- Response Properties of Hum&n ThaCamic

Neurons to High Frequency Micro-Stimulation

Sanjay Patra

A thesis submitted in confomity with the requirements For the degree of Master's of Science

Department of Physiology University of Toronto

Q Copyright by Sanjay Patra 2001

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Acknowledaements

I would like to thank my supervisor, Dr. Jonathan Dostrovsky for giving me the

opportunity to work in such an exciting and fascinating area of study. Without his

guidance I surely would not have been able to accomplish my research goals. I would

also like to thank Ron Levy for his helpful comments and Helen Belina for her technical

assistance, throughout my study. Additionally. I would like to express sincere gratitude

to my supervisory cornmittee members, Dr. Bill Hutchison and Dr. John Yeamans for

th& insight and timely feed back.

Response Properties of Human Thalamic Neurons to High Frequency MicroStimulation

Sanjay Patra, Master's of Science Degree, 2001 Department of Physiology

University of Toronto

The mechanisms of the therapeutic effect of thalamic deep brain stimulation

(DBS) are currently not well understood. The present study directly examined the

effects of electrical stimulation in the ventrolateral thalamus on the firing of nearby

neurons. Hig h frequency micro-stimulation (1 00-333Hz; current 540uA) fmm either a

nearby stimulatirtg electrode (-250um) or directly from the recording electrode was

found to produce a prolonyed inhibition lasting up to 25s in 38 out of 74 (51%) cells.

This inhibition was usually preceded by a short period of increased bursting activity

(89%). Inhibition was observed in 29 of 36 (81 %) neurons that had a bursting firing

pattern and in 9 of 38 (24%) non-burang neurons. These findings indicate that high

frequency microstimulation in thalamus can hyperpolarize the cell membrane and inhibit

the firing of thalamic neurons and that these effects may mediate the therapeutic effects - of DBS.

1. Introduction 1 .OThe Thalamus 1

1 .O. 1 Gross Thalamic Anatornical Divisions 2 1.0.2 Ultrastructure of Thalamic Neurons 3

1.1 General Concepts of Thalamic Function 5 1.2 Thalamic Cell Types and Their Intnnsic Electrophysiological

Properties 6 1.2.1 Electrophysiology of Relay Cells 7 1.2.2 Electrophysiology of Reticular Cells 10 1.2.3 Electrophysiology of lnterneurons 11

1.3 General Thalamic Synaptic Properties 12 1.3.1 Synaptic Input and Efferent Output: Relay Cells 13 1.3.2 Synaptic Input and Efferent Output: lnterneurons 16 1.3.3 Synaptic Intrdextrathalamic Connections: Reticular

Thalamus 17 1.4 Deep Brain Stimulation General Background 18

1.4.1 Role of Thalamus in Fundional Neurosurgery 20 1.4.2 The Ventrocaudal Nucleus (Vc) 21 1.4.3 The Ventral Intemediate Nucleus (Vim) 22 1.4.4 The Anterior and Posterior Ventral Oral Nucleus (Voa,Vop) 23 1 A 5 The Anterior Thalamic Nuclei 24

1.5 Parkinson's Disease (PD) 25 1 5.1 Essential Tremor (ET) 26 15.2 Tremor and DBS 27 1.5.3 Chronic Pain and DBS 28

1.6 Curtent ldeas on Mechanisms of DBS 28

,2.0 Methods 2.1 Patient Population 2.2 Stereotaxic Technique 2.3 Microelectrode Stimulation and Recordings 2.4 Cel Populations and OfRine Analysis

2.4.1 Bursting Cell Analysis

3.0 Results

4.0 Discussion 4.1 Results From Similar Studies (GPi, SNr, and STN) 4.2 Low frequency Stimulation ~ffects in the halam mus 100 4.3 100

4.3.1 Pm Inhibition Bursüng 100 4.3.2 Long Duration Inhibition 1 03

4.4 Possible Rote of Long Duration Inhibition in Therapeutic Effects Of DBS 1 06

--- - - - - 6.5 Technkar Probïems Associated with MïCrasfimufafiôn and

Recording Studies 1 08 4.6 109

4.6.1 Future Direction (Human Studies) 1 09 4.6.2 Future Direction (Animal Studies) 1 09

List of Fiaur-es and Tables

Fiaures

Figure 1.1 : Figure 1.2: Figure 1.3: Figure 1.4: Figure 1.5: Figure 1.6: Figure 1.7: Figure 1.8: Figure 1.9: Figure 1 .IO:

Nuclei of the Dorsal Thalamus Tonic and Burst Firing Modes Voltage Dependence of LTS Cyclical Buist Firing in Thalamic Relay Cells I* and IT Inactivation and Activation Curves Connections between Thalamus and Cortex Glomerular Synaptic Arrangement Thalamic Relay Cell Afferent Inputs Thalamic lntemeuron and Reticular Cell Afferent ln puts Effects of Stimulation in GPi

Figure 2.1 : Sagittal Section of Thalamus Figure 2.2: Stereotactic Frame Assembly Figure 2.3: Electrode Tiack Reconstruction ~igure 2.4: Graphical Characterization of LTS Bursts ~igure 2.5: Visual and Graphical llustrations of LTS Bursts

Figure 3.1 :

Figure 3.2: Figure 3.3:

Figure 3.4:

Figure 3.5: ,.Figure 3.6: Figure 3.7: Figure 3.8: Figure 3.9:

Effect of Hig h Frequency Stimulation from Recording Electrode Short Duration lnhibition Effects of High Frequency Stimulation h m Recording Electrode on Two Cells Effects of High Frequency Stimulation from Nearby Stimulating Electrode Effects of Stimulation on Burst Size The Effect of Current lntensity on lnhibition Effect of Current lntensity on Pm lnhibition Bursting Effect of Train Length on lnhibition Effect of Frequency on Inhibition

Figure 4.1 : Model for Pre lnhibition Bursting and Long Duration ln hibition

Tables

Table 1.1 : Summary of Thalamic Subnuclei Table 2.1 : Patient and farget Information Table 3.1: Summary Data for High Frequency Stimulation Table 3.2: Patient and Subnuclei Cell Grouping Table 3.3: Effect of Stimulation on Bursting and Non-bursting Cells

----a = - - List of Abbreviations

AC ACh AP ATNIAN BF BC CAT CCK DBS DT EMG EPSP ET GABA Glu GP GPe GPi HFS Int IPSP ISI Ki LGN Lpo LTS MD

.,-MCP MRI NA NO NOT NR P PC PBR PD PF PVG REKRN RF STN SMA SN

anterior commissure acetyl choline action potentials anterior thalamus basal forebrain bursüng cell wmputer aided tomography chole ycysto kinin deep brain stimulation dorsal thalamus electromyog ram excitatory post synaptic potential essential tremor gamma aminobutyric acid glutamate globus pallidus globus pallidus externat segment globus pallidus intemal segment high frequency stimulation intemeumn inhibitory post synaptic potential inter-spike interval kinesthetic lateral geniculate nucleus latempola ris low-threshold calcium spike mectialls dorsalis mid commisural point magnetic resonance imaging nokdrenaline nitric oxide nucleus of the optic tract no response paraesthesia posterior commissure parabrachial region Parkinson's disease projected field periventricular grey matter thalamic reticular nucleus receptive field subthalamic nucleus supplementary motor area substantia nigra

WC SlUr STT Ta vc Vim VIP Voa Vol VOP

substanfia nigra pam compacta substantb nigra pars reticulata spinothalamic tract tactile ventrocaudal nucleus ventral intemediate nucleus vasoactive intestinal polypeptide anterior ventral oral nucleus voluntary posterior ventral oral nucleus

Introduction

Electrical deep brain stimulation (DBS) is a therapeutic method applied for the

treatment of chronic pain and, rnost commonly, for movement disorders. More

recently, DBS has been applied for treatment of psychiatric neurosis (Nuttin et al., 1999)

and epilepsy. Essential tremor, Parkinson's tremor, and other symptorns related to

Parkinson's disease are the predominant movement disorders treated. Deep brain

stimulation in the human thalamus has recently been employed as the treatrnent of

choice for tremor associated with the aforementioned movement disorders. DBS is a

more versatile and safer alternative to making thalamic lesions because its parameters

can be adjusted to optimize therapeutic benefits and there are fewer adverse side

effects (reviewed by Tasker and Kiss, 1995).

It has been hypothesized that high frequency DBS (>lOOHz) results in inhibition of

the motor output of the thalamus (Gildenberg and Tasker, 1998). The mechanisms by

which this might occur, however, still remain a mystery. Knowledge of the mechanisms r

by which the therapeutic effects of DBS are mediated might provide the means to

optimize the stimulation parameters or target lOcaI'~ation when impianting DBS

electroâes. This knowledge may also provide greater insight into the pathophysiology

of the various rnovement disorders.

1.0 The Thalamus

The origin of the word thalamus is from the Greek thalamos, meaning inner brida1

chamber. This is appropriate if one considers the thalamus' central role in providing

information to the cerebml cortex. Fundionally, it can be divided into several nuclei that

---- -

contain neurons, which relay visual, auditory, and somatosensory information to the

cortex. The olfactory system represents the only pathway of sensation that does not

relay through the thalamus before it can enter the cortex. Generally, the thalamus

receives contralateral sensory information, which is then pmjected to the ipsilateral

cortex. It is located lateral to the third ventricle on each side of the head. In the

evolution of mammals, each area of the neocortex depends on a parücular thalamic

projection and the size of the neocortical area is generally related to the size of the

thalamic subnucleus size. Structurally. the thalamus is small compared to the cerebral

hemispheres (reviewed by Tasker and Kiss, 1995; Sherman and Guillery. 2001).

1.0.1 Gross Thalamic Anatomical Divisions

The classical division of the mammalian thalamus is into three divisions. The

epithalamus, donal thalamus, and ventral thalamus still serve as a fundional basis for

the descriptive anatomy. The epithalamus includes the anterior and posterior

paraventricular nuclei, the medial and lateral habenular nuclei, and the pineal gland.

These, neither project to, nor recehre input from the cerebral cortex. The ventral >-

thalamus is composed of the reticular nucleus, ventral lateral geniculate nucleus and

the nucleus of the zona incerta. The nuclei of the Fields of Forel are sometimes also

included. These nuclei receive fibers h m , but do not send fibers to the cortex. The

dorsal thalamic nuclei comprise the major part of thalamus and include the following

nudear groups: anterior, medial, intralaminar, ventral, posterior, lateral, pulvinar, medial

geniculate, and dorsal lateral geniculate (Jones, 1985). All of these groups both receive

h m , and send fibers to the cortex. The dorsal thalamus nuclei contain a certain degree

of cellular and synaptic uniformity that is lacking in the other two regions. The functional

amas of thalamic deep brain stimulation are located in the ventral tier of the dorsal

thalamic nuclei and will be more fully discussed later (Fig. 1 .l, Table 1.1).

1.0.2 Ultrastructure of Thalamlc Neurons

The morphological studies of the thalamus have identified three major types of

thalarnic cells: The thalamic relay neuron, the local circuit neuron (interneurons), and

the reticular thalamic neuron.

Almost all thalamic nuclei project to the cortex (reviewed by Sherman and Guillery.

2001 ). Thalamocortical nuclei are also referred to as first order nuclei. First order

nuclei are defined as those sending messages to the cortex about what is happening in

the sub-cortical parts of the brain, and higher order nuclei es those that provide a

transthalamic relay from one part of the cortex to another. Although in primates higher

order nuclei fomi more than half of the thalamus, Crst order nuclei are generally thought

of as the main relay cells to the cerebral cortex (Crabhee, 1999). Most of thern (85-

90%) have medium to large sized soma (area: 300-400um2), bushy, highly radial

dendritic trees, and project with high conduction velocities to medium (IV-Ill) and deep - (VI) cortical layers. About 10 - 15% of them are half the sire of the aforementioned

cells and pioject to the superficial (1-11) layers of the conesponding cortical region at

slower conduction velocities. The initial effect of afferent stimulation on thalamocortical

neurone is excitation. Glutamate andlor aspartate is the neumtransmitter that most

researchen believe to be involved in corücdhalamic projections.

Immunohistochemical studies wrnbined wiai retrograde transport of horseradish

pemxidase have shown choleycystokinin (CCKFlike and vasoactive intestinal

polypeptide (VIP)-like transmitters inside some medial and intralaminar central medial

-- --- --. neurons projeding to the cerebral cortex or striatum. The excitatory effects on the

cerebral cortex can be induced through both glutamate and VIP (Steriade and Llinas,

1988).

A simple way to classify cells in the dorsal thalamus is to distinguish cells with

axons that project to the telencephalon from those that have locally ramifying axons.

Neurons with local axonal ramifications (intemeurons) generally have spheroid or ovoid

somas with cet1 bodies around the same size (cross sectional area of soma -200 um2)

as the smallest thalamocortical relay neurons. The dendrites have vesicle wntaining

synaptic appendages. Previous studies have shown that up to 30% of thalamic neurons

in primates and carnivores have intemeuron-like properties with short axons that are

GABAergic. Recent studies suggest that this nurnber may be an underestimate. The

primary contacts of the GABAergic teminals are on thalamocortical cells (Steriade and

Llinas, 1988).

The thalamic reticular nucleus (RE) is a sheet of cells that sumund much of the

dorsal thalamus and lies betweemit and the cortex. The nucleus is bordered medially by

'the extemal medullary lamina and laterally by the intemal capsule. Because of its

position, the RE is perforated by al1 axonal tracts from the cortex to the dorsal thalamus.

Most corticothalamic axons send collaterals to the area of the RE associated with the

region of the thalamus that it receives afferents from. The collaterals are oriented

perpandicular to aie direction of the axon and parallel to the retiwlar plane. The other

major afierents to the RE nucleus corne frorn layer VI of the cortex, brainstem reticular

formation, and the basal forebrain (Crabtree, 1999). RE neurons are classified by their

medium to large soma (300400 urn2). Also, they have several thick primary dendrites

-- -

that give rise to secondary, tiny branches, covered by long appendages that project to

other RE neurons. The RE neuron axons project to thalamic neurons and not to the

cortex. Studies involving double-labeling of reticular neurons with fluorescent tracen

show that over 90% of axons originating in a given region of RE project to a distinct

thalamic group. Almost al1 of the RE neurons in the rat, cat. and monkey are

GABAergic. They not only project to thalamocortical cells but to local intemeurons as

well (Steriade and Llinas, 1988). Visual, somatosensory, and auditory secton of the RE

share many of the same organizational features (Crabtree, 1999).

1.1 .O General Concepts of Thalamic Function

The thalamus was traditionally thought of as the relay for sensory information. It

should not however, be regarded as just a set of passive relay nuclei. It operates as a

gate-master that can be cortically and sub-corücally modulated through changes in

neuronal states. These states act as intnnsic wrnponenk of thalamic networks that

mark the entrance of information into the forebrain. The main cornponents of the

networks are wmposed of thalam6-cortical, wrtico-thalamic, and cortico-reticular /"

thalamic connections. The networks can control the behavioral state that an individual .

is in, as is seen in the rhythmic oscillations of thalamic neurons that characterite the

depressed responsiveness observed in slowinrave sleep. A shift in the network

properties can push the thalamic neurons out of the oscillatory mode into a more

passive mode that responds linearîy to driving inputs, and characterizes alert wakeful

behavior (Steriade and Llinas, 1988). This will be discussed further in the pages ahead.

--- -- - 1.2.6 Thalamic Cell Types And Thek htrinsic ETec€rophysiological

ProperUes

Although the thalamus is a highly complex structure, surprisingly the

electrophysiology of thalamic neurons is relatively unifonn. It is now generally known

that al1 thalamic neurons are capable of existing in two functional states. This is

dependent on the inactivation state of a voltagedependant calcium current, Ir, which

operates via a T-type calcium channel that was first described in inferior olivary neurons

of guinea-pigs (Llinas and Yarom, 1 Q8la,b). In the tonic mode cells are able to respond

to an input with sustained firing of sodium-generated unitary action potentials and

increased excitatory inputs are reflected in increased firing rates. In the bursting mode,

cells respond in discret0 bursts with short intenpike intervals (Fig. 1.2). In the bursting

mode a larger EPSP or IPSP does not necessarily elicit larger or smaller bursts. Thus,

the input-output relationship for tonic firing is almost linear, whereas that for burst firing

is nonlinear. The neurons switch from one state to another through changes in

membrane potential, each state being dependent on different voltagegated ionic

conductances. If the cell membrane is held in a depolarired state of more than -60mV,

the neuron responds in the repetitive firing mode via the sodium action potentials.

However, at membrane potentials more negetive than -65rnV, depolarkation of the cell

results in short bursts of action potentials via an inactivating all-or-none ca2* dependent

spike (Llinas and Jahnsen. 1982 and 1984). More specifically, if the cell fs more

depolarized than -6OmV for 50-100 msec, the calcium current is inactivated.

Hyperpolarization for 50-1 00 msec de-inadvates the calcium current, which can

subsequently generate a low thmshold calcium spike (LTS). If an LTS is large enough,

-4- - il is accornpanied by a burst of action Ijofentials Cf-fa) rMRig on its cresf. The time

course of de-inactivation of the LTS channet has a sigmoidal shape between 6 5 and - 75mV. More hyperpolaiized membrane potentials are capable of generating larger

LTSs and subsequently larger bursts. Thus, there is a monotonic relationship between

LTS amplitude and the number of action potentials in a burst (Fig. 1.3). Furthemore,

the LTS can have a refractory penod of up to 200 msec (Llinas and Jahnsen, 1984).

The de-inactivation of LTS has also been shown to be time dependent. Hyperpdarizing

pulses of increasing duration pmduce graded dainactivation (Deschenes et al., 1984).

Also, shorter pulses can sum to trigger larger LTSs (Steriade et al.. 1985). It is clear

that thalamic neurons can function as relay systems or as oscillators or resonators.

1.2.1 Electrophysiology of Relay Cells

All thalamic relay cells are capable of switching between the relay and burst mode.

Besides the ionic conductance that underlies the fast sodium spikes several other

conductances are also present.

The LTS pfays an important role in relay cells. Initially, it was thought that the - location of the LTS channels was concentrated on the cell body (Deschenes et al.,

1984), but there is mounting evidence that they are preferentially distributed on the

dendrites of the relay cells (Destexhe et al., 1998; Zhan et al., 2000). Although h

inactivates very quickly, evidence in cats indicates that activation requires a moderate

rate of depolarkation. If the rate of depdarization is too slow, a thalamic relay cell can

be taken h m a hypeipdarized state in which It is fully dejnactivateâ to a depolarized

state in which lT is fully inactivated and tonic firing ensues without ever firing a low-

threshold calcium spike. The rate of rise of EPSPs generated from ionotropic receptors

-A-- Cproduce fast transient potential changes) is fast enougtr to activate tT but EPSPs

generated by metabotropic receptors (produce slower sustained potential changes) may

inactivate IT.

A voltage dependent, non-inactivating, or very slowly inactivating sodium

conductance GNap), is also present in thalamic murons. This channel generates a slow

soâium-mediated depolarking pulse which is capable of generating a slow rebound

depolarization and plays an impoitant role in the 1OHz oscillation seen in thalamic cells

(Jahnsen and Llinas, 1984a). The activation of the LTS itself leads to activation of

voltage and calcium dependent potassium conductances. They act to repolarize the

cell to its former hyperpolarized level, thereby initiating the process of IT de-inactivation.

Another conductance that can be dependent on LTS is the lh conductance. It is

activated by membrane hyperpolarization and de-activated by depolarization. I t is

sometimes called the 'sag current" because it slowly (activation time constant of ~ 2 0 0

msec) allows the membrane to drift towards more depolarized levels. The combination

of Ir, the above-mentioned potassium conductances, and lh can lead to rhythmic

#bursting, which is often seen in recardings from in vitro slice prepaiations of thalamus

(Fig. 1.4). The length of the hyperpolarkation that generates the Ih rebound

phenornenon was found to be modulated by an eady potassium cunent, lA (Jahnsen

and Llinas, 1984a). lA is generated by a voltagedependent potassium conductance (or

a combination of multiple potassium conductances since there are other members to

the IA family) in most cells in the central nervous system (Adams, 1982; Rogawski.

1985; Storm, 1990; McComick 1991). This is different from the voltage and calcium-

dependent potassium conductances previously mentioned in ternis of its kinetics and

lower activation threshold. There are a number o f similanlhs befween i~ and t. IA has

the same three states; activated, deactivated, and de-inactivated. In thalamic relay cells,

the voltage dependence of lA is similar in shape to that of lT but shifted in the

depolarized direction (Fig. 1.5) (Pape et al., 1994). Furthemore, IA is inactivated at

depolarized membrane potentials and activated via depolarization from a

hyperpolarized state. In both conductance's activation occurs at a much faster time

course than deactivation or deinactivation (McConnick, 1991 ). However, their cunents

operate in opposite directions. Because of this, the two currents would tend to offset

one another if both were acüvated at the same time. Whether a postsynaptic potential

activates one or both depends on the initial membrane voltage and the temporal

properties of the voltage changes. Both channels have slightly different temporal

properties. If the membrane starts off sufficiently hyperpolarized to completely de-

inactivate both conductances, then a depolarization would activate Ir before activating

IA. This would result in a low-threshold spike with a burst of sodium spikes Ming its

crest. The delayed IA activation would only subtly affect the LTS. Notice from figure 1.5

?hat there is a limited membrane potential range, near -70 mV, for which Ir is thoroughly

inactivateâ and IA is slightly de-inadivated. An EPSP (or other depolarizing event)

elicited from this range of membrane potentials will activate a small IA without any IT, so

only tonic firing is possible. However, the tA will serve to slow down the EPSP and

could abolish sodium spikes al1 together (McConick, 1991 ; Rush and Rinzel, 1994).

The calcium spiking can occur at a variety of frequencies, typically at 1 to 3 Hz, the

amal value depending on other parameters such as the presence of local feedback

inhibitory circuits that may becorne involved during rhythmic bursting (cm be seen at

- frequencies up to I O Hz). This burscng can onty be ïnfémpfed if a sufficienffy strong

and prdonged depolarization inactivates Ii and prevents lh from activating.

lntradendritic recordings in thalamic cells have revealed a voltagedependent high-

threshold calcium conductance that triggers all-or-none depolarizing responses that are

followed by the activation of a calciumdependent potassium conductance (GK(CS))

(Steriade, Jones and Lllnas, 1990). Phannacological deactivation (Le. via receptor

antagonists) of these channels results in tells firing at very high tonic rates.

Basecl on these conductances and in vitro studies, only rhythmic bursting should occur.

In reality three types of bursthg are observed: 1. Arrhythmic; 2. Rhythmic and

synchronized, meaning that large populations of neurons would fire with the same

rhythm and in synchrony, which occurs during slow wave sleep and certain fons of

epilepsy; 3. Rhythmic and non-synchronous. This indicates that other factors and

conductances must corne into play.

1.2.2 Reticular Cells

Reticular cells are also capable of displaying a variety of conductances besides r regular sodium action potentials (Huguenard and Prince, 1992). The low-threshold

calcium conductance in reticular cells is similar to that of relay cells. The reticular cells

can exist in both the relay and tonic modes. However, there are some quantitative

differences.

Reticular cells tend to have slower activation and inactivation kinetics than relay

cells (Huguenard and Prince, 1992). Furaieme, the inactivation of h is voltage

independent. This increases the amount of time the LTS is activated thus, making for

longer LTSs with more action potentials riding the crest. Reticular cells also need larger

depolarization for activation of IT than in relay ceL. However. this may be an

experimental artifact that is caused by the difference in cable properties between the

two cell types. Since the reticular cells have more expansive dendrites, in vitro

stimulation of the cell body may result in more current leakage. Thus, more current

would need to be applied to reticular cell bodies to depolarire the lT channels, which

have been shown to be located on the more distal dendrites, than in relay cells. One

final important distinction between reticular and relay cells is the occurrence of burst

firing. The activity of bursting cells in the reticular nucleus has not been recorded in

awake animals. Whether reticular cells exhibit both response modes during normal,

alert behavior still needs to be resolved.

It is unclear how many voltage- or ion-sensitive membrane conductances exist in

reticular cells due to the lack of published studies. For instance. the presence of IA has

not been confimed or denied. Further understanding of the intrinsic properties of

reticular cells is needed to explain their firing patterns, which can have a powerful

impact on relay cells.

- ' 1.2.3 lnterneurons

lnterneurons are much more difficult to record h m due ta their small size. There

are very few criteria to distinguish interneurons from relay cells during recordings. An

important issue is whether al1 thalamic intemeurons can exhibit action potentials. It is

possible that axonless intemeurons exist that do not fire action potentials. but clearîy

some possess axonal outputs and fire action potentials. Regardless of the issue of

action potentials. recent evidence indicates that the output from presynaptic dendritic

terininals can be acüvafed wRhouf an action pofen€îaL This will be discussed in the

following section.

Previously it was thought that interneumns did not posses calcium T channels,

however recent evidence suggests that they do. The interaction behnreen IA and Ir

tends to mask the LTS. The reason for this is the differences between voltage

dependencies and peak amplitudes of these conductances. Figure 1.58 shows the

relative overlap in the voltage dependencies of intemeurons compared to relay neurons.

The relative amplitudes, the I* to IT ratio, is much larger in intemeurons than in relay

cells, thus, IA can swamp an evoked LTS. However, m e n t studies suggest that LTS

bursting activity does occur in intemeurons so the issue is not cleariy resolved

(Sherman and Guillery, 2001).

1.3 General Thalamic Synaptic Properties

The thalamocortical neurons, intemeurons, and RE neurons make

interconnections (Fig. 1.6). Different modes of interactions among the cells account for

the difierent states of cunsciousness. The reticular nucleus may interpose between the - cartico-thalamic neurons and neurons of the dorsal thalamus. f hese circuits can not

only affect states of consciousness but sensory processing during the awake state

(Sherman, 2001 ; Crabtree, 1999).

Contacts between thalamocortical and intemeurons cm occur via 'synaptic

islands". These areas are encapsulated by glial sheets referred to as glomenili. The

glomenili contain two pre-synaptic and two post-synaptic elements. The pre-synaptic

inputs from subcortical afferents are made through their axon tenninals which contact

both the thalamocortical cell body and the presynaptic dendrite of a local intemeuron.

-Ti%e6endrites ofthe infemeumns are a h a presynaptic etemenf thaf makes

GABAergic contact with the thalamocortical dendrite. These presynaptic dendrites may

outnumber the teminals of ascending afferent axons by a factor of 4 (Jones, 1985).

The synaptic contacts of prethalamic afferent axons can also fom triads. This occurs

when the prethalamic afferent contacts not only the thalamocortical cell but an adjacent

presynaptic intemeuron dendrite as well. The extraglomerular neuropil contains

teminals of corticothalamic axons heving mund and small vesicles that make

asymmetrical synaptic contact with proximal and distal parts of the relay cell dendrites

and the dendrites of intemeurons (Steriade and Llinas, 1988) (Fig. 1.7).

One basic cornponent of thalamic cells is the presence of both ionotropic and

metabotropic receptors. The patterns of presynaptic stimulation needed to acüvate

these receptors are quite different. Single action potentials are generally able to

activate ionotropic receptors. On the other hand. activation of metabotropic receptors

requires long stimulus trains. These functional differences between the receptor types

could have implications on LTS generation. The response of an ionotropic receptor will

,-tend to generate a transient EPSP or IPSP that generally decays in 10-20 msec. This

time period is too short for de-inactivation of Ir which requires a hyperpolarization for at

least 50 msec. Metabotropic receptors however, c m produce more sustained EPSP's

and IPSP's, some lasting > 100 msec, which is ideal for Ir de-inactivation (Sherman and

Guillery, 2001 ).

1.3.1 Synaptic Input and Efferent Output: Relay Cells

Figure 1.8 shows the neurotransmitters and postsynaptic recepton for known

inputs to relay cells of the lateral geniculate nudeus (LON) and ventral posterior

nucleus. Driving inputs can be described as inputs coniaining the main information to

be relayed to the cortex. More specifically, they are the afferents that detemine the

receptive field properties of the relay cells. First order-relay nuclei primarily receive the

sub-cortical inputs (i.e. retinal inputs to the LGN) and send messages to the cortex

about what is happening in the sub-cortical parts of the brain, whereas second-order

relays primarily receive cortical inputs h m layer V. The response mode which the relay

cell is in (burst or tonic) will detemine to a large extent the type of information that is

transmitted to the cortex. The question that has not been clearly answered yet is what

capacity do driving inputs have in modulating the response mode. Indirect experimental

evidence suggests that the driving inputs do not play a role in controllhg response

mode. The theoretical reason for this is that driving inputs preferentially adivate

ionotropic and not metabotropic glutamate receptors. A train of EPSP's generated by

ionotropic glutamate receptors could, by temporal summation, produce a sustained

depolarization long enough for Ir inactivation. In the LGN for example. if the ionotropic

EPSP (generated from a single pulse retinal stimulation) lasts for 25 msec. then a

'-summation of 40 Hz is needed to inactivate IT. This is a reasonable rate for retinat

input. However, activation of retinal afferents also produces multi-synaptic lPSPs that

counteract the EPSPs reducing their latency h m 25rnsec to around 10 msec. In this

situation it is not clear that even 1OOHz stimulations would produce EPSPs sustained

enough to inactivate IT. Another important criterion is the rate of EPSP (or IPSP)

production. If it is t w fast Ir will be activated (the stimulus will act as a depolarizing

pulse typically needed to activate Ir) before it c m be inactivated. In this case. the EPSP

will have to be sustained enough to inactivate IT following the LTS burst. The

--- - aforernentioned infinafion suggesfs t h € drnrlng hputs are Iikely not to change the

response mode of relay cells. However, it is important to realize that this has only been

studied in the LON of rats. Thus, more empirical evidence is needed before this can be

generalized to other thalarnic nuclei (Sherman and Guillery, 2001).

Relay cells also receive GABAergic inputs from interneurons and the thalamic

reücular nucleus. All evidence suggests that GABA is inhibitory in the thalamus. There

are two receptor types: GABh (ionotropic), and GABAe (metabotropic). The population

of thalamic reticular cells activate both receptors types on relay cells (Fig. 1.8). There is

evidence that G A B h receptors but not GABAB are activated by interneurons. GABAA

receptors operate via a chloride channel. This does not produce very large lPSPs since

the reversal potential for the chloride ion is atound -70 to -75mV, which is close to

resting membrane potential. It does, however, clamp the membrane at that potential,

dampening subsequent EPSPs. Activation of GABAe receptors works via increasing

potassium ion conductance. Evidence suggests it increases potassium leak

conductance. This tends to move-the membrane potential to -1 OOmv and has less

<èffect on other conductances. Another major difference in the receptor types is the tirne

course of adion (Koch et al., 1 982). GABAA effects lasts for around 1 0 msec whereas

GA0h.s effeds last for up to 100 times longer (100-1 000msec) (Reviewed by Sherman

and Guillery, 2001 ).

Although GABAA recepton are unlikely to produce a shift to the bursting response

mode, due to receptor kinetics, there is evidence that this occurs. Activation of GA&

through reticular nucleus cells is capable of generating LTS bursts in relay cells. This

tias been observed in various states of sleep where large numbers of reticular cells Sre

synchrcinously in bursts. The prorongeci bursts of action Wtentials from the RE produce

a sufficientfy pmlonged IPSP to deinactivate Ir and adivate fh. This was evident even

in the presence of GABh receptor blockers.

Relay cells also receive synaptic input from cortical layer VI axons. These

synapses are generally glutarnatergic and involve both ionotropic and metabotropic

teceptors. The cortical synapses are also more distally located on the dendritic tree

than subcortical afferents (see fig 1.8). Evidence from the LON suggests that

cor&icogeniculate synapses primarily adivate type4 metabotropic glutamate recepton

on relay cells. This generates slow onset EPSPs (via potassium channels) which can

last for more than hundreds of seconds (McConnick and Von Krosigk, 1992). These

metabotropic EPSPs could result in inactivation of lT without ever activating it. pushing

the cell into the tonic mode (Gutienez et al.. 1999).

The brainstem also sends modulatory inputs to relay cells (Fitzpatrick et al., 1989).

The parabrachial region of the brainstem includes the midbrain and pontine tegmentum

surrounding the brachium conjunctivum. Cholinergie parabrachial inputs are capable of

'activating both ionotropic (nicotinic) and metabotropic (muscurinic) receptors in

thalamus, generating fast or slow EPSPs respectively (McCormick. 1992).

Metabotropic nitric oxide (NO) and noradrenaline (Fitzpatrick et al., 1989) inputs are

also part of the parabrachial pathway although their exact modes of action are less well

understood (Revieweâ by Sherman and Guillery. 2001).

The relay cells generally project to layer IV of the correspondhg cortical area. A

collateral of the axon is also sent to the RE. This is the same area in RE that sends

GABAergic axons back to that relay ceIl. Most probably al1 thalamocortical pathways

-

are topographically organized. This is most readily seen in the visual, auditory and

somatosensory pathways (Crabtree, 1999). Fuither details on the specific cortical

connections of thalamic nuclei relevant to deep brain stimulation will be discussed in the

following sections.

1.3.2 Synaptic Input and Efferent Output: lnterneurons

lntemeurons have two distinct and functionally independent innervation zones that

are electrically isolated from each other. The cell body and proximal dendrites are

functionally distinct from the other more distal dendrites (Fig. 1.9). lntemeurons have

membranes that are more leaky to cunent than relay cells. This makes transmission of

changes in distal (dendrites) membrane potentials to the cell body very weak. Sub-

cortical axons innervate intemeurons. It appean that the more distal group of teminals

receive the su bcortical afferents via the aforementioned triad formation. Su bsequentl y,

subcortical excitation of intemeuron presynaptic dendrites produce EPSPs. The

intemeuron EPSPs then cause GABA release on the adjacent relay cell dendrites.

Cholinergie parabrachial inputs have an inhibitory effect on the proximal and distal - presynaptic dendrites of interneurons via muscarinic (metabotropic ACh) receptors.

These same axons send afferents to adjacent relay cell dendrites where they have an

excitatory effect (Sherman and Guillery, 2001). Thus, the net effect of parabrachial

inputs ont0 relay cells seems to be excitatory, either directly, or through disinhibition via

interneu rons.

1.3.3 Synaptic Intralextrathalamic Connections: Reticular Thalamus

(RE)

The RE is organized in a conventional fashion, integrating synaptic inputs to

produce axonal outputs (Fig. 1.98). Figure 9B illustrates the inputs and also points out

the gaps of knowledge about reticular cell input. There are a variety of functions for the

reticular nucleus that have been proposed. Fint, it is a modulator of sensory

transmission through the dorsal thalamus. Secondly, it is an enhancer of selected

sensory transmission through the dorsal thalamus. Finally, it is a regulator of receptive

field properties of donal thalamic neurons. These functions are not considerad to be

mutually exclusive. Each of them relies on the inhibitory, or hyperpolarizing effects

exerted by reticular cells on thalamocortical relay cells (Reviewed by Crabtree, 1999).

The various sensory sectors of the RE have similar organizational features. A

thalamic reticular sector is defined as a region that receives projections from a particular

dorsal thalamic nucleus and the cortical area that is reciprocally connected to that

nucleus (Sherman and Guillery, 2001)(See Fig. 1.6). It also sends axonal projections - back to the same dorsal thalamic region. Initielly, the topographical organization of the

reticutar sectors was thought to refled a general cortical organizahn where adjacent

areas along the cortical sheet would be represented by adjacent sectors along the

reticular sheet. It is now known that functionally related cortical areas can send

projections to one reticular sector. Also, a particular reticular sector can send axons to

multiple functionally related dorsal thalamic nuclei and can receive afferents from

functionally related dorsal thalamic regions. Afferents from the dorsal thalamus provide

driving inputs (inputs determining receptive field properties) while cortical inputs tend to

-a- . - - - - be modubfory (affecthg intrinsic celluïar sfafes te. bu=€ vs fonic mode) (Reviewed by

Crabtree, 1999). Sensory cells in the dorsal thalamus and reticular nucleus fom a

network of excitatory-inhibitory interconnections. Furthemore, the sensory sectors

contain rnap that are related to inputs from cortical areas and inputs from first-order

thalamic nuclei.

1.4 Deep Brain Stimulation General Background

The discovery of electrical excitability began with an argument between two

ltalians named Luigi Galvani and Alessandro Volta. The argument was over why frog

legs twitched when placed between two different conducting metals. Galvani was

convinced that this was due to intemal electricity in the fmg whereas Volta thought the

metals themselves were producing ebctricity. Galvani finally proved with a transected

nerve (which could produce a muscle twitch in the frog upon application). that there was

indeed an intrinsic electrical potential in the frog (reviewed by Yeomans, 1990).

The first endeavor to electrically map the human brain started in 1864 by a

physician named Theodor Fritsch; He noticed that touching the cerebral cortex could - produce muscle tuvitches in the opposite side of the body. The initial mapping studies

developed out of curiosity and were analyzed further to aid in the fundional localization

of brain areas. By the earîy 1900's the first stemtaxic instruments were engineered by

Honley and Clark. and by the 1920's and 1930's Walter Rudolf Hess devised a method

for electrical stimulation of deep brain structures in a freely moving animal. This

tedinique allowed for permanent implantation of the electtodes into various brain

regions (reviewed by Yeomans, 1990). Electncal stimulation became a major t d used

to map brain regions for surgeries such as thalamotomies to treat tremor. Eventually.

chronic stimulating electrodes were used to treat chronic pain patients. Deep Brain

Stimulation in the motor thalamus was first successfully attempted to treat Parkinson's

related tremor by Benabid in 1987. Currently, implantation of chronic stimulating

electrodes into patients who suffer from chronic pain and a variety of movement

disorders such as Parkinson's disease are being employed for their very significant

therapeutic effect.

A growing number of human studies of electrical stimulation have been reported

from patients undergoing awake, open brain surgery. The evoked sensations

generated by stimulation can aid the surgeon in localking specific brain regions that

would othewise be next to impossible to separate. For example, stimulations, in the

somatosensory cortex can evoke parasthesias (tingling on the skin), in the motor cortex

can evoke twitches, and in the auditory or visual cortices can evoke sensations of

simple tones or lights. The important technique of microelectrode recordings,

developed in the 1950's. allowed neurophysiologists to record action potentials from

neurons in the brain. This information has also been valuable in identifying brain - regions as will be illustrated in the next section. Microstimulation was developed in

order to stimulate as few neurons as possible to detemine the effect that minimal

eledrical stimulation had. This technique has allowed for developing brain maps with

increased spatial resolution although the rnechanism(s) of action are still not resolved

(Yeomans, 1990).

1 A.1 Role of Thalamus in Functional Neurosurgery

Functional stereotactic neurosurgery for the treatment of pain and movement

disorden has been perfonned since the 1940's. It was developed to treat diseases by

----- L --

the most minimally invasive means (before this many of the procedures were perfonned

under open surgery). In 1947, Spiegel and Wycis perfonned the first hurnan

stereotacüc operation (in the globus pallidus and thalamus). At that time, the technique

was made possible by radiological methods that helped define intemal cerebral

landmarks. Presently. MRI and CAT sans perform this task. The stereotactic frame

allows the neurosurgeon to put the cerebral landmarks (the anterior and posterior

commissure points are the commonly used landmarks) in tenns of an X, Y, and Z

coordinate space. A microdrive mounteâ on the frame allows foi precise movement of

a micro- or macro-electrode through the person's brain (described in greater detail in

the methods section). Using this technique, the precise location of deep brain

structures can be mapped by using both receptive field and projected field information.

Microelectrodes have the advantage of being able to record from individual neurons

which allows receptive field data to be obtained. A projeded field is the area of

perceived sensation on the surface of the body when stimulating in a particular brain

region.

..- Shortly after the first stereotactic case was perfomed, lesions were made in the

ventral posterornedial nucleus and the center-median nucleus for the treatment of

thalamic pain. Since then the thalamus has been central in the neurosurgical treatment

of pain and movement disorders (Reviewed by Gildenberg and Tasker, 1998).

Hassler's nomenclature will be used from this point to describe the various sub-nuclei of

the thalamus tageteâ in functional neurosurgery and DBS (Table 1.1) (Tasker and Kiss.

1995).

L -. -

1.4.2 The Ventrocaudal Nucleus (Vc)

The main thalamic somatosensory area that receives input from the madial

lemniscus is known as the ventrocaudal (Vc, in Hasslen nomenclature) nucleus.

During intraoperative rnicroelectrode recordings this thalamic area is identified by its

cells with large amplitudes (extracellularly recorded action potentials), and by the

presence of tactile receptive fields (RFs). More specifically, cells in this area can

respond to a light touch, light brushing of glabrous skin with cotton, a hair, or a puff of

air. These cells can be rapidly or slowly adapting and their receptive fields are

contralateral exœpt for the areas of the upper and lower lip which may be bilateral. The

sue of the RFs can range from a few millimetes to centimeteis in diameter. The

somatotopy is arranged medial to laterally. Stimulation through a microelectrode or

macmelectrode in this region can evoke paraesthesia's (sensation of tingling or

numbness) in the contralateral side of the body. The locations of the evoked sensations

are termed projective fields (PFs) and are usually located in similar regions as the RFs.

When micmstimulating in the lip and manual digit regions of Vc, the threshold for such a - response can be as low as 2uA (at 300Hz 1 second stimulus trains). Larger tipped

electrodes (such as macroelectroûes) are capable of producing a wider cunent spread

and larger PFs.

Deep Bmin Stimulation is generally applied to Vc when treating chronic pain. The

stereotactic implantation of a chronic stimulating electrode is put in such a site that

stimulation produces paraesthesia in the patientas area of pain. Before the DBS

technique was applied to Vc, destructive lesions were used to treat pain. The

neumsurgeons reasoned that making lesions hem, that were somatotopographically

%.. - - -

re~ateâ to the patient's nociceptive pain, shoü~d re~ievë mat pain. ~ h e pain relief that

was recorded after such a surgery was short lived and the incidence of resulting central

pain was high. Later on it was found that lesion-making in the medial thalamus was

more effective for pain relief. Lesions in Vc also result in loss of tactile sensibility

(Tasker and Kiss, 1995).

1.4.3 The Ventral Intermediate Nucleus (Vim)

The ventral intemediate nucleus (Vim) is the main relay for the cerebellum and for

deep and kinesthetic somatosensory input. It is commonly believed that the VcNirn

border separates superficial tactile from kinesthetic input (reviewed by Tasker and Kiss,

1995). Like Vc, recordhg in Vim reveals the presence of large amplitude (extracellularly

recorded action potentials) units with high spontaneous activity. The amplitude, site

and spontaneous activity however, are not as high as in Vc (Tasker and Kiss, 1995).

The cells respond to contralateral squeezing of muscle bellies or tendons, other deep

structures, or passive joint movements (kinesthetic movements). Vim somatotopy is

medial to lateral as in Vc but not as discrete. Stimulation in posterior Vim can induce - contralateral paraesthesia just like in Vc although the PFs are larger and the thresholds

higher. This is probably due to current spread (volume wnduction) into Vc. Thus, it is

difficult to detenine the VimNc border from PF data alone. Another major factor that is

used to identii Vim in patients with Parkinsonian or Essential tremor is the presence of

tremor cells. These cells have oscillations in firing rate at the same frequency as the

peripheral tremor and will be discussed in more detail latet. In a small number of sites,

stimulation (macro more often then micro) can produce a sensation of movement

(Tasker and Kiss, 1995).

. - - -

1.4.4 The Anterior and Posterior Ventral Oral Nucleus (Voa and Vop)

The anterior ventral oral nucleus (Voa) is more dificuit to recognize

neurophysiologically in humans. The extracellulariy recorded action potential

amplitudes of the units in Voa are usually smaller than in Vc or Vim and cells don't

necessarily respond to movements. Stimulation in Voa elicits no response other than

through volume conduction (cuvent spread) into the intemal capsule at very high

currents. Voa receives some of the pallidal input from the basal ganglia (reviewed by

Tasker and Kiss. 1995).

The posterior ventral oral nucleus (Vop) is easy to identify eledrophysiologically. It

receives pallidal and cerebellar inputs. The units in this sub-nucleus have smaller

amplitudes than in Vim or Vc and respond to contralateral voluntary movements. The

voluntary cells can fire in advance of the movement. The cells in Vop also have a

medial to lateral somatotopy. In general the cells respond to contralateral movement

but in some cases to ipsilateral movement as well. Tremor cdls can also been found in

Vop. They, like Vim tremor cells, can Cre at the same frequency as the peripheral r tremor. Because they can fire in advance of their retated voluntary movement, it has

been hypothes~ed that they may be the pacemakers for tremor and other voluntary

movements. Electrical stimulation does not usually evoke sensations of movements in

the neurons' receptive field. Lesions and chronic stimulation have been applied in this

area for the treatment of dyskinesias (reviewed by Tasker and Kiss, 1995).

1.4.5 The Anterior Thalamic Nuclei

Although the anterior thalamic nuclei (ATN) share many properties with other relay

and associative thalamic nuclei they have characteristics that give them a different

---2= - -

functional significance. First, rather than being linked with neo-cortical areas, ATN are

reciprocally connected with structures such as presubicular, retrosplenial, or cingulate

cortices (reviewed by Pare et al., 1987). Second, their major afferent input cornes from

the mammillary bodies and not from motor or sensory related structures of the brain

stem and spinal cord. Thirdly, the anterior thalamic nuclei seem to be a group devoid of

inputs from the reticular thalamus. Although the cells are capable of bursting if

hyperpolarized enough, they do not exhibit spindle oscillations where populations of

neurons burst in synchrony. This is because the reticular nucleus is the generator of

spindle rhythmicity (Pare et al., 1987). Recently, this region of the thalamus has

becorne clinically important for the treatment of epilepsy. More specifically, the ventralis

anterior nucleus has been a target for chronic stimulation to prevent the propagation of

seizures. Historically, lesions were also perfomed in this region to treat psychiatrie

diseases (Tasker and Kiss. 1995).

1 S.0 Parkinson's Disease (PD)

The basal ganglia consists of the striatum, globus pallidus(GP), sub-thalamic -

/

nucleus (STN), and the substantia nigra (SN). The striatum consists of the caudate

nucleus and putamen. They shara many oganizaüonal and connecüve simüarities. lt is

thought that the putamen is the motor area and the caudate is associated with more

cognitive tasks. The striatum has three primary inputs including, dopaminergic from SN

pars compacta (SNc), glutamatergic via corticostriatal projections, and a projection from

the intralaminar thalamic nuclei. The striatal output neurons project to two major areas;

the extemal segment of the globus pallidus (GPe)(the indirect pathway) and the intemal

segment of globus pallidus (Gpi)lSN pars reüculata (SNr) cornplex (the direct pathway).

--- -

Both of these outputs are GABAergic. Ilopamine projections from SNc have an

inhibitory effect on the striatal GPe projecting neurons and an excitatory effect on the

SNrIGPi projecting neurons. GPe then projeds to STN (via GABA) which projects to

SNr and GPi (via glutamate) (Alexander and Cnitcher, 1990).

More than 500,000 Americans suffer from PD, and about 50,000 new cases are

reported annually, according to the National Institutes of Health. The numben may be

larger than what is reported, because diagnosis - especially early on - can be diffÏcult.

The syrnptoms of PD indude slowing of movements (akinesia and bradykinesia), limb

rigidity, and trernor. It is the most common of the basal ganglia diseases (Crossman,

1999). The largest patient population for DBS are Parkinson's patients. PD is caused

by degeneration of the SNc region of the basal ganglia. This results in decreased

release of dopamine from SNc ont0 the striatum. The SNc targets are twofold including

D l and 02 dopamine recepton on striatal neurons, the net result of which is

disinhibition of the GPüSNr output. This is believed to result in excessive inhibition of

thalamic targets (VoaNop) resulting in the slowing down of movements. Most patients - ' can be effedively treated with drugs for a number of years. However, in many cases

-- the effectiveness of the drug diminishes and abnorrnal involuntary movements (refened

to as dyskinesias) start to develop. For these cases neurosurgical interventions in the

basal ganglia and thalamus have been bund effective in alleviating some of the

symptoms.

1 .SA Essential Tremor (ET)

Essential tremor (ET) is a prevalent movement disorder that affects between 0.3-

1.7% of the aged population (Rautakorpi et al. 1984). ET is characterized by an action

- tremor (4-1 2Hz) which is not present at rest. €MG recordings of the limb generating the

tremor show simultaneous bursts in antagonistic muscles. PD, on the other hand,

shows altemate bursts between agonist and antagonistic muscles (Marsden 1984).

Trernor cells are also found in the VopNim region of ET patients where lesions or DBS

can anest or significantly reduce trernor (Goldman et al., 1992; Hirai et al., 1983).

The pathophysiology and possible neuronal pathways involved in ET remain

poorly understood. It is thought that tremor associated with ET is the result of a central

oscillator. The location of the oscillator has not been confimed although the inferior

olivary wmplex is a possible candidate (Hallet and Dubinsky. 1993). The net result is

an increase in firing of the deep cerebellar neurons via disinhibition (the exact circuit is

reviewed in Buford et al., 1996). There is evidence of excitatory connections from deep

cerebellar nuclei to Vim in monkeys (Hirai and Jones. 1989). Thus, increased inferior

olive activity could result in increased excitation of Vim. This is in contrast to the effects

that PD pathophysiolgy has on Vim (inhibition via GPüSNr) although both diseases can

be troated with lesions or DBS in the sarne area.

'1.5.2 Tremor and DBS

The ventral lateral thalamic nuclei are populated with cells that fire in synchrony

with tremor. Recent studies in our lab indicate that the Vim is a site where tremor cells

are concentrated and where stimulation results in optimal tremor relief. The Vim

nucleus is currently the most widely chosen sugical target for treating tremor

associated with movement disorden such as ET. Vim thalamotomy is highly effective

with over 90% of patients having satisfactory results (Koller et al., 2000). However, side

effects such as motor, sensory, cerebellar, speech, and cognitive complications can

occur. These complications are worsened when the thalamotomies are bilateral.

Because of this, dinicians have sought alternative procedures that would minimize the

unwanted side effects. DBS is now the treatment of choice for tremors associated with

PD, ET, and demyelinating (i.e. multiple sclerosis) and traumatic central nervous system

lesions.

The close temporal relationship between tremor cells and peripheral tremor

suggests that these cells could act as tremorogenic pacemakers. The trauma

associated with electrode entry into a region of tremor cells can in some cases be

enough to stop the tremor for up to several days in the coniralateral side of the body.

Furthemore, microstimulation in these same areas can evoke transient tremor arrest.

However, the mechanism(s) by which this is accomplished is still not well understood

(Reviewed by Lozano. 1998).

1.5.3 Chronic Pain and DBS

De8p brain stimulation is the treatment of last resort for patients with intractable

chronic incapacitating pain that is unresponsive to conventional therapy. Thalamic DBS

has been used to treat many nociceptive and neuropathic pain states. Neuropathic pain

results frorn damage to the pain pathway of the newous system. This type of pain c m

be responsive to sensory thalamic (Vc) stimulation and the frequencies used are

typically lower than those used to treat tremor in Vim. Nociceptive pain (sharp or

aching), such as chronic lower back pain or cancer pain, is thought to be more

responsive to pedventricular grey matter (PVG) stimulation. Some patients experience

both types of pain. making the treatment protocol more difficult (Gildenberg and Tasker,

1998).

1.6.0 Current Ideas on Mechanisms of DBS

The therapeutic effect of DBS in motor thalamus is clinically well documented for

the treatment of trernor associated with PD and ET (Lozano, 1998). The mechanism(s)

undeilying its effects however are still not well understood. In general, the beneficial

effect of thalamic (Vim) DBS mimics a lesion of the same structure. Similarly, DBS and

lesions in the internat segment of globus pallidus and STN, for treating PD, have both

yielded very similar dinical benefits. The model for PD predicts that, as a result of SNc

dopamine depletion, the STN and GPi/SNr complex are hyperactive. This implies that

any therapy directed at GPüSNr or STN should reduce the hyperactivity (Obeso et al..

2000). This has lad to the belief that DBS was somehow inhibiting surounding gray

matter structures to mediate its therapeutic effects.

There are a number of theoretical possibilities that must be considered. First, it

has been suggested that high frequency stimulation could result in neuronal

depolarization block of sodium channels due to the maintenance of a depolarized state

in the neural tissue sunounding the stimulating electrodes. More specifically, this would - have the effect of preventing sodium channels from retuming to a de-inactivated state.

which is dependant on the membrane potential retuming to a hyperpolarized state. The

kinetics of the sodium channel activation and inactivation gates have been previously

reviewed (Silverthom, 1999). During depolarization the activation gate opens and

allows sodium ions to flow thmugh. After a short period of time. the sodium channel

inactivation gate blocks hirther ion entry. Membrane hyperpolarization is needed for the

two gates to reset to their original positions. If the activation gate doesn't reset. the

ability of the sodium channel to be opened via voltage activation is prevented, and

action potential generation is blocked. Second, the neural network wuld be disrupted

(also termed 'neuronal jamrningn) by the additional nerve impulses generated by the

stimulation. Finally, stimulation might produce a net inhibition in the network by the

activation of inhibitory neurons or by other mechanisms (Benazzouz and Hallet, 2000).

The cuvent ideas on the mechanism of thalamic DBS corne from obsewing the

clinical effects of lesions and obseiving the effects of dnig micro-injection and electrical

stimulation. Recent studies, involving the micro-injection of the local anasthetic

lidocaine (sodium channel blocker) into areas with tremor cells in Vim, have shown

tremor reduction. which further strengthens the inhibition hypothesis. In most cases,

lidocaine mimicked the effects of lesions and high frequency electrical stimulation

(Dostrovsky et al., 1993). A study involving microinjections of the GA8A agonist

muscimd into the Vim of ET patients also showed tremor arrest (Pahapill et al., 1999).

The effects of DBS in the Vim are well documented. Stimulation at high

frequency, typically greater than 1 OOHz. results in tremor attenuation or tremor arrest

while the stimulator is ON. After termination of stimulation. there may be rebound

*-worsening of th8 tremor. Interestingly, slow rates of stimulation may drive the tremor.

Recent, as yet unpublished results from our lab, indicate that 50% of neurons in sensory

mtor thalamus are excited when microstimulating at low frequency (3Hz-50Hz). close

to the cell body (c250um). This study used the same glued, dual microelectrode setup

that was used in this thesis (See Methods section for more details). Short duration and

latency excitation was seen after each stimulation pulse during the stimulus trains. Due

to the presence of the stimulus artifact, at higher frequency stimulus trains (i.e. 1OOHz).

it was difficult to observe the neural activity.

- -

~ledrical stimulation causes depolarkation that generates action potentials that

propagate from the site of stimulation along the axon. The initiation of the action

potential can begin in the axon or cell body (via depolarization that is transmitted to the

axon hillock). Thus, answering the question of what neuronal element is activated

during grey matter stimulation becomes essential. The identification of which elements

might be activated can be revealed through temporal properties detemiined from the

retationship between the duration, and the threshold intensity, of a current pulse

inducing a given response (strengthduration relationship or chronaxie) (Nowak, 1998).

The studies showing large myelinated axons being the most excitable neuronal element

in cortical grey mater were first reviewed in detail by Ranck (1 975). Since then,

strength-duration measurements in the visual cortex of rats revealed that axons and not

cell bodies are the elements activated first by low threshold electiical stimulation

(Nowak, 1998). Some reœnt chronaxie data from Vim has yielded results that support

the hypothesis that DBS preferentially activates large myelinated axons and not the cell

bodies (Holsheimer et al., 2000). This study compared the strength-duration time

.-instant (chronaxie) of large myelinated axons. small myelinated axons. cell bodies.

and dendrites. The data were wllected from PD and ET patients treated by DBS in Vim

or GPi. The threshokls for tremor arrest were used to obtain the chronaxies. The study

conduded that the primary targets of stimulation in both nuclei are most probably large

myelinated axons.

Studies similar to the topic of this thesis have been done on other nuclei of the

basal ganglia. A recent study. done in the rat STN suggests that high frequency

stimulation produces a transient inactivation of voltage-gated currents (Beurrier et al.,

2001). The study involved patch-clamp preparations of rat brain slices. The study

showed long inhibitory periods (typically 5-6 mins) following prolonged (1 min stimulus

trains) high frequency (1 66-500Hz) macrostimulation and microstimulation. The group

observed that high frequency stimulation blocked tonic and bursting STN neurons with a

mechanism that does not require calciumdependent transmitter release. They

concluded the silencing effed was not mediated by activation of local networks or by

stimulation of afferents to the STN. It was still observed in the presence of blockers of

glutamatergic and GABAergic ionotropic synaptic transmission, and in the presence of

cobalt, at a concentration that totally blocked synaptic transmission. This in vitro study

shows a potential mechanism for DBS in the rat STN. However, the patch clamp

technique destroys many of the afferent inputs, making generalizations to human DBS

difficult.

Stimulation through one electrode while recording from the other has yielded some

interesüng results in other basal ganglia structures suggesting synaptic release

mechanisms. Studies in GPi showed microstimulation-induced inhibition of neuronal

-firing in hurnan patients undergoing neunisurgeiy. This was shown in nearly al1 the

cells tested at currents typically under 10 UA at frequencies as low as 5HZ (Fig. 1.1 0).

These findings suggest that microstimulation in GPi excites the GABA terminais frorn

striatal and pallidal neurons, resulting in inhibition via GABA release (Dostrovsky et al..

2000). In the monkey, a group reporteci that stimulation in GPi at therapeutic intensities

resulted in decreased firing rate but not block of activity (Boraud et al., 1996). The

dorsal thalamus (including Voa, Vop, Vim and Vc) also has a high proportion of

GABAegic afferents. The teticular thalamus projects strongly to dorsal thalamus via

GABAergic fibers. Furthemore, interneurons project to thalamocortical cells via

GABAergic fibers. The thalamus works to tum itself off through its intnnsic circuitry with

the reticular nucleus. The triadic circuitry in the thalamus, however, seems to be more

complex than in other basal ganglia structures, involving excitatory cortical and sub-

cortical inputs inputs as well (Fig.l.7).

Together these data suggest that the mechanism of DBS may be much more

cornplicated than previously thought. Studies consistently show that the rnost excitable

neuronal elements are large myelinated axons. However, cunent density decreases

with distance frorn the stimulating etectrodes tip. Thus, the neuronal elements being

excited Vary with tip distance, further complicating any interpretations.

As of yet, the effect of high frequency micro-sümulation on thalamic neurons has

not been tested. The results of this would be important since thalamic DBS is such a

widely employed neumsurgical tool for the treatment of chronic pain and tremor

associated with movement disorders. This data may aid in elucidating possible

mechanisms of thalamic DBS, and DBS in general. This thesis discusses the effects of

microstimulation on thalamic neuron firing, and its possible role in therapeutic DBS. A

recenUy developed dual electmde apparatus is used to observe the effects of high

frequency microstimulation on nearby neurons in human patients. This apparatus

allowed for stimulation through one electrode white recording from the other and is the

first in vivo study of high frequency microstimulation effects in human or animal thalamic

neurons.

-- -

Fiaure 1.1 : The figure depicts various sub-nuclei of the dorsal thalamus typically

encountered during functional stereotactic neurosurgery procedures for treatment of

movement disorders and chronic pain. The abbreviations for the nuclei are as follows:

Vc = ventrocaudal nuclei, Vim = ventralis intemedius, Vop = ventrooralis posterior, Voa

= ventrooralis anterior. Table 1 describes the afferent and efferent connections of each

nucleus in more detail.

\

1

Figure 1.1

Nuclei of the Dorsal Thalamus -

a nterior

v Iateral inferiodventral

Figure adapted from Tasker and Kiss, 1995.

- Fiaure 1.2: The cell was held at three different initial holding membrane potentials as

indicated. Each of the thtee recording traces shows a response to a 0.3nA curent

pulse. Note that when the cell is held at -55mV (A) it responds to the depolarizing

cuvent pulse with a sustained firing of unitary action potentials. 6 indicates a

membrane potential at which the LTS channels are not de-inactivated and where the

cuvent pulse does not produce an action potential. When the cell is held at -70mV (C),

the LTS channels are in the deinactivated state and are activated by the depolarizing

cuvent pulse. The LTS is accompanied by a burst of action potentials.

.- .-

Figure 1.2 ---- A..?- - - -

Tonic and burst firing modes for a relay cell from the cat's lateral geniculate nucleus recorded intracellularly in an in vitro slice

-7OmV I 0.3 nA - . f

FQum adapted h m S. Shennan and R. Guillery's Explofi'g The ~ ~ I a m u s , 2207.

- Fieure 1.3: The figure depicts the voltage dependence of the LTS and the number of

adion potentials generated by the LTS in two cells (A and 0). Note that a

depolarization pulse from more hyperpolarized membrane potentials results in a larger

LTS with more action potentials.

Figure 1.3 Voltage Dependence of LTS 30

Figure fmm Zhen el al., 2000.

Initial Membrane Holding Potential (mV)

-= -

Fiaure 1.4: The figure illustrates various voltagedependent conductances that are

thought to contribute to cyclical burst firing in thalamic relay cells. Hyperpolarization

causes both IT de-inactivation and IH activation. In activation causes lT activation and a

subsequent burst of sodium dependent action potentials. This activates various

potassium conductances that then hyperpdarize the membrane and cause a repetition

of the cycle.

' c'

1 : l~ de-inactivateci . 4: Na+/K+ siikes activated ' - 2: lh adivated 5: various 1- activated

Figureadapted from McCormick.. 33: activated 6: lh de-activated and Pape, 1900. ' -.., 7: IT inactivated -

Fiaure 1.5: The figure illustrates the difference in lA and IT deactivation and activation.

Notice that both curves have a similar shape however the voltage dependence of IA is

shifted to the depolarized direction. On the left are shown the inactivation curves

(circfes). The points for this curve were obtained by holding the membrane at various

holding potentials (plotted on abscissa) for i second and then stepping up to 42mV.

Note that the more hyperpdarized the starüng level the greater the cuvent evoked

although both currents move in opposite directions. The activation curves (squares)

shown on the right were obtained by initially holding at -1 02mV, which would completely

deinactivate the channels and stepping up to various membrane voltages plotted on

the abscissa. The greater the voltage step, the greater the evoked conductance. This

is thought to occur because larger depolarizations activate more Ir and IA channels.

Whether a postsynaptic potential activates one or both currents depends on the initial

membrane voltage and the temporal properties of the voltage channels. Notice from the

figure that there is a limited membrane potential near -70mV where lT is inactivated but

Ir is still slightly de-inactivated. During this period the cell may not fire at all. (A) Curve

"for a relay cell. (B) Curve for an intemeuron where there is more overlap between the

inactivation curves.

- - -

Fi-aure 1.6: The figure depicts the basic connections between the thalamus. reticular

nucleus, and cortex. The connections depicted are of first order thafamic nudei

depicted in a coronal section. The nuclei labeled A and B are connected with

corresponding A and B reticular sectors and conespondhg cortical regions. Note that

wrticothalarnic neurons can synapse ont0 thalamocortical neurons directly or indirectly

via RE neurons and interneurons.

Figure Connections between Thalamus and Cortex

TRN = Thalamic Reticular Nucleus

Figure adaited from Sherman and GuilIIe~y~ 2001.

- - -

Flauie 1.7: The drawing indicates the synaptic relationship typical of the majority of

thalamic nuclei. It is generally believed similar inputs are found in al1 dorsal thalamic

relay cell dendrites, although the majority of the studies have been conducted in the

lateral geniculate nucleus (LON). Notice that the subartical driving input (Tl) activates

the relay cell (R) dendrite (D) directly and via the GABAergic interneuron's (1) pre-

synaptic dendntic appendages (T2). Axons of the interneurons also teminate (F) on the

presynaptic dendrites. The sub-cortical modulatory inputs via the brain stem also make

similar dendritic contacts although they are not depicted in the figure (Sherman and

Guillery. 2001). The triadic circuit is ernbedded in a glomerulus, composed of astrocytic

processes, (G) into which a dendritic appendage (O) fmm a relay cell intnides. Outside

this, cortico-thalamic teminals (C) end on relay cell dendrites and on pre-synaptic

teminals of interneurons. H o w ~ v ~ ~ , the majority of cortico-thalarnic teminals are more

distally situated. Rt, indicates the reticular thalamic cell input (GABAergic) which also

occurs proximally on the cell body. The recticular thalamic input is a major source of

inhibition to thalamic relay cells. It should be noted that inhibitory synapses from pre- - ' synaptic intemeuron dendrites ont0 relay cells, outnumber excitatory, sub-cortical

synapses by a factor of 4 (Jones, 1985).

a- - *

Fiaure 1.8: The figure illustrates the various afferent inputs converging ont0 a relay ceIl

in the lateral geniculate nucleus. The same arrangement is thought to exist in al1 dorsal

thalamic nuclei.

to . from

Thalarnic Relay CeCl Afferent Inputs

ACh acetylcholine .

Glu glutamate Int. interneuron NO nitric oxide NA noradrenaline PBR parabrachial region TRN thalamic reticular nucleus

Figwe adapted h m Sherman end Guilleiy, Exploring me Thalamus.

1.9: The figure depicts the afferent inputs ont0 a typical thalamic intemeuron (A)

and a reticular cell (B). The conventions are the same as in Figure 1 .a. The

intemeuron has two functionally distinct innervation zones. One incfudes the cell body

and proximal dendrites when the firing of the axon is controlled (via Fi teminals;

proximal and cell body afferents). The other are the more distal F2 terminal regions

where pre-synaptic contact to relay cells are made. There are a lot of questions as to

what type of innervation the reticular cells receive. Thus, besides the established areas

of cortical, brainstem (mainly parabrachial) and w-lateral input via thalamic relay cells,

not much is known. The abbreviations are as in figure 1.8 plus: BF, basal forebrain;

NOfl nucleus of the optic tract.

Figure 1.9

Thalamic lnterneuron and Reticular Cell Afferent Inputs

Questlon marks indicate afferent connections that are not well established.

Figure adapted from Sherman and GuiIIery, 200 1.

-A&- c

Fiaura 1.1 0: The figure depicts results from recent experiments involving dual electrode

recordings and stimulations from the intemal segment of globus pallidus (GPi). Note

how micro-stimulation produces inhibition following each stimulus pulse (A). Part B

depicts a 1OOmsec interval of the same cell during a 300Hz stimulus train. Note that the

cell did fire occasionally during this period but not during the first 1.3 seconds of

stimulation. Thus, increased stimulus frequency caused more pronounced inhibition.

This suggests that microstimulation may activate the GABAergic aflerents from the

striaturn and GPe resulüng in the release of GABA onto GPi cells. Since the motor

thalamus also receives a substantial amount of GABAergic afferents from the thalarnic

reticular neurons, intemeurons. and Gpi, DBS effects here may also be mediated

through the release of GABA.

(Table 1: Summary of thalamic subm Hassler Nomenclature Types of celis Mrents L ~ o (iateropoiaris) medium sire 7

iclei being studied

-6

area 4.6 and possiMy 3a

Table adapted fmm Taker and Kiss, 1995.

. . - *

Chapter 2

Methods

2.1 Patient Population

Studies were perfonned on 13 patients undergoing functional stereotactic

neurosurgery. In nine of the patients the target was within the thalamus. In the

remaining four patients the STN was the target. In these cases soma of the electrode

tracks passed through the thalamus. Most of the patients were operated on for

alleviating chronic pain or movement disorciers. Table 2.1 shows patient groupings and

surgical target areas. The functional stereotaxic neurosurgeries were perfomed either

to make a lesion or implant a DBS eledrode. Depending on the surgical target various

groupings of cells were encountered. In the STN cases, thalamic cells were generally

cdlected from more medial (1 0.5-1 2mm h m the midline) and anterior (Thalamic

reticular nucleus, Voa, Vop) portions of the thalamus relative to the Vim and Vc cases

(generally 14.5-1 5mm from midline). Figure 2.1 shows the various thalamic subnuclei in

a sagittal section 14.5mm from the midline. The procedure for chronic nociceptive pain r

treatment targeted the periventricular grey matter (PVG). Electrode tracks dunng this

procedure recorded from th8 medialis dorsalis (MD) regkn which is medial to Virn and

Vc (-5mm from the midline) (See Introduction figure 1 A). The procedure for epilepsy

treatment targeted the anterior nucleus (AN) which is located medial (-6mm from

midline) and anterior to Vc and Virn (See Introduction figure 1.1). Cells in this area are

distinct h m the rest of the thalamic nuclei studied. because they do not receive input

frorn the thalamic reticular nucleus (see Introduction section 1.4.5). Some of the data (7

patients) analyzed for this study were obtaind previously. The procedures were

perfomed in accordance with the Human Experimentation Cornmittee of the University

of Toronto and the Toronto Western Hospital, and patients gave free and informed

consent. The chief neutosurgeon (Dr. Andres Lozano) and the neurosurgery residents

and fellows were responsible for conducting the MRI scans, setting up the stereotactic

frame, inserting the guide tube. and implanting the DBS electrode. Dr. Jonathan

Dostrovsky, Dr. Karen Davis, and Dr. Bill Hutchison were responsible for the

rnicroelectrode recordings and stimulations.

The PD patients generally stopped their regular medication for at least 12 hours

pnor to surgery. This caused the symptoms to be more pronounced which allowed for

more precise localization of effective surgical targets. All patients rernained awake

during the procedure. This was important because their participation was required in

determining receptive fields (RFs) and projected fields (PFs). two components essential

in mapping out the various regions of the brain. Furthemore, sleep can change the

intrinsic physiological properties of brain regions. particularly in the thalamus where

sleep can cause cells to switch into a bumt-finng mode and cause tremor arrest. - ' 2.2 Stereotaxlc Technique

The methods used for stereotadic neurowrgeiy have been previously ieuiewed

(Lenz et al., 1988; Gildenberg and Tasker, 1998; Hutchison et al.. 1998). The

stereotaxic technique allows for the precise location of intemal landmarks in ternis of X,

Y. and Z coordinates to be detemined. An MRl-compatible. arc-centered Leksell

stereotactic frame (see figure 2.2) was fitted on the patient's head. The coordinates for

the anterior commissure (AC) and posterior commissure (PC) were detennined relative

to the stereotaxic frame using a magnetic resonance imaging (MRI) scan. A cornputer

program was used to produce sagittal brain sections based on standard sagittal

sections from the Schaltenbrand and Wahren Atlas (Schaltenbrand and Wahren, 1977).

These were stretched or shnink to fit with the patient's ACIPC distance. In the

operating Riom, the patient was placed in a semi-sitting position. The frontal scalp was

washed, shaved, and painted with betadine solution. Under local anesthesia, a 3 cm

linear parasagittal incision was made 2cm from the midline (Lozano, 1998). A burr hole

that was in approximately the same parasagittal plane as the microelectrode trajectory

and target was then made (also under local anesthesia) by the neurosurgeon. A thin

walled guide tube was then positioned 10-1 5mm above the target. The guide tube was

necessary to protect the fine microelectrode tip before introduction into the brain tissue

and ensure minimal deviatkn of the electrode (Lenz et al., 1988). The microelectrodes

were then advanced through the brain tissue using a hydraulic microdrive.

2.3 Microelectrode Stimulation and Recordings

Physiological corroboration of the selected anatomical target was performed using

single-unit recording and stimulation using high-impedance microelectrodes. Details of r

the microelectrode setup and assembly have been previously descnbed (Lenz et al.,

$988; Hutchison et al., 1998; Oodmvsky et al., 2000) and will only briefly be addressd.

Single andlor multi units were recorded using Paryîene-coated tungsten

microelectmdes with an exposed tip length of 15dOpm. The electrode tips were plated

with gold and platinum to d u c e the irnpedance to a few hundred WI. The electrodes

were then givsn a bubble test, where the eledrode was immerseâ in saline solution

while a low OC voltage (3-5 V) was given. If the electrode is properly insulated this will

result in bubble formation only at the tip. The signals were filtered and amplified using

- the Guideline System GS3000 (Axon lnstruments, Foster city, CA). The two channels

of neuronal data were recorded on analog videotape (VR-100 digital recorder,

Instnitech Corp., Port Washington, NY). Singleunit recordings were necessary in order

to determine receptive field data that was vital in localizing the deep brain stnictures.

Two different rnicroelectrode configurations were used in the study. In some

cases Iwo microelectrodes were fixed together with glue (tips approximately 250uM

apart) and driven as one unit. Either electrode could be used to stimulate or record

from. This arrangement allowed for recording from one microelectrode while stimulating

from the other (stimulation was, biphasic. monopolar, with a .15msec pulse width). In

th8 other configuration. two independently controlled rnicroelectrodes were attached to

separate microdrives. This setup allowed movement of one electrode with respect to

the other. In this setup the rnicroeledrodes were further apart (about 650um-1 mm).

Using the above setups, the effects of microstimulation on neuronal activity could be

examined in 3 ways:

' A. Stimulation could be carried out diredly from the recording electrode and the

effects on neuronal activity following the stimulus train could be recorded. The

amplifier could not record for the 150-200msec period following the stimulus

train. In this case the electrode tip was in close proximity to the cell.

B. In the fixed configuration, stimulation thmugh one electrode was perfomed while

simultaneously recording h m a nearby (-250um away) electrode.

--- -- -

C. Finally, using the independent configuration. stimulation effects h m further

away (~650urn) could be observeâ.

The rnicroelectrode signal was monitored by listening to an audio monitor and by

observing the associated spike train displayed on the touch sensitive monitor on the GS

3000. The screen displays firing rate and a spike triggered waveform from which single

unit action potentials could be visualizeâ. The signal was also fed into an audio channel

(recorded on videocassette) that was later used to digitize the recording using the

computer program Spi ke 2 (Lenz et al.. 1 988).

Along the course of each electrode track the cells encountered were tested for

sensory responses. Electrical stimuli (1 sec, 300Hz) were delivered to determine

affects on tremor and whether sensations could be elicited. For thalamic surgeries the

initial target was usually the somatosensory portion of the dorsal thalamus (Vc). Cells in

Vc were identified by tactile receptive fields, Le. responding to light touching.

Furthemore, the Vc region was identified by the presence of high amplitude recordings

r of extracellular action potentials. and cells in this region tended to have a higher

spontaneous activity than in the adjacent Voa, Vop. and Vim nuclei. The Vim region,

which is anterior to Vc, was localized by the presence of cells with responsiveness to

passive joint movements. In tremor patients, Vim also contained tremor cells that Zred

in synchrony with the peripheral tremr. The Vop (anterior to Vim) and Voa (anterior to

Vop) regions were identified by the presence of cells that responded to voluntary

movements. Trernor cells could also be found in the Vop region (Fig. 2.3) (see

Introduction section 1.4, for review of the properties of thalamic sub-nuclei). The

&cePtive field (RF) and projected field (PF) data were usad to make reconstructions of

the electrode tracks (Fig. 2.3). The electrode track reconstnictions were compared to a

cornputer generated map of the stereotaxically detemiined anatomy (corresponding to

the location of the electrode) and were used for final localization of the cells. Thus, the

PF and RF data along with the Schaltenbrand and Wahren Atlas reconstructions were

used to identify the various thalamic nuclei.

2.4 Cell Populations and Offline Analysis

The recordings from both electrodes were stored on magnetic tape. Off-line

analyses of the digitized recordings were perfonned with a CEDI401 Spike 2 data

acquisition system. For most of the patients. individual units were discriminated using

waveform template rnatching (Spike 2 For Windows, 1998). The size and shape of the

action potential were used as criteria for the discrimination. In the remaining patients,

units were discriminated with a window discriminator.

2.4.1 Bursting Cell Analysis

- Bursting cells were detenined either visually or based on a wmputer script that

identified and quantified bursts based on their interspike intenrals. This automated

analysis identified a burst according to the following critena:

4. The maximum initial interval to identify the stait of a burst was 6msec.

2. The maximal interspike interval for a given burst was 1 Smsec. lnterspike intervals

larger than 15 msec are generally not associated with bursts.

3. The minimum burst duration was 2 mec.

4. The minimum number of spkes per burst was 2.

--

The script then computed and

activity of each cell. This was used

displayed descriptive statistics for the bursting

for graphical characterization of LTS bursts. Low

threshold calcium spike bursts were charaderized by graphing components of their

interspike intervals (see figure 2.4), or visually, by recognizing the characteristic LTS

pattern (see figure 2.5). The size of each inter-spike interval (ISI) gets larger with each

su bsequent ISI within a given burst. For example, if there was a burst with 4 spikes, the

ISI between the first two spikes would be smaller than the ISI between the second and

third spikes (decreasing ISI length as the ordinal number increases). This is shown by

plotting the ISI vs ordinal number as shown in figure 2.4. A second criterion that was

used involved comparing the Rrst ISI between various bursts. As the number of ISIS in

a burst increases (Le. larger bursts), the first ISI gets smaller. Thus, larger bursts have

smaller first lSl intervals. This is depicted in the regression plots in figure 2.4. Both of

these observations are consistent with LTS generated bursts (Lu, SM. et al., 1992;

Tsoukatos, J. et al.. 1997; Radharkrishnan et al.. 1999; Ramcharan, E.J. et al., 2000).

This burst analysis was carried out for both pre stimulus (Fig 2.4A) and post stimulus

T(Fig. 2.4 8) bunts. The majority of the LTS bursting cells were identified visually after

observing consistency betwan graphical and visual interpretations. Figure 2.5 shows

the pattern of a LTS burst. Almost al1 of the bursting cells rewrded showed a LTS

bursting pattern. Only one bursting cell was identified as a non-LTS bursting cell.

Long duration inhibition was arbitrarily defined as inhibition lasting at least 1

second. This period was defined by the absence of action potentials. In cells with

bursts before the start of inhibition, the inhibitory period was defined as starüng from the

end of the last acüon potential. In cells with irregular firing patterns, post stimulus

--- -- -

inhibitory periods were difficult to separate from random periods of silence. In these

œlls. periods of spontaneous pauses in firing were in the same order of magnitude as

the inhibitory periods, making it difficult to define inhibitory periods. Thus, cells that did

not have regular Rring patterns were not included in the results. In cells that were

included, the short spontaneous silent periods were very srnall compared to the periods

of inhibition typically observed in this study (see figure 3.2).

-Y A = -

&ure 2.1 : The anterior and posterior commissure points were determined through a

high resolution MRI scan. The figure depicts a map of the human thalamus in the

parasagittal plane 14.5mm lateral to the rnidline. Vol = area with cells that respond to

voluntary movements. Ki = area with cells that respond to kinesthetic movements.

Figure 2.1 - - L A -

Sagittal Section of Thalamus

- . L A -

Fraure 2.2: The figure illustrates the siereotactic frame assernbly. The microeledrode

carrier is attached after the frame is fitted on the skull. The arc centered frame pemiits

any target site to be reached from the same hole in the skull by changing the angles

and X, Y. and Z coordinates of the arc.

-- L-

Figure 2.3: The figure depicts receptive field (RF) and projected field (PF) data from

cells encountered through a single electrode trajectory during stereotactic surgery for

chronic pain. Cells that respond to kinesthetic inputs are generally thought to exist in

Vim. The Vop region has cells that respond to voluntary movements. The ventral

caudal region (Vc) is the tactile relay. Electrode reconstructions such as this were used

to help detennine the location of the cells.

Figure 2.3 Electrode T m k Reconstruction s21 14-" 1

wam, wh bwofse

ce4 leg buttocüs wann 20 40

AQAnlrikr Comnrbun -Connilrui. mC=rnCaimirunlPdn(

lndkaws lOCllf/on wh.n œll w.r raIuktid

-- Fiaure 2.4: The figure depicts the graphical characterization of LTS bursts. The size of

eabh inter-spike interval (ISI) gets larger with each subsequent ISI within a given burst

(increasing ISI as the ordinal number increases). This is shown in the ISI vs Ordinal

number plots. Furthemore, as the number of ISIS between bursts increases, the first

ISI gets smaller. This is depicted in the regression plots. Both of these observations

are consistent with LTS generated bursts. This was camed out for both pre stimulus (A)

and post stimulus (B) bursts.

Figure 2.4

Graphical Characterization of LTS Bursts. Length of ISI vs Ordinal Number

Length of Fint ISI vs Number of ISlss in the Burst

- - -

~ i ~ u r e 2.5: The figure depicts the pattern of an LTS generated burst from an in vivo

recording. It is quite easy to Mentify an LTS burst visually because of this characteristic

pattern.

Figure 2.5 --- -2- --= -

Extracellular methods used for visual and graphical characteriration of a low- threshold calcium burst

The duration of the first ISI decreases as a function- of total ISl's in a given burst

Furthermore, B the duration of a particular ISI within a burst increases as function of m n w a m w

a 2 C U ; N ~ J I m )

ordinal number r A mtniza(icr) 4an4Q(11u~ + s r n m c a ,

L

Table 2.1: Patient and Target Information I

Patient #

359 375 402 404 363 2242

Condition

Chronic Pain Chronic Pain Chronic Pain Chronic Pain Parkinsons Parkinson's

2243 Parkinson's Bil DBS, STN 3

DBSILesion, Target

DBS, Vc DBS, PVG DBS, Vc DBS, Vc DBS, Vim

Bil DBS, STN

2249 2250 364

1

373 367 374

Total Cells Tested

4 5 5 5 5 3

Parkinson's Parkinson's

Epilepsy Cerebellar Tremor Essential Tremor Essential Tremor

Bil DBS, STN Bil DBS, STN

Anterior Group DBS, Vim DBS, Vim

Lesion, Vim Totals

6 10 3 1 23 1

74

~hapter 3

Results

The effects of high frequency (100-333Hz) electrical stimulation, delivered from a

recording electrode or a nearby electrode, were examined in 74 neurons. Of these, 19

were from chronic pain patients, 27 from Parkinson's patients. 24 from Essential Tremor

patients, three from an epilepsy patient and 1 from a cerebellar tremor patient. Table 1

lists the number of cells tested in each patient. Cells were recorded from various

thalamic subnuclei including: Vc, Vim. Voa and Vop. Table 3.2 shows how many cells

were tested in each nucleus in each of the 3 major patient groups.

High frequency stimulation (100-333Hz) frequently resulted in long duration

inhibition (>l sec) that was usually preceded by a short period of bursting. An exemple

illustrating this effect is shown in Fig. 3.1. A total of 38 out of 74 cells showed this long

duration inhibition following the high frequency stimulation. Tables 3.1 and 3.2 list the

incidence of inhibition according to patient type and nucleus. In almost al1 the cells that

showed this effect the inhibitory par id was at least Mo seconds in duration. In some -

cases the inhibition lasted as long as 25 seconds. Only two of the 74 cells showed

inhibition of less than one second (see Fig. 3.2).

There were 3 different conditions under which the stimulations took place. First,

stimulations were carried out directly from the recording electrode in which case the cell

was in very close proximity to the stimulating electroâe (e.g. Figs. 3.1, 3.3). When this

type of experiment was carried out, stimulation cunents above 10 UA were rarely used.

At this pmximrty higher current intensities can kill the neuron being tested. This type of

stimulation was examined on a total of 44 cells and resulted in long duration inhibition in

- -

21 (48%) of the cells. The effects of stimulation deliverd from a nearby elecîroûe

(-250um) that was glued to the recording electrode were examined in 28 cells. This

type of stimulation resulted in long duration inhibition in 18 (64%) cases. Under these

conditions, stimulation currents of up to 1OOuA were tested. An example from one of

these cases is shown in Fig. 3.4. Finally. stimulations were perfomed using two

independently controlled microelectrodes (>650um) at cunents up to IOOuA. None of

the 8 cells that were tested in this way showed long duration inhibition or any other

effect.

The long duration inhibition was usually preceded by a short period of bursting

(pre-inhibition bursting) activity. Pre-inhibition bursting was seen in 33 of 38 (87%)

neurons that showed long duration inhibition. In cells that were bursting before the

stimulus was applied. the post stimulus bursts were generally larger than the bursts

prior to the stimulation. Larger post stimulus bursts were seen in 16 of 18 (89%)

bursting neurons that showed long duration inhibition. An example of pre-inhibition

bursting is shown in figure 3.5. Even though the pre-inhibition bursting period was not

'Ôbserved in a small number of cells. their occurrence could not be ruled out in these

cases because they occurred only with stimulation through the recording electrode. In

these cases the amplifier remained tumed off for a period of about 200 ms following the

end of the train. Thus, if bursting had occurred within 200 ms of the tenination of the

train, it would not have been detected.

Long duration inhibition was obsewed in most neurons that had a bursting firing

pattern (29 of 36 bursting cells tested, 81 %) but much less ftequently in cells that were

not bursting (24%). Table 3.3 shows the breakdown of effects according to nucleus and

==- --

whether the cells were bursting or no€. AJmosf amofthe burstihg celfs exhibiteci LTS

bursts.

The effeds of current intensity, train length. and frequency of stimulation on the

length of the inhibitoiy period were systematically tested in 15 cells. The effect of

varying cunent intensity was tested on a total of 6 cells. In al1 6 of these cells there was

increased duration of inhibition with increased stimulus current. Figure 3.6 shows an

example of the effects of increasing the intensity of the stimulus current. In two of the

cells, the number of pre-inhibition bursts decreased with increasing current. However,

the number of spikes per burst increased with increasing stimulus current. This is

illustrated in figure 3.7. The other cells didn't show any difference.

Six cells were tested for the effects of varying train length in the range 0.1 sec to

4sec. Al 6 cells showed longer duration inhibition with increased train length.

However, in some cases (4 of the cells) the amount of pre-inhibition bursting decreased

as a function of train length although the duration of inhbition increased. An example

showing the effects of increasing train duration is shown in Figure 3.8.

Three cells were tested for the effed of stimulus fiequency on inhibition. In al1 3

cases increasing the frequency in the range from 10 to 333Hz resulted in increased

duration of inhibition. Figure 3.9 shows an example of the effect of increasing train

frequency on the inhibition. In one of the cells, increasing frequency caused a decrease

in the number of bursts. This is illustrateâ in figure 3.9. The other cells did not show

any difference.

---- - Fliaure 33: The effect of high frequency sttmulatton detivered from the same etedrade

that was recording the cell. The duration of this type of inhibition was typically longer

than 2 seconds.

Figure 3.1 'l

Effect of high frequency stimulation from recording electrode on a bursting cell in the thalamus.

I

1 2 seconds 1 sec, 1 OOHz, 5uA. 0.1 Smsec pulse width Stimulus train bursting d l , MD, 37 SOI

- &-..A FiauFe 3.2: HQfi fiequency sfimutation producecf short duratton inhibition (clsec) in onfy

2 out of 74 cells tested. An example of this type of inhibition is depicted in the figure.

Figure 3.2

333 HZ, 0.1 5msec pulse width, ~ U A , 1 sec Stimulation Causing Inhibition

400 msec inhibitory period

1 second

ceIl35906 VoaNop

- - hure 3.3: Example of effects of stimulation on cell Rtng when stimulating directly from

the recording electrode at close proximity.

Cells 224201 a and 224201 b (smaller) are depicted before, and following several 200 Hz

5uA stimulation trains. 60th of the cells are LTS bursting cells. The bursts are depicted

at a smaller time interval in figure 3.5. Following stimulation there is an increase in the

sire of each burst after which there is a long inhibitory period of up to 8 sec. Note that

the srnaller unit takes tess time to recover.

Effects of several 500msec, 200Hz,5uA, 0.1 Smsec pulse width stimulus trains on two bursting cells.

smaller cell l

~ a r ~ e r cell

5 seconds - VoaNop ceIl224201

A - -

mure 3.k The figure depicfs the eRéc€of high fiequency stimulation fiom a nearby

stimulating electrode on a thalamic bursting cell. Notice the long duration inhibition of

the smaller unit following the stimulus train. The stimulating electrode was

approximately 250um away from the recording electrode.

Figure 3.4

Effects of stimulation from a nearby stimulating electrode

100Hz, IOuA, 0.15 msec pulse width

- 2 seconds

\ / DT, Celi 36734

-= -- - FiKm 3 S me iiiusfration shows aie efect ofstiimuhtion on bursting. The majority of

cells (33 of 38; 87%) that showed long duration inhibition showed bursting activity

preceding the onset of the inhibitory period. For cells that were bursting before the

stimulus train, the post stimulus bursts were larger than pre stimulus bursts. This was

observed in 16 of 18 (89%) cells. In this example the average burst size before

stimulation was 2.25 spikes per burst. Following stimulation, the average burst size was

4 spikes per burst.

--.. .

Figure 3.5 - ---.------.. - -

The Effects of Stimulation on Burst Size

Pre-Stimulus Burst Post-Stimulus Burst The average size of The average size of bursts for this cell was bursts following a 100Hz 2.25 spikes per burst 5uA stimulation was

4 spikes per burst Bursting CeII VoaNop (0.15 msec pulse width)

-- Fiqure 3.8: The figure depicts the effect of incteasing current on the inhibitory period of

a bursting cell. The stimulation trains were given at 50Hz and were 1 second in

duration. A similar effect was observed in 6 cells.

'/ Figure 3.6

The Effect of Current lntensity on Length of lnhibitory Period Following a 500msec 50 Hz Stimulus (0.15msec pulse width) Train

2 sec

Cell224201, V W o p

mz -- Figure 3.7: This figure shows the effect of currenf on bursf sue (events per burst) and

the total number of bursts in two neurons in Vim following IOOHz stimulus trains.

lncreasing current caused larger bursts but a lower number of total bursts. Note how

the effect is not as pronounced in the smaller unit.

Figure 3.7 -A---- 2

The Effect of Current On Pre Inhibition Bursting

1 OOHz, O. 1 Smsec pulse width, 500msec stimulus trains

4 seconds Bursting cell, Vim

Average eventslburst 3.9 5.5

Average eventdburst 3.5 3.8

Larger Cell Average for 10 UA Average for 20uA Smaller Ce11

L

Average for 10 UA Averaae for 20uA

# of Bursts 5.5 3

# of Bursts 4.5 4

- A

Fiaure 3.8: The effect of train length on the inhibitory period is depicted for a bursting

cell. Stimulation with longer train lengths resulted in longer inhibitory periods in 6 cells

that were tested. In 4 of these cells, increasing train length duration caused a decrease

in pre inhibition bursting activity.

Figure 3.8

The effect of train length on inhibitory period following 200Hz,3uA stimulations (0.1 5msec pulse width)

500 msec train I 2 seconds 1000 msec train

n -- Fiaure 3.9: The figure illustrates the effects of increasing frequency on the inhibitory

period of two bursting cells (also depicted in figure 3.5). This response was seen in 3

cells that were tested. Note the effect of stimulus frequency on pre inhibition burst

activity of the larger cell. lncreasing the frequency of stimulation resulted in a smaller

number of bursts.

Figure 3.9 ---LA a---- 2 -

An example of the effects of increasing frequency on the inhibitory period of two bursting cells

second stimulus train 5uA stimulation period 0.1 Smsec pulse width

- 2 Seconds Bursüng Cell VoaNop

'F requency of Stimulation (Hz) 10

I

20 50 100

Average # of Bursts IAverage eventshurst 5.5 5

2.5 4

2 2

4 3

Table 3.' Summar Patient # --F

I Note that loi

t . . I

r of Data For High Frequency Stimulation rn

Cerebellar Tremor Essential Tremor Essential Tremor Totals

Percent % 75 80 20 O 80 67 O 50 40 I

O

Condition

Chronic Pain Chronic Pain Chronic Pain Chronic Pain Parkinsons Parkinson's Parkinson's Parkinson's Parkinson's Epilepsy

1 _

23

g tem inhibNion was erbitraniy defined as inhibition a t e r than 1 sec

1 74

Total Cells Tested

4 5 5 5 5 3 3 6 10 3

O 17

Cells Showing lnhi bition

3 4 1 O 4 2 O 3 4 O

O 74

O 38

O 51

-- - -

I~able 3.2 Patient and Subnuclei Cell Grouping

ûther nudei included dorsal areas, above Vc, Virn, and VopNoa, along with a few cells frorn the anterior, medial, and reticular thalamus

Patient Group

PD ,Pain Other

Other patients included ET, EQilepsy and Cerebellar trernor.

Total

27 19 28

# inhibited

13 8 17

VG Total

O , 1 9

# inhibited O O 3

Vim Total

O 7 4

# inhibited O 3 2

VopNoa Total

24 6 1

Other I

# inhibited 13 1 1

Total 3 5 14

# inhibited O 4 11

Table 3.3: Effect of Stimulation on Butsting and Non-BurstingCells Location of c e b vc Vim Vop/Voa OLher Total

Bursting Cells Total #

1 4 16 15 36

NonBursting Cells Total #

9 7 15 7 38

# Inhibied

O 4 13 12 29

% lnhibited

O 100 81 80 81

# Inhibited

3 1 2 3 9

% Inhibition

33 14 13 43 24

Discussion

This study has shown that high frequency stimulation (HFS) results in long

duration inhibition of thalamic neurons (51 %) following the stimulus train (Table 3.1).

Typically, the inhibitory periods were longer than 2 seconds and in many cases above 5

seconds. These effeds were predominantly seen following high frequency (>100Hz)

stimulation at close proximity (less than or equal to 250 pm) to the cell. Furthemiore,

the long duration inhibition was seen primarily in bursting cells (81 % in bursting cells

wmpared to 24% in non-bursting cells). These findings are summarized in tables 3.2,

3.3, and 3.4. The length of long duration inhibition was found to be dependent on

current intensity, train length, and frequency of stimulation. This is the first study of its

kind done in animal or human thalamus.

4.1 Results From Similar Studies (GPi, SNr, and STN)

Recently, similar studies to the topic of this thesis have been done in our /--

laboratory in other deep brain structures of the basal ganglia. In these studies, high and

low frequency stimulation was carried out in GPi. STN, and SNr and the effects were

different than those described in this thesis for thalamus. Two main types of

observations were made. First. the effeds of single pulse stimuli during low frequency

trains on neuronal firing patterns were observed. Second, effects of high frequency

stimulus trains on neuron finng were observeci following the termination of the stimulus

train. Low frequency shidies in GPi showed inhibition of neumn firing following each

stimulus pulse. The inhibitory effects were seen using the fixed (-250pm apatt) and

independent (-650pm apart) microelectruâe assemblies. The duration of inhibition

obsenred in GPi experiments was ver- short (typically <50msec, Fig 1 .IO). That study

suggested that the inhibition was mediated by GABA release from incoming afferents of

striatal andior extemal pallidal (GPe) origin (Dostrovsky et al., 2000). The effects of low

frequency stimulation in SNr were similar to those in GPi. High frequency

microstimulation in GPi and SNr typically caused short duration inhibition (Le. a few

hundred msec) following termination of the stimulus train. This is much shorter than

that obsewed in thalamic cells. The effects of microstimulation in GPi and SNr are

consistent with the theoretical mode1 for the pathophysiology of PD.

Theoretically, inhibition of STN would cause less excitation of the SNrlOPi

cornplex and improve parkinsonian symptoms. The therapeutic effects observed from

STN DBS are consistent with this. However, low frequency microstimulation in STN did

not consistently produce any effects. This may be due to differences in the ratio of

excitatory to inhibitory terminais compared with GPi and SNr. High frequency

stimulation (50-200Hz) in the STN resulted in inhibition (58% of cells tested) following r the stimulus train. This inhibition ranged from 50 to over Sûûmsec, which was

considerably shorter than the inhibitory periods observed in this thesis for thalamic

stimulation. For some of the STN cells the pattern of inhibition consists of inhibition

followed by rebound excitation and subsequently followed by more inhibition. This is

consistent with animal studies and suggests that inhibition in STN is mediated by

hyperpolarization. Since sodium action potentials are observed during the inhibitory

periods, the inhibitory periods are most likely not a result of depolarization block.

Together, these findings show that high frequency microstimulation in GPi. SNr. and

STN results in inhibition of cell finng. However, the type of inhibition observed in these

structures is cleariy diffwent from the effects observed following high frequency

stimulation in the thalamus.

4.2 Low Frequency Stimulation Effects in the Thalamus

Interestingly, recent evidence from our lab indicates that low frequency stimulation

produces short latency and short duration excitation (~50msec) of some (-50%)

thalamic cells following each stimulus pulse. Furthemore, in a number of these cells

there is a short period of inhibition (typically c100msec) directly following the initial

excitation. The excitation could be the result of glutamate release following excitation of

glutamatergic afferents. However, the stimulation should also have activated

GABAergic afferents. Presumably. at low frequency, the net excitatory effect was larger

than the inhibitory.

4.3.1 Pre Inhibition Bursting

The predominant effect following high frequency stimulation was long duration

inhibition that was pteceded by a short period of bursting activity. A number of points r

regading the pattern of bursting prior to long duration inhibition should be considered.

Them was cormistently (in 89% of cells test& showing long duration inhibition) an

increase in LTS burst size prior to the onset of inhibition compared to burst size before

stimulation (see figure 3.5). Since the LTS behaves in an Tll or none' manner, the only

way to account for larger bursts is via greater lT de-inactivation which is mediated

throug h hyperpolarization. This finding suggests that the membrane becornes

hyperpolarized by the stimulation. This effect could be mediated either through

stimulation causing the release of GABA from afferent tenninals or initial excitation of

the recorded cell. causing activation of postsynaptic conductances (K+ channels or other

ion channels) leading to hyperpolarization (Sherman and Guillery. 2001). Since direct

stimulation effects on the cell body or dendrites seem unlikely given that large

myelinated fiben have been shown to be the most excitable neuronal element

(discussed further below), excitation of the cell body may be mediated through

antidromic activation. Depolarization of the cell body via antidromic activation could

activate the slow potassium channels required for the falling phase of the action

potential (Silverthom, 1998). Activation of these channels would hyperpolarize the cell

after the end of the stimulus train. However. the time course of hyperpolarization

mediated by these potassium channels rnay be too short for LTS de-inactiwation. On

the other hand, high frequency stimulus trains could activate the GABAergic fiber

terminals. More specifically, electrical stimulation could excite the afferent terminals of

the RE and intemeurons. The depolarization would cause GABA release into the

synaptic cleft and subsequent hyperpolarization in the thalamocortical relay cells. This

could explain the large rebound LTS bunts described in the results. LTS de-

%activation requires hyperpdarimtion of at least 50-100msec in duration. Metabotropic

receptors generally have higher activation thresholds than ionotropic receptors and are

capable of producing lPSPs long enough (100-1000msec) to de-inactivate IT. This

could account for why high frequency stimulus trains are needed to generate the long

duration inhibition (this is in contrast to the previously described low frequency

stimulation effects in thalamus which are hypothesized to result in the net activation of

ionotropic glutamatergic synapses). If the ratio of excitatory to inhibitory metabotropic

recepton favors the inhibitory ones, there would be a net inhibitory effect. Closer

p d r n i t y of the stimulation electrode may be able to better activate metabotropic

receptors by activating a larger number of afferent tenninals. This would cause more

transmitter release and increased membrane hyperpolarization through spatial

summation and may explain why stimulation effects in the thalamus were only obseived

at close distances. It should be noted that the metabotropic GABA8 receptors and the

LTS generating calcium channels are located in similar dendritic areas. Thus, the

metabotropic GABAe receptors on relay cells would be in an ideal position to de-

inactivate the LTS channels, via sustained hyperpolarization. At the high frequencies

that are generally required to elicit this response, temporal summation of the ionotropic

GABh receptor could also contribute to de-inactivation of Ir indirectly. However,

GABAr, receptors activate a chloride channel that generates a transient JPSP (10 ms

latency) which tends to hyperpolarize the membrane to no more than -70 to -75 mV

(the equiiibrium potential for the chloride ion). Although, this would prevent the

generation of further EPSP's due to efferent input, it would be unlikely to strongly de-

inactivate lT by itseL GABAe receptors, which work through activation of K* leak

rcurrents, drive the membrane to around -IO0 mV for 100-1000msec. This is ideal for Il

de-inactivation and In activation. This would subsequently generate a strong rebound

LTS such as those seen following high frequency stimulus trains (See Fig. 3.5). RE

cells, as a population, are capable of acüvating both GABA receptor types but

intemeurons are probably only capable of activating the GAB& receptor although a

more thorough investigation is needed (Reviewed by Sherman and Guillery, 2001).

The anterior thalamic nuclei do not receive afferents from the thalamic reticular

nudeus. Interestingly, the cells tested in this area did not show long duration inhibition

or increased burst activity. This rnay be because sustained hyperpolarizations could not

be generated due to the lack of ineravation of the GABb receptor via the thalamic

reticular nucleus.

Stimulus frequency. intensity, and duration of train length al1 had an effect on burst

activity and resulted in shorter periods of bursting prior to the inhibitory period. This

suggests that other membrane conductance(s) may be acüvated at higher stimulation

thresholds that shorten the pre inhibitory period bursting.

4.3.2 Long Duration Inhibition

The long duration inhibitory effects of high frequency stimulation could result from

a number of different mechanisms. The fact that a period of LTS bursts occun after the

stimulation, and before the onset of inhibition, indicates that the inhibition is not

occurring through depolarization block of sodium channels. The results clearly show

robust action potential lring in a large portion of the cells prior to the onset of inhibition.

Another fact that has to be considered are the lengths of the inhibitory periods (typically

>2 seconds and up to 25 seconds in some cases). It is unlikely that local inhibitory - circuits via the reticular thalamus would produce inhibitory periods of this length.

Another observation that needs to be considered is that the stimulation effects were

only seen at stimulation sites that were in close proximity to the cell when compared

with stimulation studies of other basal ganglia structures (STN, SNr, and GPi). This

suggests that the neuronal elements that are being excited are in close proximity to the

cell. More specifically, in order for the stimulation effects to be observed, it may be

necessary to excite a larger proportion of afferents converging onto a cell. Another

reason for close proximity may be due to the excitation of the cell body via antidromic

activation (see below). Thalamic cells generally have the same electrophysiological

properties. Interestingly, long duration inhibition was typically observed in bursting tells

where it was dependent on stimulation frequency, current intensity, and stimulus train

length. Cells in the relay mode tend to be at more depolarized levels. Thus, the current

and frequency needed to cause hyperpolarization in these cells may be higher.

There is a slowly inactivating forrn of the IA channel (see Introduction) called IAs

which could hyperpolarize the membrane for periods long enough to stop action

potential generation for the lengths observed in this study (McCormick, 1991). As

discussed in the introduction, the family of IA currents have similar kinetics to lT (de-

inactivated by hyperpolarization and activated by subsequent depolarizations) although

both currents move in opposite directions and have opposite effects (IT is an inward

cunent mediated by Ca ''entry into the cell and lA is an outward current mediated by K'

exiting from the cell). The long duration inhibitory periods following the burst excitation

could be caused by activation of these voltage dependent K+ channels. They could

generate a moderately hyperpolarized membrane potential where IT is not strongly de- - ' inacüvated but hyperpolarized enough to prevent sodium action potentials via small

EPSPs or In. At such a membrane potential In may not be activated. Thus, long

duration inhibition could be a result of the slow inactivation time course of IA channels

that would hold the membrane at a potential where Ir is inactivated and sodium spikes

can not be generated. Studies in thalamic interneurons have shown that IA can

ovemde the LTS response completely (Pape et al., 1993). In fact, the I A ~ (subtype of IA

current) current has been mathematically modeled to cause inhibitory periods of up to

10 seconds in thalamic relay cells (Rush and Rinzel, 1994). Using this model, high

L

fiequency stimulus trains would cause a prolonged (repetitive) GABA release. This

would generate a prolonged hyperpolarization leading to de-inactivation of both IT and

1 . Depending on the relative proportion of lT and IA, IA could mask lT and stop bursting

activity. Theoretically, further hyperpolarization would saturate IA de-inactivation but

increase IT de-inactivation, and bursting activity would return (Rush and Rinzel, 1994).

Figure 4.1 illustrates this possible mechanism. First. as indicated during time period 1,

high frequency microstimulation would cause GABA release. Next, during time period

2, hyperpolarization would cause de-inactivation of lT and IA. Time period 3 indicates a

hyperpolarization level where IT masks (Ir is large enough to generate an LTS in the

presence of IA activation) any effects of IA aithough both are de-inactivated. Time period

4 indicates a membrane potential where there is not enough hyperpdarization for the IT

cunent to overcome the In current (both cunents would be deinadvatad).

Alternatively, the inhibition in time period 4 could be a result of a membrane potential

too depolarized for IT de-inactivation but hyperpolarized enough for lA de-inactivation.

An altemative mechanism for long du ration inhibition, for which some evidence

Thas recently been obtained from the STN, involves stimulation mediated blocking of

voltage gated ion channels (Beurrier et al., 2001). The study, done in the STN of rats

showed that long duration high fmquency macro and microstimulation (1 00-500Hz)

produced prolonged inhibition of neuronal Aring (up to 5mins) that was independent of

synaptic transmission. The study suggests that blockage of voltage gated ion channels

mediates the inhibitory effect of high freguency stimulation. The STN study showed

blockage of IT during the inhibitory period. However, the results of this thesis show LTS

bursting activity before the onset of inhibition suggesting that the inhibitory periods in

both studies are mediated through different mechanisms.

4.4 Possible Roie of Long Duration Inhibition in Therapeutic the

Effects of DBS

The findings of this thesis provide strong evidence that high frequency stimulation

can tead to hyperpolarization and long duration inhibition of neuron firing. There are a

number of studies that suggest that inhibition could be the mechanism underlying the

clinical effects of DBS. Microstirnulating at high frequencies in areas with tremor cells

(Vim and Vop) can cause tremor arrest. Furthermore, high frequency (100-1 8OHz) DBS

in simiiar regions (Virn) also causes tremor arrest, as is clinically used for treatment of

Parkinsonian and Essential tremor (reviewed by Lozano, 1998). In addition,

thalamotomies (thalamic lesions) in Vim are also used as clinical treatments for tremor.

Studies of injecting inhibitory dnigs into Vim show tremor reduction and tremor arrest.

More specifically, injection of muscimol (GABA agonist) (Pahapill, 1 999) and lidocaine

(sodium channel blocker) into Vim; have been shown to produce tremor arrest. In most /-

cases lidocaine mimicked the effects of thalamic lesions and microstimulation in similar

amas (Dostrovsky et al., 1993). Chronaxie siudies in Vim indicate that large myeiinated

fibers are the primary candidates for mediating trerpor arrest (Holsheimer et al., 2000).

Furthermore. chronaxie studies invoMng single pulse DBS stimulation (that could briefly

excite or inhibit ongoing €MG) of Vim during ongoing muscle contractions supports the

notion of preferential activation of myelinatad axons (Ashby and Rothwell, 2000). These

studies suggest that the long duration inhibitory effects may be mediated by excitation

of large myelinated fiber teminals that cause GABA release. This type of inhibition may

mediate the therapeutic effects of DBS in Vim either by stopping tremorgenic thalamic

cells or by disrupting the tremor circuitiy.

In spite of this, there are differences in DBS stimulation parameters that need to be

taken into consideration. First, the diameter of the stimulating electrode needs to be

considered. The DBS electrode has a much larger diameter tip than a microeledmde.

Thus, although more cunent (delivered over a much larger area) is delivered through

the DBS electrode, a rnicroelectrode delivers a higher cuvent density (over a very small

area). A DBS electrode would stimulate a much larger area of the thalamus, which

could result in different effects on cell firing. Using a macrostimulating electrode, which

has an electrode tip doser to the size of a DBS electrode, may partly resolve this issue.

Furthemore, DBS electrodes stimulate continuously and tremor stops quickfy once the

stimulus train starts. In addition, once the DBS electrode is turned off tremor retums

quickly. However, due to the stimulus artifact problern, it is not known what happens to

cell firing during long, high frequency stimulus trains. According to the results of a few

cells which were tested for increasing stimulus train lengths. increasing train length - ' produced longer inhibitory periods. According to the previously described model (Fig.

4.1). prolonged stimulation would result in continued release of GABA resulting in

sustained membrane hyperpolarization. Although both IT and IA would be de-

inactivated, they would not get activateâ due to the sustained hyperpolarization. At the

end of the stimulus train In could generate a depdarization that would activate the

conductances.

4.5 Technical Problems Associated With Y icrostimulation and

Recording Studies

The techniques used in these types of microstimulation and recording studies have

potential problems and limitations. When conducting studies in human patients there is

only a short time that can be devoted to examine the effects of stimulation. Because of

this, frequency and current thresholds could not be obtained for a large portion of the

cells. Another limitation results when stimulating from the same electrode that is being

used to record from (studied on 44 cells). This does not allow for obseiving effects

during the stimulus train and, thsre is a 150-2OOmsec period after the delivery of the

stimulus train when no recordings can be made. This prevents determining short

latency effects when using just the recording electrode. Using the dual electrode set up

(studied on a total of 28 cells), this problem can be partly solved. In this setup, neurons

can be recorded during the stimulus train and immediately following it. However,

another problem, involving the stimulus artifact is present. Typically, stimulus artifacts r

last 1 msec. At high frequencies, such as 1 OOHz the stimulus artifact obscures the

recording for -10% of the train. This increases for larger currents and higher

frequencies making it difficult to decipher the neuron's firing pattern during the stimulus

train. This limits the results to observing effects after the stimulus train. Another

problem involves analyzing populations of cells that have slow or irregular firing

patterns. It is difficult to define periods of inhibition in these cells because inhibition is

hard to distinguish fmm spontaneous pauses in the cell's firing. These cells were not

included in the study although they may constitute a signifiant portion of thalamic

neurons that may or may not have different response properties to high frequency

microstimulation.

4.6.1 Future Direction (Human Studies)

There are a number of Mure experiments that can be done in humans to confin

the interpretation of the results for this study. Drug injection studies, using GABA

agonists, could be performed to see if similar inhibitory periods can be generated by

micro injecting GABA locally. This would help confimi or refute the possibility of the

effects being mediated by GABA. Furthemore, GA& or GABAe receptor antagonists

could be used during high frequency stimulation to see if they block the inhibitory effect.

Also, stimulation of tremor cells could be done to see if they are inhibited in the same

manner as the cells tested in this study. A study using long stimulation trains (around 1

min) should be carried out to detenine the effects of prolonged stimulus trains as is the

case for therapeutic DES. Chronaxie studies should also be done to detemine what

neuronal elements are eliciting the long duration inhibitory effects. Macrostirnulation,

which is similar to DBS stimulation, should be done to observe the effects of wider r current spread. Finally, a newly developed microdialysis technique. can be used to

skidy the effect of DBS on neurotransmitter levels.

4.6.2 Future Direction (Animal Studies) .

A study using long stimulus trains should be done to determine if synapse

independent (using blockers of synaptic transmission such as cobalt), long (over 1 min)

inhibitory periods similar to the STN study (Beurrier et al., 2001 ; described above) can

be generated. Chronaxie studies in monkeys, which can be perforrned in more detail

than in humans, wouW confinn or refute that large rnyelinated fibers are the excitable

--- - elements eliciting long duration inhibition. Furthermore, the intracellular effects of high

frequency stimulation c m be obseived using intracellular recordhg methods.

Figure 4.1

Model For Stimulus Generated lnhibitory Period

r -

1. High frequency microstimulation causing GABA release.

-

3. Large rebound LTS bursts generated via I, de-inactivation (at this hyperpolarited membrane potential the IT current swamps any effects mediated by IA) (See Fig. 3.5).

2. Hyperpolarization via GABA causing 1, deinacüvation and 1, de-inactivation

4. Membrane potential where there is not enough hyperpdarkation for the IT wrrent to overcome the lA airrent (bgth currents would be dainacüvated) thus no bursting is observed. Alternatively. the inhib'ion coutd be the result of a membrane potential too depolarUed for I, de-inatiivation but hyperpolarized enough for IA

1 de-inactivation. 1

2 seconds 1

bursting ceIl37501

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