Interneurons in the mouse visual thalamus maintain a high degree of retinal convergence throughout postnatal development
© Seabrook et al.; licensee BioMed Central Ltd. 2013
Received: 21 September 2013
Accepted: 6 December 2013
Published: 21 December 2013
The dorsal lateral geniculate nucleus (dLGN) of the mouse thalamus has emerged as a powerful experimental system for understanding the refinement of developing sensory connections. Interestingly, many of the basic tenets for such developmental remodeling (for example, pruning of connections to form precise sensory maps) fail to take into account a fundamental aspect of sensory organization, cell-type specific wiring. To date, studies have focused on thalamocortical relay neurons and little is known about the development of retinal connections onto the other principal cell type of dLGN, intrinsic interneurons. Here, we used a transgenic mouse line in which green fluorescent protein (GFP) is expressed within dLGN interneurons (GAD67-GFP), making it possible to visualize them in acutely prepared thalamic slices in order to examine their morphology and functional patterns of connectivity throughout postnatal life.
GFP-expressing interneurons were evenly distributed throughout dLGN and had highly complex and widespread dendritic processes that often crossed eye-specific borders. Estimates of retinal convergence derived from excitatory postsynaptic potential (EPSP) amplitude by stimulus intensity plots revealed that unlike relay cells, interneurons recorded throughout the first 5 weeks of life, maintain a large number (approximately eight to ten) of retinal inputs.
The lack of pruning onto interneurons suggests that the activity-dependent refinement of retinal connections in dLGN is cell-type specific. The high degree of retinal convergence onto interneurons may be necessary for these cells to provide both widespread and local forms of inhibition in dLGN.
KeywordsDorsal lateral geniculate nucleus Interneuron Retinogeniculate pathway Retinal convergence
The dorsal lateral geniculate nucleus (dLGN) serves as the primary relay of visual information to cortex. Underlying the faithful relay of retinal signals are the precise patterns of connectivity between retinal ganglion cells (RGCs) and thalamocortical relay cells. Initially, projections from both eyes terminate diffusely in dLGN and single relay cells receive input from a dozen or so RGCs [1–3]. However, the retinogeniculate pathway undergoes a period of activity-dependent refinement in which projections from the two eyes segregate to form non-overlapping eye-specific domains and the number of functional retinal inputs onto relay cells is reduced to a few, to reflect the adult-like pattern of convergence . Far less is known about the pattern of retinal convergence onto interneurons, the other principal cell type present in dLGN. These local-circuit neurons are involved in feedforward inhibition onto relay cells and play a role in contrast gain control, shaping receptive fields of relay cells, and altering the temporal precision of retinal inputs [5, 6].
Typically, the study of retinal convergence has been performed in acute thalamic slice preparations where synaptic responses of dLGN cells are evoked by electrical stimulation of the optic tract. However, using this approach to assess retinal convergence onto interneurons is difficult since interneurons comprise a very small percentage of the total population of dLGN cells in mouse [2, 7] and they are not readily distinguished under differential interference contrast (DIC) optics. Here we overcame these obstacles by using transgenic mice that express enhanced green fluorescent protein (GFP) in γ-aminobutyric acid (GABA)ergic interneurons (GAD67-GFP) . Such cell-type specific visualization via GFP allowed us to readily target interneurons for in vitro recordings and test whether the age-related pruning of retinal inputs onto dLGN cells varies by cell type.
Using the GAD67-GFP mouse we were able to readily identify and target dLGN interneurons across a wide range of postnatal ages. We found that these GFP-expressing neurons were distributed evenly throughout dLGN and possessed the hallmark structural and functional features reported for rodent interneurons [11–17]. Most importantly, our results revealed that dLGN interneurons in mouse maintain a high level of retinal convergence throughout postnatal development. Even after 4 weeks of age, a single interneuron receives input from as many as eight to ten RGCs. This is in stark contrast to the pattern of convergence for developing relay cells which experience a fourfold to sixfold decrease, so by 3 weeks of age a single relay neuron receives input from only one to three RGCs [1–3]. It is important to note that our quantification of the number of retinal inputs a given interneuron receives is an estimate of retinal convergence. Indeed, our estimates may be influenced by such non-synaptic factors as ionic driving force and/or the activation of voltage-gated conductances. While such non-linearities could affect EPSP amplitude their potential impact would be similar across cells. We also acknowledge that there are a number of ways to assess retinal convergence and at least for the published studies pertaining to dLGN relay cells, they yield similar estimates [1–3]. Of notable significance, is that the EPSP amplitude by stimulus intensity plots of interneurons increased in a graded manner. A graded function reflects a high level of convergence, whereas a step-like one, a low level of convergence [1–3]. Interneurons maintain a graded function throughout development while relay cells show a change with age, from graded to step-like, suggesting retinal inputs onto relay cells are pruned during early postnatal life.
Such differences in the adult pattern of convergence are consistent with some of the known functional and structural features of these cell types. Compared to relay cells, interneurons have larger receptive fields [18–20] and tend to have a disproportionately higher number of retinal synapses compared to non-retinal ones [21, 22]. Unlike relay cells, which provide the primary excitatory drive for visual cortical neurons, interneurons inhibit the activity of relay cells through complex synaptic arrangements that involve both conventional axonal (F1) as well as dendritic (F2) terminals [5, 23]. Global inhibition encompasses large sectors of dLGN and seems to require coordinated input from several RGCs converging onto a single interneuron . Such activation is needed in order to engage both F1 and F2 terminals that are distributed throughout the extensive processes of a given interneuron. Additionally, a more local form of inhibition can be accomplished via the direct activation of an isolated dendritic F2 terminal that makes contact with a single relay cell [25, 26]. In this context, a single interneuron could have hundreds of these elements dispersed throughout their dendritic fields [5, 26] potentially receiving input from many RGCs. Such high levels of convergence are even more likely when one considers that interneurons have highly complex and expansive dendritic fields [13, 15–17] that can even extend across eye-specific domains.
Perhaps the most remarkable aspect of these results is the apparent lack of age-related retinal pruning onto interneurons. It is widely believed that such refinement is mediated by spontaneous retinal activity [4, 27]. In developing dLGN relay cells, retinal activity evokes large excitatory postsynaptic potentials that activate plateau-like depolarizations that are mediated by high threshold, L-type Ca2+ channels [2, 3]. The Ca2+ influx through L-type channels has been linked to cAMP response element-binding protein (CREB)-related signaling cascades proven to be critical for the refinement of retinogeniculate projections into segregated eye-specific domains [28, 29]. While interneurons are reported to have L-type Ca2+ activity , we failed to detect retinally evoked plateau potentials. Thus, an intriguing possibility that warrants further testing is whether these events are the candidate mechanisms responsible for cell-type specific refinement.
Experiments were performed on GAD67-GFP mice (JAX, stock no. 007677, Bar Harbor, ME, USA) ranging in age from postnatal day (P) 7 to 33. The GAD67-GFP founder line was on a pigmented background (C57BL/6 × CB6F1/J). All analyses conformed to National Institutes of Health (NIH) guidelines and protocols, approved by the University of Louisville and Virginia Commonwealth University Institutional Animal Care and Use Committees.
In vitro slice physiology and intracellular filling
To examine the synaptic responses evoked by optic tract stimulation, we adopted an acute thalamic slice preparation, which preserves retinal connections and intrinsic circuitry in dLGN [1–3, 10, 23]. Mice were deeply anesthetized with isoflurane vapors and decapitated. Individual (300 μm thick) sections were cut in the parasagittal plane using methods described elsewhere [1, 3, 23]. Sections containing dLGN were placed into a recording chamber and maintained at 32°C and perfused continuously at a rate of 2.0 ml/min with oxygenated artificial cerebrospinal fluid (ACSF; 124 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2.0 mM MgSO4, 26 mM NaHCO3, 10 mM glucose, and 2 mM CaCl2 (saturated with 95% O2/5% CO2), pH 7.4).
In vitro recordings were performed in the whole-cell current-clamp configuration with the aid of DIC and fluorescence optics on a fixed-stage, visualized recording apparatus (Olympus, EX51WI, Shinjuku, Tokyo, JP). Patch electrodes (3 to 7 MΩ) made of borosilicate glass were filled with a solution containing: 140 mM K-gluconate, 10 mM hydroxyethyl piperazine-ethanesulfonic acid (HEPES), 0.3 mM NaCl, 2 mM MgATP, 0.1 mM NaGTP, pH 7.25. Neuronal activity was digitized (10 to 20 kHz) through an interface unit (National Instruments), acquired and stored directly on the computer, and analyzed by using commercial software (Strathclyde Electrophysiology Software, Whole Cell Analysis Program, WinWCP V3.8.2, Glasgow, Scotland, UK). In some cases, the membrane properties and firing characteristics of interneurons were examined by recording the voltage responses to intracellular injections of square-wave current pulses.
To evoke synaptic activity in dLGN, square-wave pulses (0.1 to 0.3 ms, 0.1 to 1 mA) were delivered once every 20 s through a pair of thin-gauge tungsten wires (0.5 MΩ) positioned in optic tract. Stimulating electrodes were connected to a stimulus isolation unit (World Precision Instruments, A360) that received input from a computer controlled, multichannel pulse generator (World Precision Instruments, PulseMaster A300, Sarasota, FL, USA). Estimates of retinal convergence were determined by EPSP amplitude by stimulus intensity plots [2, 3]. These were constructed by first determining the minimum stimulus intensity needed to evoke a postsynaptic response. Once the single fiber response was determined, current intensity was increased in small increments (0.5 to 1.0 μA) until a response of maximal amplitude was consistently reached . A change in amplitude that was equal to or exceeded the value that corresponded to the single fiber response was used to distinguish one input from another. For each intensity value a minimum of five responses were obtained. It is important to note that we saw no evidence of retinally evoked inhibition in our recordings (however, see [14, 15]), nor did we see a change in resting membrane levels even when the highest stimulus intensities were used. To further verify this we compared our recordings performed in normal ACSF with some performed in the presence of 20 μM bicuculine and 10 μM 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP) to block GABAA-mediated and GABAB-mediated activity. There was no significant difference in the number of retinal inputs between cells recorded in the presence or absence of these GABA blockers (t test, P >0.5; mean retinal inputs ± SEM; P11 normal ACSF, 8 ± 1 vs P11 ACSF with GABA antagonists, 7 ± 1; n = 6 cells for both groups).
During some of the recordings a 0.1% to 0.2% biocytin solution containing (in mM): 130 K-gluconate, 10 HEPES, 8 NaCl, 2 MgATP, 0.1 NaGTP, pH 7.25 was included in the patch pipette and neurons were filled by passing alternating positive and negative current pulses (± 0.5 nA, 200 ms) through the recording electrode. After recording, these slices were fixed overnight with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.2 and then incubated for 24 h in a 0.1% solution of Alexa Fluor 647 conjugated to streptavidin (Invitrogen, Carlsbad, CA, USA) dissolved in PBS with 0.1% Triton X-100. Slices were washed with PBS and then mounted with ProLong Gold antifade reagent (Invitrogen).
Cell density measurements of interneurons in dLGN
The overall density of interneurons was determined by counting GFP positive cells within the boundaries of dLGN. These measurements were obtained from 2 to 3 sections corresponding to the middle of dLGN (n = 3 mice; P7, P9, and P26). We also examined whether interneurons showed a preference between binocular and monocular regions of dLGN. To accomplish this we made ipsilateral eye injections of cholera toxin subunit B (CTB) conjugated to Alexa Fluor 594 (Invitrogen) to label uncrossed retinal projections within dLGN . The spatial extent of the ipsilateral patch was then used to delineate monocular and binocular segments of dLGN.
Slow outward rectifying K+ conductance
Artificial cerebrospinal fluid
Analysis of variance
cyclic adenosine monophosphate
cAMP response element-binding protein
Cholera toxin subunit B
Differential interference contrast
dorsal lateral geniculate nucleus
Excitatory postsynaptic current
Excitatory postsynaptic potential
Enhanced green fluorescent protein
Hyperpolarization activated mixed cation conductance
Hydroxyethyl piperazine-ethanesulfonic acid
Rebound low threshold Ca2+ spike
Adenosine 5′-triphosphate magnesium salt
guanosine 5′-triphosphate sodium salt hydrate
Phosphate buffered saline
Retinal ganglion cells
Resting membrane potential
Standard error of the mean
ventral lateral geniculate nucleus.
This work was supported by NIH Grant EY012716 (WG). We thank Rana El-Danaf and Jennifer Rios for their help with confocal imaging of interneurons.
- Chen C, Regehr WG: Developmental remodeling of the retinogeniculate synapse. Neuron. 2000, 28: 955-966. 10.1016/S0896-6273(00)00166-5.View ArticlePubMedGoogle Scholar
- Jaubert-Miazza L, Green E, Lo FS, Bui K, Mills J, Guido W: Structural and functional composition of the developing retinogeniculate pathway in the mouse. Vis Neurosci. 2005, 22: 661-676.View ArticlePubMedGoogle Scholar
- Dilger EK, Shin HS, Guido W: Requirements for synaptically evoked plateau potentials in relay cells of the dorsal lateral geniculate nucleus of the mouse. J Physiol. 2011, 589: 919-937. 10.1113/jphysiol.2010.202499.PubMed CentralView ArticlePubMedGoogle Scholar
- Guido W: Refinement of the retinogeniculate pathway. J Physiol. 2008, 586: 4357-4362. 10.1113/jphysiol.2008.157115.PubMed CentralView ArticlePubMedGoogle Scholar
- Sherman SM: Interneurons and triadic circuitry of the thalamus. Trends Neurosci. 2004, 27: 670-675. 10.1016/j.tins.2004.08.003.View ArticlePubMedGoogle Scholar
- Wang X, Sommer FT, Hirsch JA: Inhibitory circuits for visual processing in thalamus. Curr Opin Neurobiol. 2011, 21: 726-733. 10.1016/j.conb.2011.06.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Arcelli P, Frassoni C, Regondi MC, De Biasi S, Spreafico R: GABAergic neurons in mammalian thalamus: A marker of thalamic complexity?. Brain Res Bull. 1997, 42: 27-37. 10.1016/S0361-9230(96)00107-4.View ArticlePubMedGoogle Scholar
- Chattopadhyaya B, Di Cristo G, Higashiyama H, Knott GW, Kuhlman SJ, Welker E, Huang XJ: Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical period. J Neurosci. 2004, 24: 9598-9611. 10.1523/JNEUROSCI.1851-04.2004.View ArticlePubMedGoogle Scholar
- Jurgens CW, Bell KA, McQuiston AR, Guido W: Optogenetic stimulation of the corticothalamic pathway affects relay cells and GABAergic neurons differently in the mouse visual thalamus. PLoS One. 2012, 7: e45717-10.1371/journal.pone.0045717.PubMed CentralView ArticlePubMedGoogle Scholar
- Krahe TE, El-Danaf RN, Dilger EK, Henderson SC, Guido W: Morphologically distinct classes of relay cells exhibit regional preferences in the dorsal lateral geniculate nucleus of the mouse. J Neurosci. 2011, 31: 17437-17448. 10.1523/JNEUROSCI.4370-11.2011.View ArticlePubMedGoogle Scholar
- Rafols JA, Valverde F: The structure of the dorsal lateral geniculate nucleus in the mouse. A golgi and electron microscopic study. J Comp Neurol. 1973, 150: 303-332. 10.1002/cne.901500305.View ArticlePubMedGoogle Scholar
- Parnavelas JG, Mounty EJ, Bradford R, Lieberman AR: The postnatal development of neurons in the dorsal lateral geniculate nucleus of the rat: a Golgi study. J Comp Neurol. 1977, 171: 481-499. 10.1002/cne.901710405.View ArticlePubMedGoogle Scholar
- Webster MJ, Rowe MH: Morphology of identified relay cells and interneurons in the dorsal lateral geniculate nucleus of the rat. Exp Brain Res. 1984, 56: 468-474.PubMedGoogle Scholar
- Williams SR, Turner JP, Anderson CM, Crunelli V: Electrophysiological and morphological properties of interneurones in the rat dorsal lateral geniculate nucleus in vitro. J Physiol. 1996, 490: 129-147.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhu JJ, Uhlrich DJ, Lytton WW: Burst firing in identified rat geniculate interneurons. Neuroscience. 1999, 91: 1445-1460. 10.1016/S0306-4522(98)00665-4.View ArticlePubMedGoogle Scholar
- Zhu JJ, Uhlrich DJ, Lytton WW: Properties of a hyperpolarization-activated cation current in interneurons in the rat lateral geniculate nucleus. Neuroscience. 1999, 92: 445-457. 10.1016/S0306-4522(98)00759-3.View ArticlePubMedGoogle Scholar
- Perreault MC, Qin Y, Heggelund P, Zhu JJ: Postnatal development of GABAergic signalling in the rat lateral geniculate nucleus: presynaptic dendritic mechanisms. J Physiol. 2003, 546: 137-148. 10.1113/jphysiol.2002.030643.PubMed CentralView ArticlePubMedGoogle Scholar
- Levick WR, Cleland BG, Dubin MW: Lateral geniculate neurons in cat: retinal input and physiology. Invest Ophthalmol. 1972, 11: 302-311.PubMedGoogle Scholar
- Wróbel A: Inhibitory mechanisms within the receptive fields of the lateral geniculate body of the cat. Acta Neurobiol Exp. 1982, 42: 93-107.Google Scholar
- Mastronarde DN: Nonlagged relay cells and interneurons in the cat lateral geniculate nucleus: receptive-field properties and retinal inputs. Vis Neurosci. 1992, 8: 407-441. 10.1017/S0952523800004934.View ArticlePubMedGoogle Scholar
- Montero VM: A quantitative study of synaptic contacts on interneurons and relay cells of the cat lateral geniculate nucleus. Exp Brain Res. 1991, 86: 257-270.View ArticlePubMedGoogle Scholar
- Van Horn SC, Erisir A, Sherman SM: Relative distribution of synapses in the A-laminae of the lateral geniculate nucleus of the cat. J Comp Neurol. 2000, 416: 509-520. 10.1002/(SICI)1096-9861(20000124)416:4<509::AID-CNE7>3.0.CO;2-H.View ArticlePubMedGoogle Scholar
- Bickford ME, Slusarczyk A, Dilger EK, Krahe TE, Kucuk C, Guido W: Synaptic development of the mouse dorsal lateral geniculate nucleus. J Comp Neurol. 2010, 518: 622-635. 10.1002/cne.22223.PubMed CentralView ArticlePubMedGoogle Scholar
- Acuna-Goycolea C, Brenowitz SD, Regehr WG: Active dendritic conductances dynamically regulate GABA release from thalamic interneurons. Neuron. 2008, 57: 420-431. 10.1016/j.neuron.2007.12.022.View ArticlePubMedGoogle Scholar
- Govindaiah G, Cox CL: Metabotropic glutamate receptors differentially regulate GABAergic inhibition in thalamus. J Neurosci. 2006, 26: 13443-13453. 10.1523/JNEUROSCI.3578-06.2006.View ArticlePubMedGoogle Scholar
- Crandall SR, Cox CL: Local dendrodendritic inhibition regulates fast synaptic transmission in visual thalamus. J Neurosci. 2012, 32: 2513-2522. 10.1523/JNEUROSCI.4402-11.2012.PubMed CentralView ArticlePubMedGoogle Scholar
- Torborg CL, Feller MB: Spontaneous patterned retinal activity and the refinement of retinal projections. Prog Neurobiol. 2005, 76: 213-235. 10.1016/j.pneurobio.2005.09.002.View ArticlePubMedGoogle Scholar
- Dolmetsch RE, Pajvani U, Fife K, Spotts JM, Greenberg ME: Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science. 2001, 294: 333-339. 10.1126/science.1063395.View ArticlePubMedGoogle Scholar
- Pham TA, Rubenstein JL, Silva AJ, Storm DR, Stryker MP: The cre/creb pathway is transiently expressed in thalamic circuit development and contributes to refinement of retinogeniculate axons. Neuron. 2001, 31: 409-420. 10.1016/S0896-6273(01)00381-6.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.