexes (e.g. Deuchars et al., 2000). Some of the data presented here have appeared in abstract form (Jones and Jordan, 2001). Male Sprague – Dawley rats (300 – 360 g) were anaesthetized (pentobarbital sodium, 60 mg kg 1, i.p.), neuromuscularly blocked (gallamine, 8 mg kg 1 or a-bungarotoxin, 150 Ag kg 1, i.v.), and artificially ventilated. Anaesthesia and neuromuscular block were monitored and maintained. All the experiments were carried out under a UK Home Office Licence, in accordance with the regulations of the UK Animals (Scientific Procedures) Act, 1986. Single-unit activities of NTS neurones were recorded using a glass recording electrode attached to a seven-barrelled ionophoretic electrode. The recording electrode was filled with 1.5% Neurobiotin in 0.5 M NaCl (resistance 15– 30 MV), and the other barrels were filled with AMPA (20 mM), NMDA (20 mM), DNQX (20 mM), and AP-5 (20 mM). All drug solutions were made according to manufac-
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 2 2 - X
G.A. Jones et al. / Autonomic Neuroscience: Basic and Clinical 98 (2002) 12–16
turer’s guidelines, diluted in 0.9% saline, and adjusted to pH 8 –8.5 for ionophoresis. Extracellular signals were amplified using an Axoclamp 2A (Axon instruments) and filtered (0.1 –3 kHz). Neurones that responded to electrical stimulation of the vagus nerve (1 ms, 50– 500 AA, 0.6 –1 Hz) were tested before, during, and after application of AP-5 or DNQX (20 – 120 nA), antagonists of NMDA, and non-NMDA receptors, respectively. AMPA and NMDA (0 –100 nA) were applied before, during, and after application of the antagonists to establish effective blockade of the appropriate receptors. Second-order neurones and presumed polysynaptic neurones could be differentiated on the basis of the variability in the latency of their response to the electrical stimulation of the vagus nerve (Se´voz-Couche et al., 2000). Differences between second-order and polysynaptic neurones were not examined in this study. Following drug application, vagally evoked neurones were subjected to positive current pulses (200 ms, 50% duty cycle, 1 – 15 min) delivered through the recording electrode at sufficient amplitude to entrain the cell to fire with the positive current (0.1 – 10 nA). Current amplitude was continuously adjusted to maintain entrainment but limit possible damage to the neurone. Recordings were often maintained subsequent to juxtacellular labelling with no discernible difference in the physiology of the neurone. Subsequent to juxtacellular labelling, neurones were left for at least 2 h, after which, animals were perfused via the ascending aorta with 200 ml heparinised physiological saline followed by at least 500 ml of fixative (4% formaldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were post-fixed in the same fixative for at least 2 days at 4 jC. To determine the morphology of juxtacellularly labelled neurones, 50-Am slices of the brainstem were cut on a Vibratome and incubated for 2 –3 days in ExtrAvidin conjugated to horseradish peroxidase (Sigma) diluted, 1:1000, in immunobuffer (10 mM Tris, 10 mM phosphate buffer, 0.9% saline, 0.05% merthiolate, and 0.03% Triton, pH 7.4). Neurones were then visualised using a glucose oxidase Ni-DAB method (Llewellyn-Smith et al., 1993). To detect immunoreactivity for glutamate receptor subunits in labelled neurones, brainstems were dehydrated rapidly in ethanol and then infiltrated with and embedded in polyethylene glycol (Murphy et al., 1998). Serial sections, 10 Am thick, were cut on a rotary microtome into phosphate buffer and then washed in immunobuffer. After a blocking step in 10% normal horse serum in Tris-phosphate buffered saline containing 0.3% Triton X-100 (TPBS-Triton), the sections were incubated for 24 h at room temperature in rabbit anti-GLUR2,3 (1:250; Chemicon, Temecula, CA) or rabbit anti-NMDAR2a,b (1:500; Chemicon) antiserum made up in the same diluent, which also contained Alexa Fluor 488-streptavidin (Molecular Probes, Eugene, OR). After several washes, the sections were exposed overnight to Cy3anti-rabbit immunoglobulin (Jackson ImmunoResearch, West Grove, PA) in TPBS-Triton containing 1% serum,
13
washed, and mounted serially on untreated slides in buffered glycerol. Sections were viewed with an Olympus widefield AX70 fluorescence microscope and images were collected with a Hamamatsu Orca cooled CCD camera (Hamamatsu City, Japan). Using a seven-barrelled compound glass electrode (Fig. 1), it was possible to apply glutamate receptor agonists and antagonists to vagal-evoked NTS neurones and test their responses to these drugs (Fig. 2). A total of 25 NTS neurones was recorded. Nineteen responded to stimulation of the vagus nerve. Of the 21 neurones tested, all were excited by AMPA, as were all 15 tested with NMDA (Fig. 2). DNQX blocked the response to AMPA in the 10 neurones tested (Fig. 2) and produced inhibition of vagus nerve-evoked activity in 6/6 neurones (not shown). AP-5 blocked the NMDA response in all four neurones tested (Fig. 2), but did not attenuate the vagus nerve-evoked response in 2/2 neurones (not shown). Following completion of the pharmacological studies, neurones were filled juxtacellularly with Neurobiotin. As previously described by Pinault (1996), entrainment of neuronal firing with the positive current pulse was an essential prerequisite for successful filling (Fig. 3A). Over 80% of the neurones (17/21) were recovered after entrainment, filling, and visualisation with Ni-0DAB. Some neurones were successfully retrieved after entrainment for as little as 1 min. However, detection of neuronal morphology improved with increasing entrainment time so that neurones entrained for 2 min or more showed excellent filling of cell bodies, axons,
Fig. 1. Diagram of a seven-barrelled compound electrode used for extracellular recording, juxtacellular labelling, and ionophoresis. Electrodes were fabricated by bending the tip of a seven-barrelled glass electrode and gluing it to a single-barrelled recording electrode. This was reinforced with epoxy resin and metal bars. Tips were separated by 0 – 10 Am. The multibarrelled electrodes were broken back to between 7 and 15 Am and the single-barrelled to around 1 Am. Scale bar, 10 Am.
14
G.A. Jones et al. / Autonomic Neuroscience: Basic and Clinical 98 (2002) 12–16
Fig. 2. Effects of ionophoresis of glutamate receptor agonist and antagonists on vagal-evoked NTS neurones. Panels show continuous ratemeter records (1s bins) illustrating the excitatory effects of AMPA and NMDA on a vagal-evoked NTS neurone before (top left), during (top right), and following (bottom left) the application of AP-5. The bottom right panel shows the ionophoresis of AMPA and NMDA during the ionophoresis of DNQX. The time between panels is not continuous; antagonists were applied for 1 – 2 min. The bars represent drug ejections and the currents are specified.
Fig. 3. Entrainment and juxtacellular labelling of vagal-evoked NTS neurones. (A) Three discontinuous raw traces showing the activity of a neurone during a successful entrainment process. Top panel, the current intensity is not sufficient to entrain the neurone and activity is not related to current ejection. Middle panel, the current amplitude is great enough to entrain the cellular activity to the positive-going phase of current ejection. Note the increase in noise during the transition to entrainment indicated by the arrow. Lower panel, the cell has been successfully entrained and the current intensity lowered so as to avoid damage to the neurone. (B) Photomicrograph of a single, vagally evoked NTS neurone that was recovered after entrainment, juxtacellular labelling, and processing for morphological characterisation. The neurone was filled with Neurobiotin and visualised with ExtrAvidin-HRP and a Ni-DAB reaction. Scale bar, 20 Am. (C) Camera lucida reconstruction of the neurone shown in B. The reconstruction was done using a 40 objective. In both B and C, the axon is indicated by the arrow.
G.A. Jones et al. / Autonomic Neuroscience: Basic and Clinical 98 (2002) 12–16
15
Fig. 4. Immunohistochemistry for the NMDA receptor subunits 2a,b in a vagally evoked, juxtacellularly labelled NTS neuron. (A) Visualisation of Neurobiotin in the vagal-evoked neuron. The soma and a dendrite have been well filled. The nucleus of the neuron is marked with a star. (B) Immunoreactivity for NMDAR2a,b in the neuron. Staining appears as a fluorescent network around the neuronal nucleus (star).
and dendrites (Fig 3B). Indeed, camera lucida reconstructions of neurones filled for 5 min or more have axons that can be traced for several hundred microns (Fig 3C). Juxtacellularly labelled neurones were not as readily detected with Alexa Fluor 488-streptavidin as with ExtrAvidin-HRP. Recovery was consistently obtained with the fluorescent tag only when neurones had been entrained for at least 3 min. Combining detection of the juxtacellular label with Alexa Fluor 488-streptavidin (Fig. 4A) and immunohistochemistry for the glutamate receptor subunits, GLUR2,3 and NMDAR 2a,b (Fig. 4B) allowed us to determine the receptor phenotypes of vagally evoked NTS neurones whose excitatory amino acid pharmacology had been characterised. The GLUR2,3 immunoreactivity filled the neurones (not shown), whereas immunoreactivity for NMDAR2a,b appeared as an anastomosing network around neuronal nuclei (Fig. 4B), probably due to concentration of the antigen in the Golgi apparatus. The critical aspects of the immunohistochemical method were the use of 10-Am sections and a Cy3-labelled secondary antibody so that visualisation of immunoreactivity for glutamate receptor subunits was optimised. The results of this study prove that it is possible to (1) functionally identify a neurone in vivo on the basis of its response to nerve stimulation, (2) define its receptor profile pharmacologically in vivo, (3) juxtacellularly label the neurone, and then (4) either demonstrate its somatodendritic architecture with avidin – peroxidase or its expression of amino acid receptor subunits with fluorescence immunohistochemistry. In addition to nerve stimulation, responses to other physiological manipulations, such as administration of phenyl biguanide into the right atrium or intravenous injection of phenylephrine, can be measured before juxtacellular labelling (Jones and Jordan, unpublished). Compared to intracellular recording, the juxtacellular labelling method is fast, reliable, and noninvasive. Excellent anatomical results can be obtained provided the activity of the targeted neurone is entrained with the positive current pulses from the electrode and entrainment lasts long enough to adequately fill the cell (in our hands, about 2 min for detection with ExtrAvidin-
HRP and 3 min for detection with Alexa Fluor 488-streptavidin). Furthermore, combining a multibarrelled pipette with a pipette filled with Neurobiotin allows the neuronal response to a variety of agonists and antagonists to be tested. Juxtacellular labelling has already proved to be very useful for studying central autonomic neurones (for example, see Schreihofer and Guyenet, 1997; Verberne et al., 1999; Kirouac and Pittman, 1999; Llewellyn-Smith et al., 2001; Aicher et al., 2001). Our addition of pharmacology and complementary detection of receptor subunits to the technique should make it even more powerful.
Acknowledgements This work was supported by the Wellcome Trust (UK), the National Health and Medical Research Council of Australia, and the National Heart Foundation of Australia. We thank Carolyn Martin and Lee Travis for the expert technical assistance and Chris Guillebaud for doing the neuronal reconstruction.
References Aicher, S.A., Schreihofer, A.M., Kraus, J.A., Sharma, S., Milner, T.A., Guyenet, P.G., 2001. Mu-opioid receptors are present in functionally identified sympathoexcitatory neurons in the rat rostral ventrolateral medulla. J. Comp. Neurol. 433, 34 – 47. Deuchars, J., Li, Y.-W., Kasparov, S., Paton, J.F.R., 2000. Morphological and electrophysiological properties of neurones in the dorsal vagal complex of the rat activated by arterial baroreceptors. J. Comp. Neurol. 417, 233 – 249. Jones, G.A., Jordan, D., 2001. Single unit recordings, ionophoresis and histological labelling of nucleus tractus solitarii neurones in vivo: a new development in juxtacellular labelling. J. Physiol. 533, 83 – 84. Jordan, D., Spyer, K.M., 1986. Brainstem integration of cardiovascular and pulmonary afferent activity. Prog. Brain Res. 67, 295 – 314. Kirouac, G.J., Pittman, Q.J., 1999. Identification of barosensitive neurons in the mediobasal forebrain using juxtacellular labeling. Am. J. Physiol. 276, R1766 – R1771. Llewellyn-Smith, I.J., Pilowsky, P., Minson, J.B., 1993. The tungstate-sta-
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