Anatomical and Electrophysiological Clustering of Superficial Medial Entorhinal Cortex Interneurons

66 Pages·2017·2.4 MB·English

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Research Article: New Research | Neuronal Excitability
Anatomical and Electrophysiological Clustering of Superficial Medial
Entorhinal Cortex Interneurons
Anatomical and electrophysiological clustering of MEC interneurons
Joan José Martínez1,2, Bahar Rahsepar2 and John A. White2
1Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA
2Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
DOI: 10.1523/ENEURO.0263-16.2017
Received: 31 August 2016
Revised: 19 September 2017
Accepted: 29 September 2017
Published: 6 October 2017
Author contributions: JJM and JAW conceived of the project. JJM collected and analyzed the
electrophysiological and anatomical data. BR performed immunocytochemical analyses and follow-up
electrophysiological experiments. JJM, BH, and JAW wrote the manuscript.
Funding: National Institutes of Health
R01 EB016407
Funding: National Institutes of Health
R01 MH085074
The reports no competing financial interests.
Funding sources: This work was supported by the National Institutes of Health (R01 MH058074 and R01
EB164017, to JAW).
Correspondence should be addressed to: John A. White, Department of Biomedical Engineering, Boston
University, 44 Cummington Mall, Room 403, Boston, MA 02215 USA. Email: [email protected]
Cite as: eNeuro 2017; 10.1523/ENEURO.0263-16.2017
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Copyright © 2017 Martínez et al.
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1 Anatomical and electrophysiological clustering of superficial
2 medial entorhinal cortex interneurons
3
4 Joan José Martínez1,2, Bahar Rahsepar2, and John A. White2
5
6 1 Department of Bioengineering, University of Utah, Salt Lake City, UT 84112 USA
7 2 Department of Biomedical Engineering, Boston University, Boston, MA 02215 USA
8
9 Abbreviated title: Anatomical and electrophysiological clustering of MEC interneurons
10
11 Number of words (abstract): 224
12 Number of words (significance statement): 89
13 Number of words (introduction): 587
14 Number of words (discussion): 3165
15
16 Number of figures: 10
17 Number of tables: 2
18 Number of text pages: 44
19
20
21 Correspondence should be addressed to:
22 John A. White
23 Department of Biomedical Engineering
24 Boston University
25 44 Cummington Mall, Room 403
26 Boston, MA 02215 USA
27 Email: [email protected]
28
29 Author contributions: JJM and JAW conceived of the project. JJM collected and
30 analyzed the electrophysiological and anatomical data. BR performed
31 immunocytochemical analyses and follow-up electrophysiological experiments. JJM, BH,
32 and JAW wrote the manuscript.
33 Acknowledgements: We thank Dr. Mark A. Kramer for helpful discussions regarding
34 clustering methods, Dr. Matt Wachowiak for microscope use and Dr. Alessandra
35 Angelucci for help with cell reconstruction methods. Dr. Fernando R. Fernandez helped
36 with experimental techniques, advised on data analysis, and provided useful critiques of
37 the manuscript.
38 Conflicts of interest: The authors acknowledge no competing financial interests.
39 Funding sources: This work was supported by the National Institutes of Health (R01
40 MH058074 and R01 EB164017, to JAW).
41
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42 Abstract
43 Local GABAergic interneurons regulate the activity of spatially-modulated
44 principal cells in the medial entorhinal cortex (MEC), mediating stellate-to-stellate
45 connectivity and possibly enabling grid formation via recurrent inhibitory circuitry.
46 Despite the important role interneurons seem to play in the MEC cortical circuit,
47 the combination of low cell counts and functional diversity has made systematic
48 electrophysiological studies of these neurons difficult. For these reasons, there
49 remains a paucity of knowledge on the electrophysiological profiles of superficial
50 MEC interneuron populations. Taking advantage of GAD2-IRES-tdTomato and
51 PV-tdTomato transgenic mice, we targeted GABAergic interneurons for whole-
52 cell patch-clamp recordings and characterized their passive membrane features,
53 basic input/output properties and action potential shape. These
54 electrophysiologically characterized cells were then anatomically reconstructed,
55 with emphasis on axonal projections and pial depth. K-means clustering of
56 interneuron anatomical and electrophysiological data optimally classified a
57 population of 106 interneurons into four distinct clusters. The first cluster is
58 comprised of layer 2- and 3-projecting, slow-firing interneurons. The second
59 cluster is comprised largely of PV+ fast-firing interneurons that project mainly to
60 layers 2 and 3. The third cluster contains layer 1- and 2-projecting interneurons,
61 and the fourth cluster is made up of layer 1-projecting horizontal interneurons.
62 These results, among others, will provide greater understanding of the
63 electrophysiological characteristics of MEC interneurons, help guide future in vivo
64 studies, and may aid in uncovering the mechanism of grid field formation.
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65 Significance Statement
66 Despite the critical role that entorhinal inhibitory interneurons play in
67 computation and grid cell formation, the electrophysiological properties of this
68 inhibitory interneuron population remain largely uncharacterized. This study
69 describes systematically the electrophysiology and anatomy of the inhibitory cells
70 in the medial entorhinal cortex and introduces a grouping framework for the
71 population. This framework divides the interneuron population into four clusters,
72 based on differences in their axonal projections and electrophysiological
73 properties. These findings confirm and extend findings from previous anatomical
74 studies and will inform future studies of medial entorhinal interneurons.
75 1 Introduction
76 By modulating the activity of principal neurons, interneurons play a crucial
77 role in the spatial navigation function of the superficial medial entorhinal cortex
78 (Beed et al., 2013; Buetfering et al., 2014; Couey et al., 2013; Domnisoru et al.,
79 2013; Fuchs et al., 2016; Garden et al., 2008; Pastoll et al., 2013; Varga et al.,
80 2010). Among other findings, recent studies have demonstrated that MEC
81 GABAergic interneurons mediate stellate-to-stellate cell communication (Couey
82 et al., 2013; Pastoll et al., 2013) and that background inhibition of principal cells
83 is stronger in superficial layers of the MEC than the deeper layers (Woodhall et
84 al., 2005). Grid cell computation work has implemented inhibition-dominated
85 network models to simulate spatial navigation mechanisms (Burak and Fiete,
86 2009; Pastoll et al., 2013; Thurley et al., 2013). Nevertheless, there is uncertainty
87 as to whether interneurons provide location-dependent input onto grid cells, with
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88 Buetfering et al. (2014) providing in vivo data that supports at least parvalbumin
89 positive interneurons not exhibiting such spatial variability and computational
90 studies of the MEC (Shipston-Sharman et al., 2016; Solanka et al., 2015)
91 contending that other interneuron groups may be providing such a role .
92 Despite their importance, electrophysiological data for GABAergic
93 interneurons remain scarce (Fuchs et al., 2016; Gloveli et al., 1997; Wolansky et
94 al., 2007). The characterization of superficial MEC interneurons has been difficult
95 for two reasons: the low proportion of interneurons (approximately 10%)
96 compared to principal cells (Gatome et al., 2010) and the relative physiological
97 and anatomical diversity of cortical interneuron populations (Buzsáki et al., 2004;
98 DeFelipe et al., 2013; Maccaferri and Lacaille, 2003; Whittington and Traub,
99 2003). Previous research suggests that the superficial MEC is anatomically
100 diverse, containing at least 7 anatomical categories as defined by soma depth
101 and dendritic morphology (Canto et al., 2008). The anatomical differences in the
102 interneuron population are likely to coincide with different roles within the local
103 cortical circuit (Kepecs and Fishell, 2014). Despite the anatomical categorization
104 of MEC interneurons, the combination of low cell counts and functional diversity
105 has made systematic electrophysiological studies difficult with only a few such
106 studies having been attempted (Ferrante et al., 2016; Fuchs et al., 2016;
107 Gonzalez-Sulser et al., 2014). Limited data are available on the firing pattern of
108 basket cells and chandelier cells, both of which have generally lumped together
109 using their common molecular identifier parvalbumin (Wouterlood et al., 1995),
110 but these data do not include passive membrane features, basic input/output
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111 measures or action potential characterization. Furthermore, the
112 electrophysiological properties of remaining cell types in the superficial MEC
113 have remained largely unknown (Gloveli et al., 1997; Wolansky et al., 2007).
114 This study takes advantage of recent developments in transgenic
115 techniques that specifically label GABAergic interneurons in order to
116 systematically characterize the superficial MEC interneuron population both
117 electrophysiologically and anatomically. Acute brain slices were harvested from
118 GAD2+ and PV+ labeled transgenic mice and whole cell patch clamp techniques
119 were used to measure a variety of electrophysiological features. Post-hoc
120 anatomical reconstruction was then conducted using fluorescence staining and
121 2-photon imaging to couple each interneuron’s electrophysiological profile with its
122 MEC localization and axonal tree distribution. We find that superficial MEC
123 interneurons can be grouped into four separate groups that have distinct
124 anatomical and electrophysiological profiles. These categories include deep-
125 projecting layer 2/3 slow-firing interneurons, layer 2/3 projecting fast-spiking
126 interneurons, layer 1/2-projecting interneurons and layer 1-projecting superficial
127 interneurons. Our results complement recently published data on the relationship
128 between electrophysiological properties and molecular markers (Ferrante et al.
129 2016). Together, these two papers represent a large step toward a complete
130 characterization of medial entorhinal interneurons.
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132 2 Materials and Methods
133 2.1 Electrophysiology
134 All electrophysiology experiments were conducted according to protocols
135 approved by the Institutional Animal Care and Use Committees of the university.
136 Brain slices were harvested from 18-35 day old transgenic mice of either sex.
137 Because no effects of age were evident in our data set, data were not classified
138 by age of the animal. Two transgenic strains were used: cre-dependent GAD2-
139 IRES-tdTomato transgenic mice (Taniguchi et al., 2011, The Jackson
140 Laboratories, strain 010802), which labeled glutamic acid decarboxylase 2 gene
141 (GAD2) expressing cells and thus facilitated targeting of GABAergic cortical
142 interneurons; and PV-tdTomato transgenic mice (Hippenmeyer et al., 2005, The
143 Jackson Laboratories, strain 008069), which labeled all parvalbumin (PV)
144 expressing cells and thus facilitated targeting of the specific PV+ genotype in
145 inhibitory interneurons. These mice were anesthetized with isoflurane and
146 decapitated. The brain was then harvested, chilled in sucrose-substituted artificial
147 cerebrospinal fluid (ACSF, units in mM, 185 sucrose, 2.5 KCl, 1.25 NaH PO , 10
2 4
148 MgCl , 25 NaHCO , 12.5 Glucose, 0.5 CaCl ), and cut parasagittally into 300 μm
2 3 2
149 thick slices using a vibrating microtome (Vibratome VT1200; Leica, Buffalo Grove,
150 IL). Slices were incubated for 15 minutes in ACSF (units in mM, 125 NaCl, 2.5
151 KCl, 1.25 NaH PO , 10 MgCl , 25 NaHCO , 25 Glucose, 2 CaCl ) at 37°C, and
2 4 2 3 2
152 then allowed to recover for at least 30 minutes at room temperature. For
153 recordings, slices were transferred to a heated (32-34°C) slice chamber (Warner
154 Instruments) that is mounted on an upright microscope stage (Olympus BX53;
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155 Olympus, Tokyo, Japan) and perfused with 95/5 percent O /C0 ACSF.
2 2
156 GAD2+/PV+ neurons were visualized using fluorescence and whole-cell patch
157 clamp clamped using patch pipettes (5-6 MΩ) fabricated from borosilicate glass
158 (1.5 O.D. 1.1 I.D.; Sutter Instruments, Novato, CA) and filled with artificial
159 intracellular fluid (ICF, units in mM, 120 K-Gluconate, 5 MgCl , 0.2 EGTA, 10
2
160 HEPES, 20 KCl, 7 di(tris) phosphocreatine, 4 Na ATP, 0.3 Tris-GTP) loaded with
2
161 biocytin (1% by weight) for post-hoc reconstruction. Presented data were not
162 corrected for the junction potential, which we measured as 11.6 mV. Electrode
163 resistance in the bath was ~ 5 M(cid:58)(cid:17)(cid:3)(cid:55)he pipette capacitance was compensated
164 for automatically by the MultiClamp 700B / CV-7 system. The bridge balance was
165 applied to correct for series electrode resistance under whole-cell patch clamp (~
166 10 M(cid:58)). Cells were patched for at least 30 minutes to ensure complete biocytin
167 fill. Following electrophysiological trials, brain slices were perfused in 4%
168 paraformaldehyde for 16-24 hours, then washed in phosphate-buffered saline
169 (units in mM, 137 NaCl, 2.7 KCl, 10 Na HPO , 1.8 KH PO ) three times for 15
2 4 2 4
170 minutes each and stored in 4° C for later staining.
171 2.2 Post-hoc anatomical reconstruction
172 Brain slices were incubated for 3 hours in a PBS solution containing 3
173 μg/mL streptavidin Alexa 488 (Molecular Probes) and 2% Triton X-100 (by
174 volume). Slices were then washed in PBS three times for 15 minutes each and
175 mounted on slides using a Mowiol mounting medium. At least 24 hours after
176 mounting, slides were imaged using a two-photon microscope (Ultima Intravital,
177 Bruker Corporation), with excitation wavelength set to 810 nm and a 520 nm low-
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178 pass filter. Alexa 488-filled cells were localized in the brain slice and a z-stack of
179 585 μm x 585 μm raster-scanned images was acquired, covering the entire
180 range of the soma and neuronal processes (usually 100-200 μm). This z-stack
181 was projected onto a single composite image and endowed with a dark-cell, light-
182 background look-up table to aid axonal visualization (see Figures 1, 6-9). In order
183 to describe the anatomical features of each neuron, soma depth was measured
184 and the extent of the axonal tree was described with a rectangular approximation
185 using the z-projected image (see Figure 1).
186 2.3 Electrophysiological protocols and data analysis
187 We used established methods to characterize the electrophysiological
188 features of patched cells. All electrophysiological protocols were conducted in
189 current clamp and were performed within 30 minutes of breaking into the cell to
190 begin the whole cell patch clamp recording.
191 2.3.1 Input resistance, time constant, sag ratio, and resting membrane
192 potential
193 A bias current was applied in current clamp to hold the cell to -70 mV. Five
194 one-second negative current pulses (with a 2 second rest time) were injected to
195 hyperpolarize the cell to approximately -80 mV (between -20 pA and -50 pA,
196 depending on input resistance). The resulting steady-state hyperpolarization from
197 -70 mV was divided by the applied current to calculate the input resistance. To
198 determine the membrane time constant, a single-order exponential was fit to the
199 membrane response (from start to peak polarization) to the hyperpolarizing
200 current. The sag ratio was determined by dividing the maximum voltage
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201 hyperpolarization (the sag) by the steady state hyperpolarization during the pulse.
202 The resting membrane potential was measured for every cell by placing the cell
203 in current clamp, injecting no current, and measuring the membrane potential.
204 2.3.2 Action potential (AP) half-width, AP rise time, and spike
205 afterhyperpolarization (AHP)
206 A depolarizing bias current was injected to elicit action potentials just above
207 firing threshold. The average of 50-100 total action potentials recorded in a 30-40
208 second recording was used to describe the action potential shape. First, the AP
209 half-width was determined by determining the AP height from the action potential
210 upstroke (defined as the point at which the second derivative of the action
211 potential with respect to time was at its maximum) to the peak and calculating the
212 duration between passing the half-height on the depolarizing phase and passing
213 the half-height on the hyperpolarizing phase is the AP half-width. The AP rise
214 time was calculated as the time required for the action potential to go from 20%
215 of its total height to 80% of its total height. The afterhyperpolarizing potential
216 (AHP) was measured as the membrane potential difference between the AP
217 upstroke initiation and the most hyperpolarized membrane potential immediately
218 following the AP.
219 2.3.3 Firing threshold
220 The cell was hyperpolarized to -80 mV and a 50 pA/s current ramp for 10-
221 20 seconds was applied, depending on the input resistance. The firing threshold
222 was determined as the membrane potential at the upstroke of the first action
223 potential in the ramp.
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