DISCHARGE PROPERTIES OF NEURONS RECORDED
IN THE SOMATOSENSORY CORTEX OF THE MOUSE
Discharge Properties of Neurons Recorded in the Somatosensory Cortex of the Mouse
Natal´ı Barros-Zulaica1,b, ´Angel Nu˜nezb, Alessandro E.P. Villa1,∗
aNeuroheuristic Research Group, HEC Lausanne, University of Lausanne, Switzerland
bDepartamento de Anatom´ıa, Histolog´ıa y Neurociencia, Facultad de Medicina, Universidad Aut´onoma de Madrid, Madrid, Spain
Abstract
Here we present a study of the firing pattern distribution in the barrel cortex not only along the different layers, but also along the antero-posterior and medio-lateral directions. Performing extracellular recordings in the barrel cortex with a multi-electrode array with four recording points and in Urethane anesthetized mice. We found significant differences between antero-posterior and medio-lateral firing pattern distribution of four types of firing patterns according to their autocorrelogram. Bursting cells where placed mostly in the more posterior and lateral part of the barrel cortex, while regular firing neurons where placed mostly in the middle of the medio-lateral direction and in the infra-granular layer. No significant difference was found in the distribution in the dorso-ventral direction. These findings set the idea for the first time that processing of information in the barrel cortex is not uniform, neither inside the column nor in the whole volume of the barrel cortex.
Keywords: Barrel cortex, Spontaneous firing pattern, Autocorrelograms, Fano Factor, 1. Introduction
Rodents use the whiskers on their snouts for exploring the environment seeking for objects, making maps of the surround- ings and even performing fine-grain texture discrimination. In order to transmit all this important information to the subse- quent brain nuclei the sensory innervation of each whisker fol- licle is quite high.
Sensory information from whiskers is sent from the whisker follicle to the contralateral area of the thalamus through two different pathways: the lemniscal and the paralemniscal path- ways. In the lemniscal pathways, receptors are connected with the sensory principal trigeminal nucleus into the brain stem and send projections to the ventro-postero-medial thalamus nucleus (VPM). On the contrary, paralemniscal pathway source from the interpolar section of the rostral area in the spinal trigeminal nucleus at the brain stem level and send projections to the me- dial area of the posterior thalamus nucleus (POm) and to a little area of the VPM [1].
These projections from thalamus target the primary so- matosensory cortex (SI), mainly in layer IV, which is con- formed to clusters of neurons (barrel). Each cluster is related to one whisker and this area is called the barrel cortex [2].
It is known that the somatosensory barrel cortex is com- posed of local circuits heavily interconnected by vertical and horizontal projections [3, 4, 5]. In the lemniscal pathway, the barrel cortex receive a strong innervation from VPM mainly to layer IV and also to layers III and VIa, while in paralem- niscal pathway the POm sends projections to layers I and Va
∗University of Lausanne, Tel. +41-21-6923594, Internef 138.1, CH-1015 Lausanne, Switzerland
[1, 2, 3, 4, 5, 6, 7]. This vertical organization is linked hori- zontally by prominent projections within layer II/III and layer V [8, 9, 10, 11, 12].Sensory cortices have a laminar architecture with specific functions of each layer in information processing. In rat, sensory processing is performed by means of different re- sponse properties that differ according to the cortical layer and the cell type [13, 14].
It is known that in the neocortex there are three principal types of firing patterns: regular spiking (RS), intrinsically burst- ing (IB) and fast spiking (FS) [15, 16]. RS cells are pyramidal and stellate cells that trigger sodium action potentials in a sus- tained manner during the application of a depolarizing current pulse. They are placed in all cortical layers except in layer I. IB cells are pyramidal cells principally from layer V that surpris- ingly do not receive direct thalamo-cortical inputs. They pos- sess unusually thick apical dendrites that rise to layer I. They generate a burst of 3-5 action potentials riding on a calcium spike in response to an intracellular depolarization. FS cells are characterized by short duration action potentials, an ability to fire tonically at high frequency (>250 Hz) and a relative lack of spike frequency adaptation when long-lasting depolarizing current pulses are applied to these neurons; they are GABAer- gic interneurons [17]. Although the laminar distribution of the functional firing pattern has been little described, nothing is known about the superficial functional distribution.
The complexity of connectivity pattern and the variety of numerous types of neurons that built the somatosensory cor- tex makes it difficult to study. This is why despite of all the researches made in the lasts years about the mouse barrel cortex[18, 19, 20, 21, 22, 23], little is known about the basis of the functional activity of neurons in this area. In our study we recorded the activity of neurons during spontaneous periods in
barrel cortex.
2. Material and Methods 2.1. Animals
All animal procedures were performed in accordance with the Ethics Committee of the Universidad Autonoma de Madrid, and with Council Directive 86/609/EEC of the European Com- munity. Mice were group housed with a 12 h light/dark cycle and had free access to food and water. Every effort was made to minimize the number, and suffering, of the animals used. 2.2. Electrophysiological recordings
Experiments were performed on 9 urethane-anesthetized (1.2 g/kg i.p.) adult C57BL/6 WT mice (3 - 6 months old) weigh- ing 25 - 30g. Animals were placed in a Kopf stereotaxic device in which surgical procedures and recordings were performed. The body temperature was maintained at 37 C. An incision was made exposing the skull and a small hole was drilled in the bone over the barrel cortex. Single-unit recordings in the BC (A 0 - 2 mm, L 3 - 4 mm from bregma and V 0.3 - 1.2 mm from dura) [24] were made through a multielectrode Neuronexus or Mi- croLIQUID longitudinal array of four Iridium Oxid electrodes (15 µm electrodes diameter; 200 µm separation between elec- trodes; 1.5 - 2 MΩ impedance). Unit firing was filtered (0.3 - 5 kHz), amplified via an AC preamplifier (DAM80; World Pre- cision Instruments, Sarasota, USA), visualized on an oscillo- scope, digitally recorded in WAV format (44,100 Hz sampling rate, 16 bit), and stored for post hoc analysis. The files were analyzed offline using a spike-sorting program [25, 26]. From one to five cells were detected from each single electrode. The spike trains were digitally stored for time series analysis. 2.3. Statistical analysis
Spike trains were analyzed by time series renewal density plots scaled in rate units (spikes/s) to evaluate statistical prop- erties of single-unit discharges during spontaneous activity. For each histogram, the 99% confidence limits were calculated, as- suming that spikes occurred following a Poisson distribution. The Fano factor (equal to 1 if data follow Poisson process) was used to characterized the variability of the spike train. The bust- ing index indicates the grade of bursting of the cell, if the value is between [0, 1] it means that the cell fires more “bursting”. We also computed the Intra-burst frequency (IBF) and the av- erage burst duration (ABD) (Table 1) [? ]. We calculated the Fisher stadistic test in order to determine if the functional distri- bution of the different firing patterns was significant respect to the others. This test was performed with R Project of Statistical
sions using five pulses of 5 µA for 10 s at intervals of 10 s were induced at the top and at the bottom of the electrode array. Mice were given a sublethal dose of 8 µl/g ketamine/xylazine and perfused with 100 ml of 0.9% NaCl, followed by 100 ml 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed after perfusion and cut of coronal sections at 50 µm thickness with a Leica freezing microtome. Sections were mounted on two parallel slide series for cresyl violet staining [Figure 1].
Figure 1: Histological analysis. A: Microphotographs of coronal sections of the superficial and deep recordings areas stained with cresyl violet showing a representative electrode penetration in the barrel cortex.B: Enlargement of the previous panel emphasizing the electrolytic lesions within the barrel cortex at the supragranular (top) and infragranular (down) layers. Scale bar is 1 mm.
3. Results
In this study of spontaneous activity in the mouse barrel cor- tex under Urethane anesthetized condition we found four differ- ent types of firing patterns according to their autocorrelograms: irregular (IRR) or type I, bursting cell (BC) or type II, large bursting cell (LBC) or type III and regular (REG) or type IV (Fig. 2A, B, C and D) (Table 1). Most of the recording cells (48%, n=50) showed a REG activity, which is characterized by a constant probability to spike, showing a flat autocorrelogram (Fig. 2D), a median firing rate of 1.3 spikes/s and a median Fano factor close to 1 (0.7), meaning that the spikes distribu- tion for this type of firing pattern is almost Poissonian. The second more abundant (25%, n=26) type of firing is BC that is characterized by an autocorrelogram with a hump close to time zero (Fig. 2B) with a median firing rate of 1.8 spikes/s. We also found that the 14% (n=15) of cells showed a LBC type of fir- ing pattern characterized by an autocorrelogram with big hump close to time zero (Fig. 2C) and a median firing rate of 1.6 spikes/s. The rest of the recorded cells (13%, n=14) presented an IRR type of firing pattern with an autocorrelogram charac- terized by a narrow peak very close to time zero that decay fast to a flat form (Fig. 2A) and a median firing rate of 1.1 spikes/s. A study of all recorded cells distribution in the different stereotaxic directions is shown in Figure 2 E, F and G. In the
Cell Type IRR1 BC2 LBC3 REG4 (TYPE I) (TYPE II) (TYPE III) (TYPE IV) N = 105 14 26 15 50 (100%) (13%) (25%) (14%) (48%) Firing rate 1.1 1.8 1.6 1.3 (spikes/s) (1.3±0.2) (1.2±0.1) (1.8±0.2) (1.5±0.1) Fano factor 1.1 1.6 2.1 0.7 (1.0±0.1) (1.8±0.1) (3.3±0.7) (0.8±0.1) Bursting index 10.5 1.9 0.7 - (11.6±1.6) (1.6±0.1) (0.6±0.1) IBF 43 20 41 - (38±4) (22±5) (47±5) ABD 29 115 160 - (36±5) (111±5) (170±12)
Table 1: Discharge properties of spike trains recorded in the somatosensory cortex of the mouse. Statistics are described in the text. In this table is possible to see the median and the mean (± standard desviation) values of the firing rates, the Fano factor, the busrting index, the IBF and the ABD of all the recorded neurons
analysis of Fisher exact test showed significant differences (p- value < 0.05) between all the sectioned areas (p-value=1.44e- 05). A pair wise post hoc analysis showed that mostly signifi- cant differences could be found within each layer between the different antero-posterior areas. Is possible to observe that most of the cells with a BC or LBC firing pattern are placed more posterior. IRR cells are placed almost all of them, in the third antero-posterior subdivision, while REG cells seems to be uni- formly distributed along the antero-posterior direction, however the number of REG neurons increase in the infra-granular layer. For the distribution along medio-lateral and dorso-ventral di- rections (Fig. 2F) the Fisher exact test showed that the cell firing pattern distribution was significantly different between segmented areas (p-value=0.00624). The post hoc analysis re- vealed that again the main differences could be found within layers along the medio-lateral direction. Is also possible to see that most of the BC and LBC are located in more medial than lateral. IRR cells are placed mostly in the middle section along the medio-lateral direction. REG cells are mostly placed in the middle of the medio-lateral direction but specially in the infra- granular layer, as we saw previously.
In the figure of the antero-posterior versus medio-lateral directions (Fig. 2G) the Fisher exact test showed again a big significance between the different distribution groups (p- value=2.941e-06). The post hoc analysis revealed that the main differences could be found in the antero-posterior direction. Ac- cording with the previous distribution diagrams BC and LBC could be found in the more posterior and more lateral area of the barrel cortex. IRR cells are localized in the middle area of the barrel cortex with some preference of being more posterior and medial. REG cells are mostly localized in the middle section of the medio-lateral direction with a tendency of being distributed medial and posterior. Notice that the medio-lateral coordinate is measured from 3.65 to 2.5 mm, while in the Figure 2F it is set from 3.65 to 2.2 mm. Electrophysiological recordings were performed from 3.65 to 2.2 mm, however recordings from 2.2 to 2.5 mm where placed deeper and they are not represented in the diagram. Consequently, to fix these recordings points into
IRREGULAR (TYPE I)
BURSTING (TYPE II) LARGE BURSTING (TYPE III) REGULAR (TYPE IV)
Figure 2: Firing patterns according to their raster plots, autocorrelograms and one example of a spike form (A, B, C, D) and their dictribution in the barrel cor- tex (E, F G).A: Irregular firing pattern or Type I. B: Busrt firing pattern or Type II.C: Large Burst firing pattern or Type III. D: Regular firing pattern or Type IV.E: Cell type distribution according to antero-posterior and dorso-ventral recoording coordinades. F: Cell type distribution according to medio-lateral and dorso-ventral recoording coordinades.G: Cell type distribution according to antero-posterior and medio-lateral recoording coordinades. The shapes of barrel cortex S1 were selected according to Kirkcaldie 2012 [27] (E, G) and Paxinos and Franklin 2001 (F) [24]
4. Discussion
The main finding of this study is that is possible to describe a functional distribution of cells according to their autocorrelo- gram and that this distribution is not homogeneous in the barrel cortex. We found that most of the cells fired in a regular manner with a median firing rate of 1.3 spikes/s and with a Fano factor close to 1 which means that these cells fired following a Pois- son distribution or randomly. These type IV or REG cells are mainly located in the middle area of the barrel cortex and most of them are placed in the infra-granular layer. Another impor- tant finding was that most of the type II and type III cells, the ones that fired in bursts, are placed mostly in the more posterior and lateral zone of the barrel cortex.
do with thalamo-cortical projections, because it is known that these neurons receive a lot of projections from the thalamus [17] while REG neurons in supra-granular layer do not receive any thalamic projection [15]. BC and LBC are placed mostly in infra-granular and more posterior, these neurons seems to match the previously described IB pyramidal neurons that are mainly placed in layer V and VI and that do not receive direct thalamic projections. For sure they have an specific work in processing sensory information and connecting infra-granular layer with layer I [29]. It is interesting to observe how type I IRR cells are located in a very specific area practically dis- tributed only in the middle of the barrel cortex. It is possible to hypothesize that type II and III neurons role is mainly to syncronized neuronal cortical activity through thalamo-cortical inputs [30] keeping some reverberant base activity, while REG and IRR cells maybe process the information by their own and after processing they share the process with the adjacent cells.
The fact that there are no significat differences between cell distribution in the layers means that inside the cortical column the process of information does not depend on the cell distri- bution. However, the different distribution along the directions, antero-posterior and medio-lateral give the idea that probably the different functional firing rates not only have a processing function but also a trasnmission function [31]. One possibility is that the neuronal distribution change according to the projec- tion pattern. For example, posterior barrel cortex areas project to other cortical areas that need to send the information differ- ently than more anterior barrel cortex areas [32]. On the other hand, it is well known that there is a cortical representation of the whiskers on the barrel cortex [1]. Our data indicate that the different cortical cell types described here are not homogeneous distributed on the barrel cortex that may means that according to the sensory input, barrel cortex uses different neuronal types to processes sensory information. This point is crucial for sen- sory plasticity because single spikes or bursting discharges may induce long-term facilitation [33].
5. Conclusions
Our findings suggest that there is a cell distribution in the bar- rel cortex depending on the firing pattern of the neurons. This distribution is significantly different in the antero-posterior and medio-lateral directions giving the idea that the spreading of the processed information depends on the way neurons fired. As this is the first study performed in this issue of cell distribution according to their firing pattern in the barrel cortex we suggest that more research should be done in order to better understand the problem.
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