Michael Brecht

In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain

Abstract. A blind patch-clamp technique for in vivo whole-cell recordings in the intact brain is described. Recordings were obtained from various neuronal cell types located 100–5,000xa0µm from the cortical surface. Access resistance of recordings was as low as 10xa0MΩ but increased with recording depth and animal age. Recordings were remarkably stable and it was therefore possible to obtain whole-cell recordings in awake, head-fixed animals. The whole-cell configuration permitted rapid dialysis of cells with a calcium buffer. In most neurons very little ongoing action potential (AP) activity was observed and the spontaneous firing rates were up to 50-fold less than what has been reported by extracellular unit recordings. AP firing in the brain might therefore be far sparser than previously thought.

Temporal binding, binocular rivalry, and consciousness.

Cognitive functions like perception, memory, language, or consciousness are based on highly parallel and distributed information processing by the brain. One of the major unresolved questions is how information can be integrated and how coherent representational states can be established in the distributed neuronal systems subserving these functions. It has been suggested that this so-called binding problem may be solved in the temporal domain. The hypothesis is that synchronization of neuronal discharges can serve for the integration of distributed neurons into cell assemblies and that this process may underlie the selection of perceptually and behaviorally relevant information. As we intend to show here, this temporal binding hypothesis has implications for the search of the neural correlate of consciousness. We review experimental results, mainly obtained in the visual system, which support the notion of temporal binding. In particular, we discuss recent experiments on the neural mechanisms of binocular rivalry which suggest that appropriate synchronization among cortical neurons may be one of the necessary conditions for the buildup of perceptual states and awareness of sensory stimuli.

Dynamic representation of whisker deflection by synaptic potentials in spiny stellate and pyramidal cells in the barrels and septa of layer 4 rat somatosensory cortex.

Whole‐cell voltage recordings were made in vivo from excitatory neurons (n= 23) in layer 4 of the barrel cortex in urethane‐anaesthetised rats. Their receptive fields (RFs) for a brief whisker deflection were mapped, the position of the cell soma relative to barrel borders was determined for 15 cells and dendritic and axonal arbors were reconstructed for all cells. Three classes of neurons were identified: spiny stellate cells and pyramidal cells located in barrels and pyramidal cells located in septa. Dendritic and, with some exceptions, axonal arborisations of barrel cells were mostly restricted to the borders of a column with a cross sectional area of a barrel, defining a cytoarchitectonic barrel‐column. Dendrites and axons of septum cells, in contrast, mostly extended across barrel borders. The subthreshold RFs measured by evoked postsynaptic potentials (PSPs) comprised a principal whisker (PW) and several surround whiskers (SuWs) indicating that deflection of a single whisker is represented in multiple barrels and septa. Barrel cells responded with larger depolarisation to stimulation of the PW (13.7 ± 4.6 mV (mean ±S.D.), n= 10) than septum cells (5.7 ± 2.4 mV, n= 5), the gradient between peak responses to PW and SuW deflection was steeper and the latency of depolarisation onset was shorter (8 ± 1.4 ms vs. 11 ± 2 ms). In barrel cells the response onset and the peak to SuW deflection was delayed depending on the distance to the PW thus indicating that the spatial representation of a single whisker deflection in the barrel map is dynamic and varies on the scale of milliseconds to tens of milliseconds. Septum cells responded later and with comparable latencies to PW and SuW stimulation. Spontaneous (0.053 ± 0.12 action potentials (APs) s−1) and evoked APs (0.14 ± 0.29 APs per principal whisker (PW) stimulus) were sparse. We conclude that PSPs in ensembles of barrel cells represent dynamically the deflection of a single whisker with high temporal and spatial acuity, initially by the excitation in a single PW‐barrel followed by multi‐barrel excitation. This presumably reflects the divergence of thalamocortical projections to different barrels. Septum cell PSPs preferably represent multiple whisker deflections, but less dynamically and with less spatial acuity.

Whisker movements evoked by stimulation of single pyramidal cells in rat motor cortex

Neuronal activity in the motor cortex is understood to be correlated with movements, but the impact of action potentials (APs) in single cortical neurons on the generation of movement has not been fully determined. Here we show that trains of APs in single pyramidal cells of rat motor cortex can evoke long sequences of small whisker movements. For layer-5 pyramids, we find that evoked rhythmic movements have a constant phase relative to the AP train, indicating that single layer-5 pyramids can reset the rhythm of whisker movements. Action potentials evoked in layer-6 pyramids can generate bursts of rhythmic whisking, with a variable phase of movements relative to the AP train. An increasing number of APs decreases the latency to onset of movement, whereas AP frequency determines movement direction and amplitude. We find that the efficacy of cortical APs in evoking whisker movements is not dependent on background cortical activity and is greatly enhanced in waking rats. We conclude that in vibrissae motor cortex sparse AP activity can evoke movements.

Dynamic Receptive Fields of Reconstructed Pyramidal Cells in Layers 3 and 2 of Rat Somatosensory Barrel Cortex

Whole‐cell voltage recordings were made in vivo from subsequently reconstructed pyramidal neurons (n= 30) in layer 3 (L3) and layer 2 (L2) of the barrel cortex of urethane‐anaesthetised rats. Average resting membrane potentials were well below (15−40 mV) action potential (AP) initiation threshold. The average spontaneous AP activity (0.068 ± 0.22 APs s−1) was low. Principal whisker (PW) deflections evoked postsynaptic potentials (PSPs) in almost all cells of a PW column but evoked AP activity (0.031 ± 0.056 APs per PW stimulus 6 deg deflection) was low indicating ‘sparse’ coding by APs. Barrel‐related cells (n= 16) have their soma located above a barrel and project their main axon through the barrel whereas septum‐related cells (n= 8) are located above and project their main axon through the septum between barrels. Both classes of cell had broad subthreshold receptive fields (RFs) which comprised a PW and several (> 8) surround whiskers (SuW). Barrel‐related cells had shorter PSP onset latencies (9.6 ± 4.6 ms) and larger amplitude PW stimulus responses (9.1 ± 4.5 mV) than septum‐related cells (23.3 ± 16.5 ms and 5.0 ± 2.8 mV, respectively). The dendritic fields of barrel‐related cells were restricted, in the horizontal plane, to the PW column width. Their axonal arbors projected horizontally into several SuW columns, preferentially those representing whiskers of the same row, suggesting that they are the major anatomical substrate for the broad subthreshold RFs. In barrel‐related cells the response time course varied with whisker position and subthreshold RFs were highly dynamic, expanding in size from narrow single‐whisker to broad multi‐whisker RFs, elongated along rows within 10‐150 ms following a deflection. The response time course in septum‐related cells was much longer and almost independent of whisker position. Their broad subthreshold RF suggests that L2/3 cells integrate PSPs from several barrel columns. We conclude that the lemniscal (barrel‐related) and paralemniscal (septum‐related) afferent inputs remain anatomically and functionally segregated in L2/3.

Whole-Cell Recordings in Freely Moving Rats

Intracellular recording, which allows direct measurement of the membrane potential and currents of individual neurons, requires a very mechanically stable preparation and has thus been limited to in vitro and head-immobilized in vivo experiments. This restriction constitutes a major obstacle for linking cellular and synaptic physiology with animal behavior. To overcome this limitation we have developed a method for performing whole-cell recordings in freely moving rats. We constructed a miniature head-mountable recording device, with mechanical stabilization achieved by anchoring the recording pipette rigidly in place after the whole-cell configuration is established. We obtain long-duration recordings (mean of approximately 20 min, maximum 60 min) in freely moving animals that are remarkably insensitive to mechanical disturbances, then reconstruct the anatomy of the recorded cells. This head-anchored whole-cell recording technique will enable a wide range of new studies involving detailed measurement and manipulation of the physiological properties of identified cells during natural behaviors.

Targeted whole−cell recordings in the mammalian brain in vivo

While electrophysiological recordings from visually identified cell bodies or dendrites are routinely performed in cell culture and acute brain slice preparations, targeted recordings from the mammalian nervous system are currently not possible in vivo. The blind approach that is used instead is somewhat random and largely limited to common neuronal cell types. This approach prohibits recordings from, for example, molecularly defined and/or disrupted populations of neurons. Here we describe a method, which we call TPTP (two-photon targeted patching), that uses two-photon imaging to guide in vivo whole-cell recordings to individual, genetically labeled cortical neurons. We apply this technique to obtain recordings from genetically manipulated, parvalbumin-EGFP-positive interneurons in the somatosensory cortex. We find that both spontaneous and sensory-evoked activity patterns involve the synchronized discharge of electrically coupled interneurons. TPTP applied in vivo will therefore provide new insights into the molecular control of neuronal function at the systems level.

Sub‐ and suprathreshold receptive field properties of pyramidal neurones in layers 5A and 5B of rat somatosensory barrel cortex

Layer 5 (L5) pyramidal neurones constitute a major sub‐ and intracortical output of the somatosensory cortex. This layer 5 is segregated into layers 5A and 5B which receive and distribute relatively independent afferent and efferent pathways. We performed in vivo whole‐cell recordings from L5 neurones of the somatosensory (barrel) cortex of urethane‐anaesthetized rats (aged 27–31 days). By delivering 6 deg single whisker deflections, whisker pad receptive fields were mapped for 16 L5A and 11 L5B neurones located below the layer 4 whisker‐barrels. Average resting membrane potentials were −75.6 ± 1.1 mV, and spontaneous action potential (AP) rates were 0.54 ± 0.14 APs s−1. Principal whisker (PW) evoked responses were similar in L5A and L5B neurones, with an average 5.0 ± 0.6 mV postsynaptic potential (PSP) and 0.12 ± 0.03 APs per stimulus. The layer 5A sub‐ and suprathreshold receptive fields (RFs) were more confined to the principle whisker than those of layer 5B. The basal dendritic arbors of layer 5A and 5B cells were located below both layer 4 barrels and septa, and the cell bodies were biased towards the barrel walls. Responses in both L5A and L5B developed slowly, with onset latencies of 10.1 ± 0.5 ms and peak latencies of 33.9 ± 3.3 ms. Contralateral multi‐whisker stimulation evoked PSPs similar in amplitude to those of PW deflections; whereas, ipsilateral stimulation evoked smaller and longer latency PSPs. We conclude that in L5 a whisker deflection is represented in two ways: focally by L5A pyramids and more diffusely by L5B pyramids as a result of combining different inputs from lemniscal and paralemniscal pathways. The relevant output evoked by a whisker deflection could be the ensemble activity in the anatomically defined cortical modules associated with a single or a few barrel‐columns.

Organization of rat vibrissa motor cortex and adjacent areas according to cytoarchitectonics, microstimulation, and intracellular stimulation of identified cells

The relationship between motor maps and cytoarchitectonic subdivisions in rat frontal cortex is not well understood. We use cytoarchitectonic analysis of microstimulation sites and intracellular stimulation of identified cells to develop a cell‐based partitioning scheme of rat vibrissa motor cortex and adjacent areas. The results suggest that rat primary motor cortex (M1) is composed of three cytoarchitectonic areas, the agranular medial field (AGm), the agranular lateral field (AGl), and the cingulate area 1 (Cg1), each of which represents movements of different body parts. Vibrissa motor cortex corresponds entirely and for the most part exclusively to AGm. In area AGl body/head movements can be evoked. In posterior area Cg1 periocular/eye movements and in anterior area Cg1 nose movements can be evoked. In all of these areas stimulation thresholds are very low, and together they form a complete representation of the rats body surface. A strong myelinization and an expanded layer 5 characterize area AGm. We suggest that both the strong myelinization and the expanded layer 5 of area AGm may represent cytoarchitectonic specializations related to control of high‐speed whisking behavior. J. Comp. Neurol. 479:360–373, 2004.

Whisker maps of neuronal subclasses of the rat ventral posterior medial thalamus, identified by whole-cell voltage recording and morphological reconstruction

Whole‐cell voltage recordings were made in vivo in the ventral posterior medial nucleus (VPM) of the thalamus in urethane‐anaesthetised young (postnatal day 16–24) rats. Receptive fields (RFs) on the whisker pad were mapped for 31 neurones, and 10 cells were recovered for morphological reconstruction of their dendritic arbors. Most VPM neurones had antagonistic subthreshold RFs that could be divided into excitatory and inhibitory whiskers. VPM cells comprised different classes, the most frequently occurring being single‐whisker excitation (SWE) and multi‐whisker excitation (MWE) cells. In SWE cells (36 % of VPM neurones), only principal whisker (PW) deflection evoked an EPSP and was followed by a single action potential (AP) or remained subthreshold. The depolarisation was terminated by a large, delayed IPSP. A stimulus evoked on average 0.74 ± 0.46 APs (mean ±s.d.) with short latency (8.1 ± 1.0 ms) and small temporal scatter (0.31 ± 0.23 ms dispersion of 50 % of the first APs). In MWE cells (29 % of VPM neurones), deflection of several whiskers evoked EPSPs. PW responses were either subthreshold EPSPs or consisted of an EPSP followed by one or several APs (0.96 ± 0.99 APs per stimulus). AP responses were often associated with putative low‐threshold calcium‐dependent regenerative potentials and were followed by a small delayed IPSP. AP responses had a longer latency (12.3 ± 2.6 ms) and larger temporal scatter (2.5 ± 1.6 ms) than responses of SWE cells. MWE cells had a lower input resistance than SWE cells. The elongation of dendritic arbors along the representation fields of rows and arcs in VPM barreloids was weakly correlated with the subthreshold RF elongation along whisker rows and arcs, respectively. Evoked EPSP‐AP responses exhibited a sharper directional tuning than subthreshold EPSPs, which in turn exhibited a sharper directional tuning than IPSPs. In conclusion, we document two main classes of VPM neurones. SWE cells responded with a precisely timed single AP to the deflection of the PW. In contrast, MWE cell RFs were more broadly tuned and the temporally dispersed multiple AP responses of these cells represented the degree of collective deflection of the PW and several adjacent whiskers.