J. Brendan Ritchie Joanna Szczepanik RESPONSES OF RAT TRIGEMINAL GANGLON NEURONS TO LONGITUDINAL WHISKER STIMULATION Maik C. Stüttgen, Stephanie Kullmann and Cornelius Schwarz

Figure 1. Synaptic Pathways for Processing Whisker-Related Sensory Information in the Rodent Barrel Cortex (A) Deflection of a whisker evokes action potentials in sensory neurons of the trigeminal nerve, which release glutamate at a first synapse in the brain stem (1). The brain stem neurons send sensory information to the thalamus (2), where a second glutamatergic synapse excites thalamocortical neurons projecting to the primary somatosensory barrel cortex (3). (B) The layout of whisker follicles (left, only Crow whiskers shown) on the snout of the rodent is highly conserved and is identical between rats and mice. There are obvious anatomical structures termed ‘‘barrels’’ in layer 4 of the primary somatosensory neocortex (right), which are laid out in a near identical pattern to the whiskers. The standard nomenclature for both whiskers and barrels consists of the rows A–E and the arcs 1, 2, 3, etc. The C2 whisker follicle and the C2 barrel are highlighted in yellow. (C) There are at least two important parallel thalamocortical pathways for signaling whisker- related sensory information to the barrel cortex. Neurons in the ventral posterior medial (VPM) nucleus (labeled red, left) are glutamatergic and signal information relating primarily to deflections of a single whisker. The axons of VPM neurons terminate predominantly in individual layer 4 barrels, with a minor innervation in upper layer 6 (right). Corticothalamic layer 6 neurons provide reciprocal feedback to the VPM (not shown). Neurons of the posterior medial (POM) thalamic nucleus (labeled green, left) have broader receptive fields and are tightly regulated by state-dependent control imposed by zona incerta and the cortex. The axons of POM neurons avoid the layer 4 barrels and target primarily layer 1 and 5A (right). Corticothalamic neurons in layer 5 provide a strong input to POM (not shown). (D) Neurons in the barrel cortex are reciprocally connected to other cortical areas through long-range glutamatergic corticocortical synapses. The most important pathways connect the primary somatosensory (S1) barrel cortex with secondary somatosensory cortex (S2) and primary motor cortex (M1) on the same hemisphere. Callosal projections are also present but less prominent. (A) is modified and reproduced from Neuron, Knott et al. (2002), Copyright (2002), with kind permission from Cell Press, Elsevier.

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Whisking in the Dark!

Early interest - Vincent, in Watson’s lab, noticed that rats whose whiskers were clipped showed behavioral impairments, for example on maze navigation. She concluded that whiskers are ‘delicate tactile organs, which function in equilibrium, locomotion, and the discrimination of surfaces’ Shiffman et al, 1970’s studied rats with a depth perception task, and found that rats rely on whiskers and hence tactile information rather than vision when descending from a platform. Animals with unilaterally clipped whiskers are cautious about exploring their environment, and walk with the intact whisker side along the wall Bilateral vibrissotomy results in impairment of locomotion and swimming, and behavioral ‘frustration’ Studies in the 90’s determined that rats are capable of fine texture discrimination (a task where rats were rewarded for jumping onto either rough or smooth surface platform). Carvell and Simons concluded that rat’s whiskers are a tactile organ as good as primate’s fingertips! New findings in this decade- by whisking, rats can differentiate spacing between walls, even when a difference is only 3mm. They are good at discriminating horizontal object locations. More evidence of how much information rats get from whisking-it is 10x faster than previously thought, and allow rats to move confidently in the dark-detect objects, determine texture, size and shape of objects. Barrels, barreloids and barrelettes reflect organization cortical level – but what about the first level of processing, from moving whisker to trigeminal ganglion? What’s so interesting about rat’s whiskers?

The Relevance of Stüttgen, Kullman & Schwarz (2008)  Looked at the quantitative relationship between longitudinal whisker stimulation, and activation in the TG.  Interesting conclusion: Longitudinal stimulation of TG cells provides a good method for classification into: (1) amplitude responsive, slow adapting cells, and (2) velocity responsive, fast adapting cells.

Stüttgen, Kullman & Schwarz: Methods  10 anesthetized adult female rats  To access TG, left hemisphere sucked out.  Whiskers probed for receptive field of cells in TG, then trimmed to ~5mm and attached to actuator.  Stimulation was at the same longitudinal axis as whisker.  All whiskers were caudal: whiskers 1-4 in rows A-E, and straddlers.  Stimuli: fast half cosine, 500ms plateau, then slow cosine return. 15 stimuli based on 3 amplitudes (95, 155, 285 μm) and 5 velocities (5, 22, 43, 87, 130mm/s).  Two Analyses for separating the effects of variation in peak velocity and amplitude in relation to variation in cell spike responses: (1) η2 as a measure of effect size and (2) for each neuron a multiple regression equation with number of spikes as criterion variable.  Analysis was insensitive to time windows of spike counting (which TG neurons are sensitive to; Stüttgen, et al., 2006).

Results!  33 of 38 neurons sampled responded to longitudinal stimulation (note: all 33 were responsive to the lowest amplitude used in the study, 95 μm…with enough velocity)

Example PSTHs Y axis = amplitudes X axis = velocities A: Representative slow adapting (SA) cell with high- amplitude and low-velocity responsiveness. B: Representative rapidly adapting (RA) cell with low- amplitude and high-velocity responsiveness. C: Normalized PSTHs for all neurons superimposed. Orange is amplitude- responsive neurons, green is velocity-responsive neurons Figure 1

Quantitative analysis of spike responses A: spike counts of individual units as a function of deflection velocity averaged over amplitudes. B: spike counts of individual units as a function deflection amplitude, averaged over velocities. C: scatter plot of individual units η 2 for amplitude and velocity. D: scatter plot of individual units' β weights from multiple regression analysis. E: Spike responses of all neurons to a slow high-amplitude longitudinal stimulus (285 µm, 5 mm/s). Asterisks = RA neurons. Figure 2

Three Other Characteristics That Differentiate the Two Categories  1. The two classifications exhibit different adaptation profiles along the same lines as SA and RA cells in latitudinal studies.  2. Average spike counts of AR cells (53, n = 22) were much higher than those for VR cells (2.7, n = 11).  VR and AR cells had different velocity thresholds (Fig. 2E)

Question:  Does longitudinal classification predict latitudinal classification for TG cells? Answer: yes…eventually.

AR/SAVR/RARow totals Class membership using latitudinal stimulation-adaptation in hold phase only (55% overlap) SA617 RA11718 Column totals17825 Class membership using adaptation in hold and return phases (68%) SA10111 RA7714 Column totals17825 Class membership using adaptation in hold and return phases and velocity thresholds (Overlap 88%) SA15116 RA279 Column totals17825 Class Membership Using Longitudinal Stimulation Table 1

What Explains the Remaining Discrepancies?  1. Missing responses of SA cells that require a precise direction preference.  2. SA cells are extremely sensitive to latitudinal position of stimulation (see Fig.3)…

PSTHs for cell from Fig. 1A. A: responses to rostrocaudal whisker stimulation (rostral first). Stimulus waveforms are truncated at 1 s due to fixed recording duration during calibration. B: Offset of absolute starting position by 1 mm. Figure 3

Conclusion  TG cells are highly sensitive to longitudinal stimulation.  In fact, because of this, they can be categorized into AR/SA and VR/RA cell types.  (Question: what do you think is the functional significance of this division?)