EFFECTS OF NORADRENERGIC ALPHA-RECEPTOR AGONISTS AND ANTAGONISTS ON AIR-STEPPING IN DEVELOPING RATS
By
LYNETTE TAYLOR
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1999 TABLE OF CONTENTS
page
ABSTRACT iv
CHAPTERS
1 INTRODUCTION 1
Locomotor Development in the Rat 1 L-DOPA-induced Air-stepping 5 Noradrenaline and Locomotion 9 Purpose of Current Research 15
2 MATERIALS AND METHODS 19
Subjects 19 Drugs 19 Apparatus 20 Procedure 20 Data Analysis 22 Statistics 25
3 RESULTS 26
Experiment 1A: Agonists Administered Separately 26 Experiment IB: Agonists Administered in Combination 34
Experiment 2: Antagonists / L-DOPA 39
4 DISCUSSION AND CONCLUSIONS 56
Experiment 1A: Agonists Administered Separately 56 Experiment IB: Agonists Administered in Combination 61
Experiment 2: Antagonists / L-DOPA 64 Summary and Conclusions 22
REFERENCES 75
ii APPENDIX STATISTICS TABLES 86
BIOGRAPHICAL SKETCH 89
iii Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
EFFECTS OF NORADRENERGIC ALPHA-RECEPTOR AGONISTS AND ANTAGONISTS ON AIR-STEPPING IN DEVELOPING RATS
By
Lynette Taylor
May 1999
Chairman: Carol Van Hartesveldt Major Department: Psychology
Rat pups suspended in harnesses and administered L-dihydroxyphenylalanine (l-
DOPA) engage in the highly stereotyped and coordinated locomotor behavior pattern termed L-DOPA-induced air-stepping. l-DOPA is synthesized into the neurotransmitters dopamine and noradrenaline. In spinal cord-injured humans, noradrenergic receptor agonists can help restore locomotor abilities. A detailed understanding of noradrenaline’s
locomotor effects is required to improve these clinical applications. The present research
assess the roles of the a and a noradrenergic utilizes the air-stepping paradigm to l 2 receptor subtypes in locomotion across ontogeny in the rat.
is sufficient to The first aim was to determine if a, and/or a2 receptor activation
induce air-stepping. The a, agonist cirazoline and the a2 agonist UK- 14,3 04 were
IV administered separately to rat pups suspended in the air. The a! agonist elicited a
pronounced extension of the limbs and the tail, but did not induce air-stepping. The a2 agonist induced extremely brief episodes of air-stepping. The a! agonist followed by the
elicited longer durations of air-stepping than either agonist administered a2 agonist much separately.
The second aim was to determine if a! and/or a2 receptor activation is necessary
for L-DOPA-induced air-stepping. The a! receptor antagonist prazosin and the a2
administered separately prior to l-DOPA. The a receptor antagonist idazoxan were x antagonist changed the topography of L-DOPA-induced air-stepping at low and moderate
doses and blocked L-DOPA-induced air-stepping at high doses. The a2 antagonist dose- dependently decreased the duration of L-DOPA-induced air-stepping without changing its topography.
for In conclusion, activation of both a, and a2 noradrenergic receptors is optimal
L-DOPA-induced air-stepping. Combined activation of both receptor subtypes is much more effective at eliciting air-stepping than selective activation of one subtype. Thus, in humans with spinal cord injuries, the locomotor effects of drugs that activate both a, and
be assessed, a strategy that is not currently being utilized. a2 receptor subtypes should CHAPTER 1 INTRODUCTION
Tocomotor Development in the Rat
The first three weeks of a rat’s postnatal life are characterized by rapid and significant maturation of postural support mechanisms and locomotor abilities.
Observing rat pups in their home cages, Bolles and Woods [17] noted that the predominant form of locomotion from the day of birth (postnatal day 0) until postnatal day (PD) 8 or 9 was crawling with the ventral surface of the body on the ground. At first, the forelimbs provided the major propulsive force while the hindlimbs were dragged
behind. After approximately PD 3, both forelimbs and hindlimbs propelled the rat pups forward during crawling. When rat pups could lift their bodies off the ground long enough to stand at PD 10, walking occurred for the first time. The walking improved each day and rat pups began to run by PD 13 or 14.
Altman and Sudarshan [2] described the locomotor behavior of rat pups observed
for 3 minutes in an open field. In addition to the crawling during the first postnatal week
described by Bolles and Woods, Altman and Sudarshan observed “pivoting,” a form of
motor activity involving alternate movements of the forelimbs which pivot the anterior
trunk around the hindquarters. The hindlimbs were mostly passive during pivoting, but
provided some propulsion during crawling, which became more common early in the
1 2 second postnatal week. Body weight support on the forelimbs emerged during the second week and hindlimb alternation became more common. As the ability to support body weight on the hindlimbs emerged at the end of the second week, the ventral surface of the body was supported above the ground and there was a transition from crawling to quadrupedal walking. As pups became capable of walking with full postural support, there was a significant increase in the amount and the speed of locomotion and, by PD 16,
fast running became common.
Westerga and Gramsbergen [119] described and quantified the locomotion of PD
9 to PD 19 rat pups tested in an elongated runway. The first episodes of walking with the ventral surface of the body off the floor occured at PD 10. During these episodes, the hindlimbs were hyperextended, abducted and rotated inward at the hip. At around PD 14-
15, the hyperextension, abduction and hip rotation disappeared. Also at this age, the step frequency increased and the step cycle duration decreased, reflecting a significant increase in the speed of locomotion.
The fact that there is very little spontaneous locomotion during the first week of
life does not necessarily indicate that the neural substrates for locomotion are absent. In
fact, the spinal cord circuitry for locomotor-like limb movements appears to be in place
before birth [94]. However, postural constraints and an absence of mature descending
pathways to activate these circuits may ordinarily limit the expression of locomotor
behavior during the first two weeks of life. These circuits may even be inhibited early in
their in order life, when it is advantageous for pups to remain in close proximity to dams
to suckle and thermoregulate. Even in the absence of mature descending pathways, 3 locomotor behavior has been elicited soon after birth via sensory and pharmacological activation.
Locomotor activity has been elicited in the neonatal rat by the odor of ammonia
[46] and the odor of soiled bedding from the home nest [61]. Neonatal rat pups deprived of milk and maternal contact for 22 hours exhibited marked increases in locomotor activity when placed in close proximity to their anesthetized dams [53]. Oral milk infusions delivered to food-deprived rat pups elicited diffuse behaviors such as “rolling,”
“curling” and “twisting” as well as more organized walking [52], During the second postnatal week, electrical shock to the feet elicited “wall climbing,” in which pups stood on their hindlimbs in a vertical plane with their forelimbs alternately treading against the wall [14,109],
During the first postnatal week, the dopamine receptor antagonist haloperidol and the a noradrenergic receptor antagonist phentolamine decreased behavioral activation elicited by oral milk infusions [19]. Many catecholamine agonists elicit locomotor
activity in the neonatal rat, including L-3,4-dihydroxyphenylalanine (l-DOPA)
[19,49,63,92,117], clonidine [63,86,92,93,106], apomorphine [19,63,99] and amphetamine [20,77]. The serotonin receptor agonist quipazine induced wall climbing and forward locomotion in 3-day-old rat pups [107], During the second postnatal week, haloperidol decreased wall climbing elicited by foot shock, while the catecholamine agonist amphetamine elicited wall climbing [14], These forms of behavioral activation may thus be mediated by monoamine systems. 4
Locomotor behavior has also been investigated in neonatal rats utilizing an in vitro preparation of the brainstem and spinal cord with the hindlimbs attached. The brainstem and spinal cord are dissected out with the spinal cord ventral roots still innervating the hindlimbs. In this paradigm, hindlimb “airstepping” has been induced by the application of various drugs to the artificial cerebrospinal fluid bath surrounding the brainstem or spinal cord. Separate application of dopamine, aspartate, glutamate, N- methyl-D-aspartic acid (NMDA) or serotonin to the spinal cord bath induced hindlimb airstepping [7,24,31,66,76]. Combined application of serotonin and dopamine [68] or serotonin and NMDA [59,71] also induced hindlimb airstepping. GABA antagonists,
NMDA, substance P, acetylcholine, the cholinergic agonist carbachol and serotonin applied to the brainstem bath induced hindlimb airstepping [7], The in vitro preparation has two major limitations. First, the preparation cannot be maintained for long periods of time. Second, the preparation is viable only in very young (PD 0-4) rat pups and thus cannot be utilized to study the ontogeny of locomotion beyond the first few postnatal days.
While sensory stimulation and pharmacological agents can elicit motor behaviors
in neonatal rats, the expression of coordinated locomotor activity in very young pups may be limited by their inability to support their own body weight. Several researchers have thus tested the motor capabilities of rat pups tested in water [16,23,1 12]. The buoyancy provided by the water medium relieves the pups from postural constraints. Being placed in water itself serves as an eliciting stimulus for the expression of coordinated motor 5 behavior. However, due to the neonates’ inability to remain afloat during swimming
tests, only brief observation sessions are possible.
L-DOPA-induced Air-stepping
With the above limitations in mind, the L-DOPA-induced air-stepping paradigm
was developed as an alternative method to study the ontogeny of locomotion in rat pups.
In this paradigm, rat pups are suspended in the air in harnesses with all four limbs off the
ground, eliminating the need for weight support. Unlike placing rat pups in water,
suspending rat pups in harnesses does not elicit coordinated motor behavior on its own.
Following a systemic injection of l-DOPA, however, rat pups engage in a highly
stereotyped and coordinated locomotor behavior pattern termed L-DOPA-induced air-
stepping [117].
On the day of birth, l-DOPA induces alternate stepping of the forelimbs. In older
rat pups (at PD 5, 10, 15 and 20), the predominant locomotor pattern induced by l-DOPA
is “diagonal progression,” in which one forelimb and the contralateral hindlimb step
synchronously and in alternation with the other diagonal pair of limbs. Short episodes of
“air-galloping,” characterized by synchronous stepping of the forelimbs in antiphase with
synchronous stepping of the hindlimbs, occur at PD 10, 15 and 20. At PD 10 and 15,
there are also some short episodes of “air-swimming,” characterized by alternate stepping
of the hindlimbs with the forelimbs held stationary beneath the chin, flexed at the
shoulders and extended at the elbows and wrists. Each of these air-stepping gaits is 6 accompanied by a characteristic dorsiflexed posture with head, body and tail alignment
[80,117],
The peripherally-administered l-DOPA that crosses the blood-brain-barrier is converted to dopamine by the enzyme aromatic L-amino acid decarboxylase. In the
noradrenergic nerve terminals of the brain and spinal cord, dopamine is further converted to noradrenaline by the enzyme dopamine-(3-hydroxylase. The air-stepping elicited by L-
DOPA could thus result from the actions of l-DOPA itself, dopamine, noradrenaline or some combination of these substances. In 5-day-old rat pups, blocking either dopamine
[101] or noradrenaline [115] receptors blocks L-DOPA-induced air-stepping.
Pretreatment with an aromatic L-amino acid decarboxylase inhibitor, preventing L-
DOPA’s conversion to dopamine, or with a dopamine-P-hydroxylase inhibitor, preventing dopamine’s conversion to noradrenaline, also blocks L-DOPA-induced air-
stepping [5,88], Thus, in the 5-day-old rat, the final step in the catecholamine synthesis pathway, the conversion of dopamine to noradrenaline, is necessary for L-DOPA-induced air-stepping.
In decerebrate PD 0-20 rat pups, l-DOPA induces air-stepping of the same topography as that induced in intact rat pups [60]. Thus, the forebrain and its afferent and efferent pathways are not necessary for l-DOPA to induce air-stepping. In decerebrate
PD 5 rat pups, blocking either dopamine [79] or noradrenaline [114] receptors blocks L-
DOPA-induced air-stepping, implicating the brainstem and spinal cord projections of
midbrain dopamine and pontine noradrenaline systems in this behavior. 7
When the spinal cord is transected at the midthoracic level in PD 0-20 rat pups,
isolating the caudal spinal cord from descending inputs, L-DOPA-induced hindlimb air-
stepping is disrupted [60,81]. There are several possible interpretations of this finding.
First, spinal cord transection may disrupt catecholamine synthesis in axon terminals
originating from neurons anterior to the transection site, resulting in insufficient spinal cord catecholamine receptor activation. Direct noradrenaline and dopamine receptor agonists have not been administered to spinal-transected rat pups in the air-stepping paradigm, so this hypothesis has yet to be tested.
Another possibility is that spinal cord catecholamine receptor activation is not
sufficient to induce hindlimb air-stepping in the absence of tonic descending inputs from the brain. Perhaps both catecholamine receptor activation and tonic descending inputs from brainstem regions such as the reticular formation, the vestibular nuclei or the raphe
system are necessary for l-DOPA to induce air-stepping. In support of this alternative, coadministration of l-DOPA and the serotonin receptor agonist quipazine induces hindlimb air-stepping in PD 5 rat pups with midthoracic spinal cord transections [81].
Thus, a drug that substitutes for descending input from the raphe system enables l-DOPA
to induce hindlimb air- stepping in subjects with spinal transections. In chronic spinal
adult rats supported on a platform with holes through which their hindlimbs hung freely,
precursors for the synthesis of serotonin and direct serotonin receptor agonists increased
EMG amplitude in hindlimb flexor and extensor muscles but were not reported to induce
hindlimb stepping [11]. In chronic spinal cats on a treadmill, serotonin receptor agonists
similarly increased EMG amplitude in hindlimb flexor and extensor muscles [13,96], 8
The raphe system may be just one of several brainstem regions that contribute to L-
DOPA-induced air-stepping. It is possible that loss of direct or indirect input to the spinal cord from any of these areas might bring the spinal cord circuitry for hindlimb locomotion below threshold for the initiation of air-stepping.
Two brainstem regions that have been implicated in locomotion in adult cats and
rats are the mesencephalic locomotor region (MLR) and the medioventral medulla
(MED). Electrical stimulation of the MLR of the decerebrate cat [100] or the decerebrate rat [104] induces locomotion on a treadmill. The MLR is a posterior midbrain region that projects to the MED in the cat [45] and rat [44], Electrical stimulation of the MED in the decerebrate cat [43] and the decerebrate rat [69] elicits coordinated locomotion on a treadmill. Increasing the current amplitude administered to the MLR [104] or the MED
[69] of the decerebrate rat increases the step-cycle frequency such that the gait changes from a walk to a trot to a gallop with increased treadmill speed.
Administration of various chemicals into the MLR or MED of the decerebate rat induces stepping on a treadmill. When administered into the MLR or MED, GABA antagonists, NMDA and substance P induce treadmill walking [42,69]. Cholinergic agonists injected into the MED but not the MLR also induce locomotion [69], In the in vitro brainstem-spinal cord hindlimb-attached preparation of the neonatal rat, electrical stimulation of two brainstem regions has been shown to consistently elicit airstepping.
These regions appear to correspond with the MLR and the MED of the adult rat. Higher amplitude currents increase the step-cycle frequency but do not induce a gallop [8,9]. 9
Catecholamine agonists administered into the MLR or the MED have not been
reported to elicit treadmill stepping in adult rats. Catecholamine agonists applied to the brainstem bath of the in vitro brainstem- spinal cord hindlimb-attached preparation of the neonatal rat have not been reported to induce hindlimb airstepping. It thus appears that brainstem catecholamine receptor activation is not sufficient to induce locomotor behavior in these paradigms. At the spinal cord level, however, noradrenergic receptors have long been implicated in treadmill locomotion in adult cats.
Noradrenaline and Locomotion
In the acute spinal cat, systemically-administered clonidine, an a2 noradrenergic receptor agonist, induces hindlimb treadmill stepping [10,13,38,50] while the dopamine receptor agonist apomorphine does not [10,13]. Intrathecal injection of clonidine or norepinephrine at the L5 level of the spinal cord in the spinal cat also induces hindlimb treadmill stepping [27,95]. Hindlimb afferent nerve stimulation combined with intrathecal administration of noradrenaline to the lumbosacral enlargement of acute spinal cats induces fictive locomotor activity in hindlimb nerve recordings [65].
These studies demonstrate that spinal cord noradrenergic receptor activation is sufficient to induce treadmill locomotion in the adult spinal cat. In chronic spinal adult
rats supported on a platform with holes through which their hindlimbs hung freely, however, clonidine did not elicit EMG activity in hindlimb flexor and extensor muscles
adult [11]. Although there may be a species difference, clonidine’s lack of effect in these rats may reflect the absence of a treadmill. A moving treadmill belt passively extends the 10 limbs, providing afferent input that may facilitate clonidine-induced hindlimb stepping in subjects tested on a treadmill. In the adult rats just described and in the air-stepping paradigm, an absence of similar afferent input may be an important limiting factor for the occurrence of locomotion. When rat pups with spinal transections are placed on a
stationary surface and administered l-DOPA, hindlimb stepping does occur [4],
Presumably, afferent input from the supporting surface facilitates the occurrence of stepping in these pups.
Noradrenergic projections to the spinal cord of the rat are ideally situated to modulate afferent input to the spinal cord and motoneuron output to the muscles.
Noradrenergic neurons of the locus coeruleus project to the dorsal horn, ventral horn, intermedio lateral cell column and the region surrounding the central canal of all levels of the spinal cord of the rat [91,120]. Noradrenaline in the dorsal horn mediates analgesic
effects primarily through its actions at a2 noradrenergic receptors in adult [123] and developing [57] rats. In the ventral horn, glutamate-evoked lumbar motoneuron firing
rate is increased by microiontophoretic application of noradrenaline or the noradrenergic receptor agonist phenylephrine in adult rats [121,122]. Noradrenaline
acting at a! receptors depolarizes motoneurons in in vitro preparations of the neonatal rat spinal cord [29,36,70], This a! noradrenergic receptor-mediated motoneuron depolarization resembles the “plateau potentials” that play an important role in locomotor behavior in cats and neonatal rats [18,64,67].
The dual noradrenergic innervation of the spinal cord dorsal and ventral horns
also plays a role in spinal cord reflex modulation. Electrical stimulation of the locus 11 coeruleus facilitates lumbar spinal monosynaptic reflexes elicited by dorsal root or peripheral extensor or flexor nerve stimulation in adult cats and rats [25,41]. This monosynaptic reflex (the stretch reflex elicited by group la muscle spindle afferent
activation) is normally involved in the maintenance of muscle tone during postural
support and locomotion. Noradrenaline acts at spinal cord esq receptors to facilitate and at
flexor reflex mediated by group II muscle spindle afferents in a2 receptors to inhibit the adult rats [98]. These reflexes appear to play a role in locomotor activity in adult cats, adult rats and in neonatal rats [30,39,47,58,59,66].
Noradrenergic receptor activation in the spinal cord thus modulates afferent input, motoneuron output and reflex circuits that have been implicated in locomotion. There
are, however, circumstances in which noradrenergic receptor activation in the brain seems to override this spinal cord output. In the intact cat, for example, local injection of clonidine into the locus coeruleus produces postural atonia [89]. This result may help explain the finding that clonidine facilitates treadmill locomotion in cats with complete spinal transections but decreases hindlimb weight support in cats with partial spinal cord lesions sparing some descending spinal cord inputs [97], Clonidine produces sedation in
intact adult rats tested on a stationary surface [33,87,108], perhaps as a result of an a2 noradrenergic receptor-mediated suppression of locus coeruleus activity [1,111].
Clonidine’ s behavioral effects across ontogeny in the rat closely parallel the maturation of the locus coeruleus and the noradrenergic projection pathways. Clonidine markedly increases overground locomotor activity when administered to neonatal rat pups but produces sedation when administered to PD 20 and older rats 12
[63,92,93,106,1 16]. Soon after birth, when clonidine induces overground locomotion,
there is very little spontaneous activity in the locus coeruleus [84] and thus very little neural activity to inhibit. This sporadic pattern of neural activity in the locus coeruleus becomes more like the regular, tonic, adult-like pattern by PD 20 [84], when clonidine- induced sedation replaces clonidine-induced locomotion.
In the developing spinal cord, there is a rostrocaudal pattern of innervation by
noradrenergic terminals [3,90,1 13]. During postnatal weeks 2 and 3, the adult-like
pattern of spinal cord innervation is established following significant increases in lumbar
dorsal and ventral horn innervation [90,1 13]. The density of a, and a2 noradrenergic receptors increases in the rat spinal cord [78,102] and brain [83] during postnatal weeks 2
and 3. In the frontal cortex, hypothalamus, hippocampus and mesolimbic brain areas, the
density of both receptor subtypes is low at birth and increases thereafter [55]. In the midbrain and brainstem, however, the density of both receptor subtypes is already high at birth and does not change substantially thereafter [15].
Investigations of the effects of the a2 noradrenergic receptor agonist clonidine on the initiation, modulation and recovery of locomotor abilities in spinal-transected cats have long outnumbered investigations of the locomotor effects of a, noradrenergic receptor agonists and dopamine or serotonin receptor agonists. The fact that clonidine has been very successful at eliciting locomotion in subjects with spinal transections
provides one obvious reason for focusing on clonidine and a2 noradrenergic receptor
activation in relation to locomotion. However, clonidine is not very selective for the a2
receptor subtype. For example, in rats with spinal transections, clonidine facilitates the 13 flexor reflex via its stimulation of the noradrenergic receptor subtype [62]. It thus
seems possible that clonidine facilitates locomotion by activating both a, and a2 receptor subtypes. Since researchers have recently begun to assess the locomotor effects of
clonidine in spinal cord-injured humans [12,34,40], it is becoming increasingly important to carefully delineate the contributions of specific noradrenergic receptor subtypes to clonidine-induced locomotion and to locomotion in general.
Until recently, a, noradrenergic receptor agonists that cross the blood-brain- barrier have been unavailable and studies of the locomotor effects of systemically-
administered agonists have been limited. Systemic administration of the a!
noradrenergic receptor agonist phenylephrine (an agonist that does not effectively cross the blood brain barrier) to 6-day-old rats does not affect locomotor behavior [19].
Although the blood brain barrier may not be mature enough to prevent phenylephrine
from entering the brain and spinal cord during the first postnatal week, it is not clear that
phenylephrine effectively stimulates central adrenoceptors in 6-day-old pups.
The majority of information about the possible role played by noradrenergic
receptor activation in locomotion has been derived indirectly from investigations of the
effects of centrally administered a, noradrenergic agonists and antagonists.
Noradrenaline superfused onto the isolated lumbar spinal cord in the neonatal rat
depolarizes motoneurons by acting on a, noradrenergic receptors [29]. Iontophoretic
application of low doses of noradrenaline or phenylephrine increases the firing rate of
neurons of the locus coeruleus in PD 1-14 rat pups, but not in older animals [85]. In adult
and PD 1-30 rat pups, a, noradrenergic receptor activation increases electrical activity in 14 the dorsal raphe nucleus [105]. In adult rats, a, receptor activation increases the electrical activity of the substantia nigra and ventral tegmental area [48]. Thus, there is evidence for an a! noradrenergic receptor-mediated excitation of many brain and spinal cord areas implicated in locomotion.
Cirazoline, an a, noradrenergic receptor agonist that crosses the blood brain
barrier, has recently been utilized in a number of behavioral investigations, including
investigations of ingestive behavior [32], spatial working memory [6] and the
discriminative stimulus properties of cocaine [74], In adult rats, the a2 noradrenergic receptor agonist dexmedetomidine induces a hypnotic effect, as defined by loss of the
is righting reflex [35]. This a2 receptor-mediated hypnotic response antagonized by
systemically administered cirazoline [51]. The locomotor effects of cirazoline have not yet been determined in adult or developing animals.
As previously discussed, l-DOPA must ultimately be converted to noradrenaline
to induce air-stepping in the 5-day-old rat pup [88]. The air-stepping paradigm has
proven to be a very reliable measure of locomotor behavior across ontogeny in the rat and
is thus a useful model for assessing noradrenaline’s contributions to locomotor behavior.
Though very different from the intact adult organism, intact rat pups during the first two
weeks of postnatal development are similar to adult subjects with spinal transections in
one important respect. In adult subjects with midthoracic spinal transections, the lumbar
spinal cord is isolated from descending noradrenergic projections. Before approximately
PD 15, the noradrenergic projections to the lumbar spinal cord of rat pups are not yet
mature. Thus, noradrenergic agents that effectively elicit locomotion in very young rat 15 pups may also prove to be useful in spinal adult subjects and in spinal cord-injured humans.
Purpose of Current Research
The current experiments were designed to investigate the relative contributions of
receptor subtypes to air-stepping across ontogeny in intact rat the a, and a2 noradrenergic
pups. The first aim was to determine if activation of a, and/or a2 noradrenergic receptors
is sufficient to induce air-stepping at PD 5, 10, 15 and 20. Previous research testing the
agonist clonidine in the air-stepping paradigm effects of the a2 noradrenergic receptor
sufficient to induce very brief episodes of non- suggested that a2 receptor activation was
dorsiflexed air-stepping in PD 5 and 10 rat pups and much longer-duration air-stepping at
likely PD 20 [1 16]. At the doses required to elicit air-stepping, however, clonidine most
receptors. Thus, conclusions could be drawn activated both a, and a2 noradrenergic no
about the specific roles played by either of these receptor subtypes in air-stepping. The
agonist 04 was thus utilized in the present research. more selective a2 receptor UK- 14,3
The a! receptor agonist cirazoline was also chosen for systemic administration. The
effects of these agonists administered separately and in combination were tested in PD 5,
10, 15 and 20 rat pups in the air-stepping paradigm. The predicted experimental
outcomes and their alternatives are outlined below.
It is predicted that UK- 14,3 04, like clonidine, will induce air-stepping at all ages.
it have a However, since UK- 14,3 04 is a more selective a2 agonist than clonidine, may
stronger sedative effect, particularly in the older rat pups. It is also possible that the more 16
will less effective at selective activation of the a2 receptor subtype by UK- 14,3 04 be
inducing air- stepping than the broader actions of clonidine at both and a2 receptor
subtypes. It is hypothesized that UK-14,304 will induce air-stepping less effectively in
PD 5 and 10 than in PD 15 and 20 rat pups, either because of the relative immaturity of
receptors or because of a lack of necessary tonic activity at a a2 noradrenergic l noradrenergic receptors, dopaminergic receptors or serotonergic receptors early in life.
Since the a, noradrenergic receptor agonist phenylephrine did not induce
overground locomotion in 6-day-old rat pups [19], it is expected that the a! receptor agonist cirazoline will not induce air-stepping in PD 5 rat pups. However, due to its
ability to cross the blood brain barrier, sytemic cirazoline administration may be more
effective at inducing air-stepping than phenylephrine was at inducing overground locomotion. Because there have been no previous observations of the locomotor effects
administered a agonists in older pups, it is difficult to speculate about of systemically t whether cirazoline will induce air-stepping in the older pups. Given the increased <*[ receptor density in the brain and spinal cord across ontogeny in the rat, however, it seems possible that cirazoline will more effectively induce air-stepping in older than in younger
rat pups.
Since both the noradrenergic receptor antagonist prazosin and the a2 antagonist idazoxan block L-DOPA-induced air-stepping at PD 5 [1 15], combined administration of
is expected the a, noradrenergic receptor agonist cirazoline and the a2 agonist UK- 14,304 to more effectively elicit air-stepping than either agonist administered separately. This combined receptor activation is expected to be particularly effective in the younger rat 17 pups. Given the absence of tonic activity at noradrenergic receptors in the younger rat pups relative to older pups, artificial receptor stimulation is expected to mediate more
striking behavioral changes in younger subjects. However, it is also possible that combined activation of both a noradrenergic receptor subtypes will not be sufficient to induce air- stepping in the absence of p noradrenergic, dopaminergic and/or serotonergic receptor activation.
The second aim of the current research was to determine if cq and/or a2 noradrenergic receptor activation is necessary for L-DOPA-induced air-stepping across
ontogeny in the rat. As previously discussed, the noradrenergic receptor antagonist
antagonist idazoxan block L-DOPA-induced prazosin and the a2 noradrenergic receptor air-stepping in PD 5 rat pups [115]. These antagonists’ effects have not been tested in older pups, however, and the dose-response curve obtained at PD 5 was too broad to detect possible changes in the topography of the air-stepping that might have occured following a less widely-distributed range of antagonist doses. Thus, in the current research, a more limited range of doses of each antagonist was administered to PD 5 rat pups and the effects of the antagonists were tested in older subjects. The a,
antagonist noradrenergic receptor antagonist prazosin or the a2 noradrenergic receptor idazoxan were thus administered prior to l-DOPA in PD 5, 10, 15 and 20 rat pups
suspended in the air. The predicted outcomes and their alternatives are outlined below.
Given the immense body of research demonstrating the importance of a2
is noradrenergic receptor activation in locomotion, the a2 receptor antagonist idazoxan expected to block L-DOPA-induced air-stepping at all ages. However, the vast majority 18 of previous research has been in spinal-transected subjects. In intact rats, noradrenaline
neural activity in the locus This acting at the a2 receptor subtype inhibits coeruleus [1].
present inhibition can be blocked by an a2 noradrenergic receptor antagonist [1], In the study, any such disinhibition of the locus coeruleus may override the behavioral effects of
the noradrenergic receptor antagonist spinal cord a2 receptor blockade. At PD 5, a2 idazoxan has previously been shown to block L-DOPA-induced air-stepping. However, idazoxan administered to PD 25 to 50 rat pups increases locomotor activity in an open field [103]. Idazoxan may similarly increase general activity or air-stepping in older rat pups in the present study.
Since the a, noradrenergic receptor antagonist prazosin has already been
demonstrated to block L-DOPA-induced air-stepping at PD 5, prazosin is expected to block L-DOPA-induced air-stepping or significantly alter its topography, perhaps inducing “atypical air-stepping,” at all ages. However, since previous studies have
agonist clonidine induces air-stepping, it demonstrated that the a2 noradrenergic receptor
seems possible that l-DOPA synthesized into noradrenaline that activates only a2
receptors, as it will when a! receptors are blocked, will also induce air-stepping. CHAPTER 2 MATERIALS AND METHODS
Subjects
Male and female Sprague-Dawley rats supplied by Zivic-Miller were mated in the vivarium of the University of Florida Psychology Department. The colony rooms housing the pregnant females were maintained on a 10:14 hour light-dark cycle with food and water available ad libitum. The breeding cages were checked twice daily for litter births. The day of birth was designated postnatal day (PD) 0. When possible, litters were culled to 10 pups, half male and half female, on PD 1. Pups remained with the dams until
removal at PD 5, 10, 15, or 20 for behavioral testing.
Drugs
Cirazoline hydrochloride (Research Biochemicals International) and idazoxan hydrochloride (Sigma Chemical Company) were dissolved in distilled water, with a resulting pH of approximately 6.7. UK- 14,304 (Research Biochemicals International) was dissolved in 0.1 N hydrochloric acid and diluted with saline, 0.1 M sodium phosphate dibasic, and 0.1 M sodium phosphate monobasic to a pH of approximately 6.5.
Prazosin hydrochloride (Sigma) was dissolved in glacial acetic acid and diluted with distilled water, 0.1 M sodium phosphate dibasic, and 1 N sodium hydroxide to a pH of approximately 5.9. L-3,4-dihydroxyphenylalanine (l-DOPA; Research Biochemicals
19 20
International) was dissolved in 1 N hydrochloric acid and diluted with distilled water and
0.1 M sodium phosphate dibasic to a pH of approximately 6.7.
Apparatus
Each subject was wrapped around the abdomen with an adhesive tape sling and suspended from a dowel in a glass-enclosed incubator lit from below with a fluorescent
light. Subjects were suspended with one side toward a video camera. A mirror placed below the subjects at a 45° angle provided a ventral view and a view of the contralateral side of each subject. All four limbs were inspected to ensure that movement was unhindered by the tape.
Procedure
Experiment 1 A: Agonists Administered Separately
Each suspended subject received one of three doses of the a, noradrenergic
agonist receptor agonist cirazoline or one of three doses of the a2 noradrenergic receptor
UK-14,304 or the corresponding vehicle. All injections were subcutaneous at the nape of the neck at an injection volume of 0.005 ml/g body weight. The following doses were determined separately in each age group based on the results of pilot experiments.
PD 5 rat pups received 1.0, 0.25, or 0.0625 mg/kg cirazoline, 1.0, 0.25, or 0.0625 mg/kg UK-14,304 or the corresponding vehicle. PD 10 rat pups received 1.0, 0.5, or 0.25
mg/kg cirazoline, 0.25, 0.125, or 0.0625 mg/kg UK-14,304 or the corresponding vehicle.
PD 15 rat pups received 0.5, 0.25, or 0.125 mg/kg cirazoline, 0.5, 0.25, or 0.125 mg/kg 21
UK-14,304 or the corresponding vehicle. PD 20 rat pups received 0.5, 0.25, or 0.125 mg/kg cirazoline, 0.25, 0.125, or 0.0625 mg/kg UK-14,304 or the corresponding vehicle.
A split-litter design was implemented, such that one or two subjects within a litter received a single dose of one of the agonists. There were 7-9 subjects in each experimental group. The behavior of each subject was videotaped for 90 minutes following the drug injection.
Experiment IB: Agonists Administered in Combination
One dose of the a! agonist cirazoline or its vehicle was administered to each
suspended subject and followed 20 minutes later by one dose of the a2 agonist UK-
14,304 or its vehicle. Within each age group, doses were chosen based on the results of pilot experiments in which varying doses of UK- 14,3 04 were combined with varying doses of cirazoline. The doses that induced the air-stepping that most closely resembled
L-DOPA-induced air-stepping were selected for combined administration. PD 5 rat pups received 1.0 mg/kg cirazoline or its vehicle followed by 0.5 mg/kg UK-14,304 or its vehicle. PD 10 and 15 rat pups received 0.5 mg/kg cirazoline or its vehicle followed by
0.25 mg/kg UK-14,304 or its vehicle. PD 20 rat pups received 0.25 mg/kg cirazoline or
its vehicle followed by 0.125 mg/kg UK- 14,304 or its vehicle. There were 7-9 subjects in each experimental group. The behavior of each subject was videotaped from the time of
the first drug injection until 90 minutes after the second injection. 1
22
Experiment 2: Antagonists / l-DOPA
One of three doses of the a! noradrenergic receptor antagonist prazosin, the a2 antagonist idazoxan, or the corresponding vehicle was administered and followed 20 minutes later by 100 mg/kg l-DOPA or its vehicle. The noradrenergic antagonists were
administered at an injection volume of 0.005 ml/g body weight. l-DOPA was administered at an injection volume of 0.01 ml/g body weight.
Antagonist doses were determined separately for each age group based on the results of pilot experiments. PD 5 rat pups received 0.1, 0.025, or 0.00625 mg/kg
prazosin, 0.2, 0.05, or 0.0125 mg/kg idazoxan or the corresponding vehicle. PD 10 rat pups received 0.1, 0.025, or 0.00625 mg/kg prazosin, 0.5, 0.125, or 0.03125 mg/kg idazoxan or the corresponding vehicle. PD 15 rat pups received 0.25, 0.0625, or
0.015625 mg/kg prazosin, 1.0, 0.25, or 0.0625 mg/kg idazoxan or the corresponding vehicle. PD 20 rat pups received 0.5, 0.25, or 0.125 mg/kg prazosin, 2.0, 0.2, or 0.02 mg/kg idazoxan or the corresponding vehicle. A split-litter design was utilized such that one or two subjects within a litter received a single antagonist dose. There were 7-1
subjects in each experimental group. The behavior of each subject was videotaped from
the time of the first injection until 60 minutes after the second injection.
Data Analysis
The gross behavioral characteristics of each subject were analyzed using a
computer keyboard to log the time of onset, duration, and time of offset of each episode
of several behavioral categories. When only one drug was administered to a subject, the 23 subject’s behavior was quantified for the 60 minutes immediately following the drug injection. When two drugs were administered, the subject’s behavior was quantified for the 60 minutes immediately following the second of the two injections. Because the direct noradrenergic agonists induced air-stepping after a much longer latency in the PD
20 subjects than in the younger subjects, much of the air-stepping occurred after the first
60 minutes had elapsed. The PD 20 rat pups’ behavior was thus quantified for the 90 minutes following the direct noradrenergic agonists administered separately or in combination.
Due to differing behavioral outcomes, the exact behaviors chosen for quantification depended on the drug or drug combination administered as well as the
subjects’ age. The behaviors quantified always included some combination of the
following behavioral categories:
- dorsiflexed 1) Diagonal progression stereotypic air-stepping characterized by a
posture, head, trunk and tail alignment, an absence of laterally-directed head movements
and an alternating gait of all four limbs in a diagonal progression pattern.
2) Galloping - air-stepping of all four limbs in which the forelimbs stepped in
synchrony, the hindlimbs stepped in synchrony and the forelimbs stepped in alternation
with the hindlimbs.
3) Forelimb galloping - air-stepping of all four limbs in which the forelimbs
stepped in synchrony while the hindlimbs stepped alternately.
4) Airswimming - stereotypic alternation of the hindlimbs but not the forelimbs,
with the forelimbs were held stationary under the chin. 24
5) Forelimb stepping - stereotypic alternation of the forelimbs but not the hindlimbs. During this air-stepping gait, the hindlimbs were stationary and strongly extended behind the subject.
6) One-limb stepping - stepping of only one limb.
7) Atypical air-stepping - dorsiflexed posture absent or coordination between limbs disrupted during diagonal progression.
8) Atypical airswimming - dorsiflexed posture absent during airswimming.
9) Atypical forelimb stepping - dorsiflexed posture absent during forelimb stepping.
10) Nonstereotyped activity - general movements of the head, limbs or trunk.
- extension of all four limbs, sometimes 1 1) Extension/tremor a pronounced accompanied by a body tremor.
12) Forelimb flexion - forelimbs held stationary under the chin, flexed at the
shoulders and extended at the elbows and wrists. The hindlimbs were stationary and
usually extended behind the subject.
locked in 13) Hindlimb flexion - hindlimbs stationary with hips, knees and ankles
a tightly flexed position. The forelimbs were stationary but relaxed.
under the 14) 4-limb flexion - inactive with all 4 limbs flexed, the forelimbs held
chin and the hindlimbs tightly locked in flexion.
subject otherwise 15) Tail extension - a pronounced extension of the tail, with the
completely inactive. 25
- 1 6) Relaxed inactivity characterized by an absence of movement and a nondorsiflexed posture. The head, tail and all four limbs were relaxed (neither flexed nor extended).
Statistics
Within each age group, the mean total duration of time spent engaged in each behavior was determined for each experimental group. In the agonist experiments, comparisons were made among the group means using a one-way ANOVA when the agonists were administered separately. When the agonists were administered in combination, comparisons were made using a two-way ANOVA with cirazoline dose as one factor and UK- 14,3 04 dose as the second factor. In the antagonist experiments, comparisons were made using a two-way ANOVA with antagonist dose as one factor and presence or absence of L-DOPA as the second factor. In all experiments, Duncan’s
Multiple Range Test was utilized for subsequent pairwise comparisons. When the duration of air-stepping following a given dose of one of the direct noradrenergic agonists was significantly greater than in subjects receiving vehicle injections, the mean latency to the onset of the first episode of air-stepping was calculated for that experimental group.
Subjects that did not air-step were excluded from the mean latency calculations. CHAPTER 3 RESULTS
Experiment 1A: Agonists Administered Separately
Cirazoline - PD5
The total durations of the behaviors quantified in PD 5, 10, 15 and 20 rat pups following cirazoline administration are presented in Figure 1. Although there were some episodes of diagonal progression in PD 5 rat pups, cirazoline did not significantly affect the duration of diagonal progression at any dose. Cirazoline did significantly increase the
durations of tail extension (F=10.9, dfi=3,29, p<.001), extension/tremor (F=5.9, df=3,29, p=. 003) and hindlimb flexion (F=3.2, df=3,29, p=.039). There was significantly more tail extension (p’s<.01) and hindlimb flexion (p’s<.05) in subjects receiving the high cirazoline dose than in subjects receiving the vehicle for cirazoline, the low cirazoline dose or the medium cirazoline dose. The medium (p<.01) and high (p<-05) cirazoline doses induced significantly more extension/tremor than the vehicle for cirazoline. The duration of extension/tremor was significantly longer following the medium dose than
following the low dose (p<.05).
Cirazoline significantly decreased the durations of relaxed inactivity (F=5.9,
df=3,29, p=. 003) and nonstereotyped activity (F=4.6, df=3,29, p=.009). There was less
relaxed inactivity in subjects receiving the high cirazoline dose than in subjects receiving
the low dose, the medium dose or the vehicle for cirazoline (p’s<.01). Subjects receiving
26 27
Relaxed Inactivity 4-limb Flexion Forelimb Gallop | | Airswim IX/I Nonstereotyped Activity b==j Hindlimb Flexion I 1 i Diagonal Progression Atypical Airstep [nun Tail Extension [\Z3 IXXI Extension/Tremor
a) Postnatal Day 5 b) Postnatal Day 10
Cirazoline Dose (mg/kg)
c) Postnatal Day 15 d) Postnatal Day 20
behaviors quantified following administration of cirazoline or its Figure 1 . Mean durations of differences vehicle. Note the larger time scale for PD 20 subjects. Asterisks denote significant from vehicle (0.0 mg/kg) subjects. *=p<.05; **=p<.01
a) PD 5; b) PD 10; c) PD 15; d) PD 20 28 the low (p<.05), medium (p<.01) and high (p<.01) cirazoline doses engaged in less nonstereotyped activity than subjects receiving the vehicle for cirazoline.
Cirazoline - PD 10
Although there were some episodes of diagonal progression, forelimb galloping, atypical air-stepping, hindlimb flexion and 4-limb flexion in PD 10 rat pups following cirazoline administration, cirazoline did not significantly affect the durations of these behaviors. Cirazoline did significantly increase the durations of tail extension (F=4.9, df=3,28, p=.007) and extension/tremor (F=5.1, df=3,28, p=.006). The duration of tail extension was significantly longer in subjects receiving the low (p<.05), medium (p<-01) and high (p<.05) cirazoline doses than in subjects receiving the vehicle for cirazoline.
The low (p<.01), medium (p<.05) and high (p<.01) cirazoline doses also induced significantly more extension/tremor than the vehicle for cirazoline. Cirazoline decreased the duration of nonstereotyped activity (F=6.14, df=3,28, p=.002). Subjects receiving all three cirazoline doses engaged in less nonstereotyped activity than subjects receiving the vehicle for cirazoline (p’s<.01).
Cirazoline - PD 15
Although there were some episodes of diagonal progression, airswimming,
atypical air-stepping, tail extension and hindlimb flexion following cirazoline
administration to PD 15 rat pups, cirazoline did not significantly affect the durations of
these behaviors. Cirazoline significantly increased the duration of extension/tremor
(F=10.3, df=3,27, pc.001). The medium cirazoline dose induced more extension/tremor 29 than the vehicle for cirazoline (p<.05). The high cirazoline dose induced more
extension/tremor than the medium cirazoline dose (p<.05), the low cirazoline dose
(p<.01) or the vehicle for cirazoline (p<.01). Cirazoline significantly decreased the
durations of relaxed inactivity (F=3.0, df=3,27, p=. 046) and nonstereotyped activity
(F=3.8, df=3,27, p=.021). There was less relaxed inactivity following the medium and
high cirazoline doses (p’s<.05) and less nonstereotyped activity following the medium
(p<.05) and high (p<.01) doses than following the vehicle for cirazoline.
Cirazoline - PD 20
Although there were some episodes of diagonal progression, airswimming,
atypical air-stepping and tail extension following cirazoline administration to PD 20 rat
pups, cirazoline did not significantly affect the durations of these behaviors. Cirazoline
significantly increased the durations of extension/tremor (F=l 8.5, df=3,27, p<.001) and
hindlimb flexion (F=8.1, df=3,27, p<.001). The high cirazoline dose induced
significantly longer durations of extension/tremor and hindlimb flexion than the low
dose, the medium dose or the vehicle for cirazoline (p’s<.01).
Cirazoline significantly decreased the durations of relaxed inactivity (F=5.2,
df=3,27, p=.006) and nonstereotyped activity (F=3.7, df=3,27, p=.022). There was less
relaxed inactivity in subjects receiving the medium cirazoline dose than in subjects
receiving the low dose (p<.05). There was less relaxed inactivity in subjects receiving the
high cirazoline dose than in subjects receiving the low dose (p<.01) or the vehicle for
cirazoline (p<.05). The low cirazoline dose decreased the duration of nonstereotyped
activity relative to subjects receiving the vehicle for cirazoline (p<.01). 30
IJK-14.304 - PD 5
The total durations of the behaviors quantified in PD 5, 10, 15 and 20 rat pups following UK- 14,304 administration are presented in Figure 2. Tail extension, extension/tremor, forelimb flexion, hindlimb flexion and 4-limb flexion were not induced
by UK- 14,304 or its vehicle. These behaviors were thus not quantified. In PD 5 rat pups,
UK- 14,3 04 significantly induced the diagonal progression pattern of air-stepping (F=5.8, df=3,27, p=.003) and atypical air-stepping (F-3.2, df=3,27, p=.039). The duration of diagonal progression was significantly longer following the low UK- 14,3 04 dose than following the medium dose, the high dose or the vehicle for UK-14,304 (p’s<.01). The low UK- 14,304 dose induced significantly more atypical air-stepping than the medium dose or the vehicle for UK-14,304 (p’s<.05).
The mean latency to the onset of the first episode of diagonal progression following the low UK-14,304 dose was 12 minutes. The total duration of UK-14,304- induced air-stepping and atypical air-stepping usually occurred immediately at this time
of first onset. For the remainder of the 60 minute session, subjects alternated between episodes of relaxed inactivity and nonstereotyped activity. UK- 14,304 significantly affected the durations of relaxed inactivity (F=47.2, df=3,27, p<.001) and nonstereotyped activity (F=32.8, df=3,27, p<.001). The low dose decreased relaxed inactivity (p<.01) and increased nonstereotyped activity (p<.05) relative to subjects receiving the vehicle for
UK-14,304. The medium and high doses increased relaxed inactivity (p’s<.01) and
decreased nonstereotyped activity (p’s<.01) relative to subjects receiving the low dose or the vehicle for UK-14,304. 31
| Relaxed Inactivity Diagonal Progression Atypical Airstep j HH Airswim 3-limb Airstep [77] Nonstereotyped Activity 1 I I t=j
(Minutes)
Duration
UK-14,304 Dose (mg/kg) UK-14,304 Dose (mg/kg) Day 5 10 Q ) Postnatal b) Postnatal Day
(Minutes)
Duration
UK-14,304 Dose (mg/kg) UK-14,304 Dose (mg/kg)
c) Postnatal Day 15 d) Postnatal Day 20
Figure 2. Mean durations of behaviors quantified following administration of UK- 14,3 04 or its vehicle. Note the larger time scale for PD 20 subjects. Asterisks denote significant differences from vehicle (0.0 mg/kg) subjects. *=p<.05; **=p<.01
a) PD 5; b) PD 10; c) PD 15; d) PD 20 1
32
UK-14.304 -PD 10
Although there were some episodes of diagonal progression and atypical air- stepping in PD 10 rat pups following UK- 14,3 04 administration, UK- 14,3 04 did not significantly affect the durations of these behaviors. UK- 14,3 04 significantly increased the duration of relaxed inactivity (F=44.3, df=3,28, p<.001) and significantly decreased the duration of nonstereotyped activity (F=64.9, df=3,28, p<.001). All three doses increased the duration of relaxed inactivity and decreased the duration of nonstereotyped activity relative to subjects receiving the vehicle for UK-14,304 (p’s<.01). There was more relaxed inactivity and less nonstereotyped activity in subjects receiving the medium and high UK-14,304 doses than in subjects receiving the low dose (p’s<.05).
UK-14.304 - PD 15
In PD 15 rat pups, UK- 14,304 induced very brief but significant durations of diagonal progression (F=3.5, df=3,26, p=. 028) Subjects receiving the low dose engaged in significantly more diagonal progression than subjects receiving the vehicle for UK-
14,304 (p<.05). The mean latency to the first episode of diagonal progression was 1 minutes. The total duration of UK-14,304-induced air-stepping usually occurred at the
time of this first episode, after which subjects alternated between episodes of relaxed
inactivity and nonstereotyped activity.
UK-14,304 significantly increased the duration of relaxed inactivity (F=l 14.7,
df=3,26, p<.001) and decreased the duration of nonstereotyped activity (F=125.5,
df=3,26, p<.001). All three doses increased the duration of relaxed inactivity and
decreased the duration of nonstereotyped activity relative to subjects receiving the vehicle 33 for UK-14,304 (p’s<.01). There was more relaxed inactivity in subjects receiving the medium and high doses (p’s<.01) and less nonstereotyped activity in subjects receiving
in receiving the low dose. the medium (p<.05) and high (p<.0 1 ) doses than subjects
1 IK-14.304 - PD 20
UK-14,304 significantly induced diagonal progression (F=3.2, df=3,28, p=.036) and 3-limb airstepping (F=10.5, df=3,28, p<.001) in PD 20 rat pups. The duration of diagonal progression was significantly longer following the medium and high doses than following the vehicle for UK- 14,304 (p’s<.05). The high dose induced significantly more
3-limb airstepping than the low dose, the medium dose or the vehicle for UK-14,304
(p’s<.01). Although there were some episodes of airswimming and atypical air-stepping,
UK- 14,3 04 did not significantly affect the durations of these behaviors.
The mean latency to the first episode of diagonal progression was 30 minutes following the medium UK-14,304 dose and 58 minutes following the high UK-14,304 dose. The 30-60 minutes preceding the onset of UK-14,304-induced air-stepping was predominated by relaxed inactivity and, to a lesser extent, nonstereotyped activity. The durations of relaxed inactivity (F=7.8, df=3,28, p<.001) and nonstereotyped activity
(F=13.1, df=3,28, p<.001) were significantly affected by UK-14,304. The low and medium doses decreased the duration of relaxed inactivity relative to subjects receiving the vehicle for UK-14,304 (p’s<.05) or the high UK-14,304 dose (p’s<.01). The duration of nonstereotyped activity was longer in subjects receiving the low dose than in subjects receiving the vehicle for UK- 14,3 04 (p<.05). The high dose decreased the duration of 34 nonstereotyped activity relative to subjects receiving the low dose, the medium dose or the vehicle for UK-14,304 (p’s<.01).
Experiment IB: Agonists Administered in Combination
Cirazoline / UK-14.304 - PD 5
The total durations of the behaviors quantified in PD 5, 10, 15 and 20 rat pups
following combined administration of the noradrenergic agonists and/or their vehicles are
presented in Figure 3. At each age, there were four possible drug combinations: the
vehicle for cirazoline followed by the vehicle for UK- 14,3 04, the vehicle for cirazoline
followed by UK- 14,3 04, cirazoline followed by the vehicle for UK- 14,3 04 or cirazoline
followed by UK-14,304. Subjects receiving cirazoline followed by the vehicle for UK-
14,304 will be referred to as the cirazoline alone group. Subjects receiving the vehicle for
cirazoline followed by UK- 14,304 will be referred to as the UK- 14,304 alone group.
The F values, degrees of freedom and p values are reported in the appendix for all
behaviors in which there were significant interactions between cirazoline and UK- 14,3 04
at the four different ages. In PD 5 rat pups, diagonal progression, forelimb galloping,
atypical air-stepping and forelimb flexion were induced in significant amounts only when
cirazoline was followed by UK-14,304 (p’s<.01). Tail extension was significantly
induced only by cirazoline alone (p<.01). Thus, for each of these behaviors, there were
significant main effects by cirazoline and UK- 14,3 04 and significant interactions.
There were significant main effects by cirazoline but not UK- 14,304 on the
durations of extension/tremor (F=16.0, df=l,28, p<.001) and hindlimb flexion (F=6.4, A
35
Airswim Inactivity Hindlimb Flexion . J ] Relaxed J FFFFffl 4-limb Flexion Gallop 1 1 1 V/A \/ Nonstereotyped Activity II II Diagonal Progression K\l Atypical Airstep Tail Extension 1 1 1 1 1 1| Forelimb Gallop Atypical Airswim |yVI Extension/Tremor Forelimb Flexion
(Minutes)
Duration
Cirazoline Dose/UK— 1 4,304 Dose (mg/kg)
Postnatal Day 10 a) Postnatal Day 5 b)
(Minutes)
Duration
1 4,304 Dose (mg/kg) Cirazoline Dose/UK- 1 4,304 Dose (mg/kg) Cirazoline Dose/UK—
c) Postnatal Day 15 d) Postnatal Day 20
Figure 3. Mean durations of behaviors quantified following cirazoline and UK-14,304. Note the larger time scale for PD 20 subjects. Asterisks denote significant differences from subjects receiving both vehicles (VEH/VEH group). *=p<.05; **=p<.01
a) PD 5; b) PD 10; c) PD 15; d) PD 20 36 df=l ,28, p=.016). Cirazoline thus significantly induced these behaviors whether followed by UK-14,304 or its vehicle. There were significant main effects by cirazoline and UK-
14,304 on the durations of relaxed inactivity and nonstereotyped activity, as well as significant interactions. Relative to subjects receiving both vehicles, the duration of relaxed inactivity was increased by UK- 14,3 04 alone (p<.01), unaffected by cirazoline alone and decreased by cirazoline followed by UK-14,304 (p<.01). UK-14,304 alone, cirazoline alone and cirazoline followed by UK-14,304 decreased the duration of nonstereotyped activity relative to the subjects that received both vehicles (p’s <.01).
Cirazoline / UK- 14.304 - PD 10
In PD 10 rat pups, airswimming and forelimb flexion were induced in significant amounts only when cirazoline was followed by UK-14,304 (p’s<.01). Atypical air- stepping and extension/tremor were significantly induced only when cirazoline was followed by the vehicle for UK-14,304 (p’s<.01). Thus, for each of these behaviors, there were significant main effects by cirazoline and UK- 14,304 and significant interactions.
There were significant main effects by cirazoline but not UK- 14,3 04 on diagonal progression (F=17.5, df=l,30, p<.001), forelimb galloping (F=34.6, df=l,30, p<.001), tail extension (F=7.9, df=l,30, p=.008) and 4-limb flexion (F=8.0, df=l,30, p=.008).
Cirazoline thus significantly induced these behaviors whether followed by UK- 14,304 or
its vehicle.
There were significant main effects by cirazoline and UK- 14,3 04 on the durations of relaxed inactivity and nonstereotyped activity, as well as significant interactions.
Relative to subjects receiving both vehicles, the duration of relaxed inactivity was 37 increased by UK- 14,3 04 alone, decreased by cirazoline alone and decreased by cirazoline followed by UK-14,304 (p’s<.01). UK-14,304 alone, cirazoline alone and cirazoline followed by UK- 14,3 04 decreased the duration of nonstereotyped activity relative to the
subjects receiving both vehicles (p’s<.01).
Cirazoline /IJK-14.304 -PD 15
In PD 15 rat pups, diagonal progression, airswimming, galloping and forelimb
flexion were induced in significant amounts only when cirazoline was followed by UK-
14,304 (p’s<.01). Thus, for each of these behaviors, there were significant main effects
by cirazoline and UK- 14,3 04 and significant interactions. Although there were some
episodes of atypical airswimming in subjects receiving cirazoline followed by UK-
14,304, the duration of this behavior was not significantly different from subjects
receiving both vehicles.
There were significant main effects by cirazoline but not UK- 14,304 on the
durations of atypical air-stepping (F=9.8, df=l,27, p=. 004) and extension/tremor (F=29.6,
df=l,27, p<.001). Cirazoline thus significantly induced these behaviors whether followed
by UK-14,304 or its vehicle. There was a significant main effect by UK-14,304 but not
cirazoline on the duration of tail extension (F=6.2, df=l,27, p=. 018). UK-14,304 blocked
the tail extension otherwise present in subjects receiving the vehicle for UK- 14,304.
There were significant main effects by cirazoline and UK-14,304 on the duration
of relaxed inactivity, as well as a significant interaction. Relative to subjects receiving
both vehicles, the duration of relaxed inactivity was increased by UK- 14,304 alone
(p<.01), unaffected by cirazoline alone and decreased by cirazoline followed by UK- 38 14,304 (p<.01). There was a significant main effect by UK-14,304 but not cirazoline on the duration of nonstereotyped activity, along with a significant interaction. UK- 14,304 decreased nonstereotyped activity when preceded by the vehicle for cirazoline or by cirazoline (p’s<.01). Subjects that received UK-14,304 preceded by cirazoline, however, were more active than subjects that received UK- 14,304 alone (p<.01).
Cirazoline / UK- 14,304 - PD 20
In PD 20 rat pups, airswimming, atypical air-stepping and forelimb flexion were induced in significant amounts only when cirazoline was followed by UK- 14,3 04
(p’s<.01). Thus, for each of these behaviors, there were significant main effects by cirazoline and UK- 14,3 04 and significant interactions. There were significant main
14,304 effects by cirazoline (F=4.3, df=l,29, p=.044) and UK-14,304 (F=8.6, df=l,29, p=.006) on the duration of diagonal progression, with no interaction. Thus, cirazoline and UK-
induced small amounts of diagonal progression whether administered alone or in combination.
There were significant main effects by cirazoline but not UK-14,304 on the durations of extension/tremor (F=17.4, df=l,29, p<.001) and relaxed inactivity (F=16.4, df=l,29, p<.001). Cirazoline increased the duration of extension/tremor and decreased
14,304 the duration of relaxed inactivity whether followed by UK- 14,304 or its vehicle. There
were significant main effects by UK- 14,304 but not cirazoline on the durations of
galloping (F=7.8, df=l,29, p=.008) and tail extension (F=4.6, df=l,29, p=.037). UK-
increased the duration of galloping and decreased the duration of tail extension
whether preceded by cirazoline or its vehicle. 39
Experiment 2: Antagonists / L-DOPA
Prazosin / l-DOPA - PD 5
Subjects of all ages receiving prazosin or its vehicle followed by the vehicle for L-
DOPA alternated between episodes of nonstereotyped activity and relaxed inactivity throughout the entire 60 minute session. l-DOPA preceded by the vehicle for prazosin induced significant durations of air-stepping at all ages. The primary L-DOPA-induced air-stepping gait was diagonal progression. l-DOPA elicited significant durations of 3- limb airstepping, airswimming and galloping at PD 15 and significantly induced galloping at PD 20.
The mean durations of each of the typical air-stepping gaits quantified following
prazosin and L-DOPA administration to PD 5, 10, 15 and 20 rat pups are presented in
Figure 4. In this figure, “total airstep” represents the sum of the typical air-stepping gaits
elicited at each age. Because air-stepping was not significantly induced by prazosin followed by the vehicle for l-DOPA, the means are presented only for those subjects that received l-DOPA. The mean durations of the atypical air-stepping gaits and all other
non-airstepping behaviors are presented in Table 1. The F values, degrees of freedom and p values are reported in the appendix for all behaviors in which there was a significant interaction between prazosin and L-DOPA.
In PD 5 rat pups, l-DOPA preceded by the vehicle for prazosin induced diagonal progression, 3-limb airstepping, atypical air-stepping and extension/tremor. When L-
DOPA was preceded by prazosin, it also elicited episodes of forelimb airstepping, atypical forelimb airstepping and 1-limb airstepping. The data are not shown for 3-limb 40
O o l_ QD
Prazosin Dose (mg/kg) with 100 mg/kg L— DOPA Prazosin Dose (mg/kg) with 100 mg/kg L— DOPA
a) Postnatal Day 5 b) Postnatal Day 10
30 Jg| Total Airstep LXJ Diagonal Progression 3— Limb Airstep 25 - 21 l-H-Hl Airswim
20 -
15
o a 10
x X I X X mm 0.0 .015625 .0625 0.25
Prazosin Dose (mg/kg) with 100 mg/kg L— DOPA Prazosin Dose (mg/kg) with 100 mg/kg L— DOPA c) Postnatal Day 15 d) Postnatal Day 20
Figure 4. Mean durations and standard deviations of typical air-stepping gaits quantified following prazosin and l-DOPA administration. Note the different time scales at the different ages. Asterisks denote significant differences from vehicle (0.0 mg/kg) subjects. Crosses denote **=p<.01 ++=p<.01 significant differences from next lower prazosin dose. *=p<.05; ; +=p<.05;
a) PD 5; b) PD 10; c) PD 15; d) PD 20 i 1 H — 1• < I• —t i » <
41
c o o o D. OO in sq o +1 +1 +1 o o o o o o o o o © o o 'T ^ ^ +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 + £ g £ —'incoOOOOOOOOOOOO E & behaviors 2 - of c g C3 5 s- (minutes) r-H Os ^r in 00 CN m CN c X ’ ’ 00
fc • ~ the 4—* c/3 rS + ^3 on + < — in © ^t; * CN CN so ON 4 Nonstereotyped sd cn 00 00 SO m in 00 ON r— OO • T3 § 4-1 4-1 4-1 4-1 > m +1 +i +1 +1 00 “hi +1 + 1 B .2 in r- Os ON 00 so in r- 00 +1 4-1 4-1 “h a 't: - —* + | l-DOPA 4 - « S o so CN N ON SO
-2 . « cd Cl* *— in 73 3 O prazosin in in CN CD CN CN SO in § Sv in S oo cl, © in SO m CN m 1,2 CN CN »— so m CN in ^ II oo © o TO 00 Dose © © O t-j © © o r-H © © © CN © CN J of E © © o o © © cd o © © © o © d> © 13§ ^ 1 eS) wo o in © s s in <— '— CN v Effects Table a Age Q Q Q Q h© r Oh Oh Oh Oh s 42 and 1-limb airstepping, each of which occurred for less than a minute in all experimental groups. The significant main effects and/or interactions for each quantified behavior were as follows.
Very brief episodes of 3-limb airstepping were induced by L-DOPA (F=7.6, dfM,55, p=.007), with no effect by prazosin and no interaction. There were significant main effects by prazosin and l-DOPA and significant interactions between them for each of the following behaviors: total air-step, diagonal progression, forelimb airstepping, 1- limb airstepping, atypical air-stepping, atypical forelimb stepping, extension/tremor and relaxed inactivity. The significant interactions reflect the fact that prazosin affected the
durations of these behaviors when it preceded L-DOPA, but not when it preceded the vehicle for l-DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA.
In subjects receiving l-DOPA, all three prazosin doses significantly decreased the durations of total airstep and diagonal progression relative to subjects receiving the vehicle for prazosin (p’s<.01). Subjects in the medium prazosin dose group engaged in significantly less total airstep and diagonal progression (p’s<.01) than subjects in the low prazosin dose group. Subjects in the high dose group engaged in significantly less total airstep (p<.05) than subjects in the medium dose group. The high prazosin dose significantly decreased the duration of extension/tremor relative to subjects receiving the vehicle for prazosin, the low prazosin dose or the medium prazosin dose (p’s<.01).
In subjects receiving l-DOPA, all three prazosin doses significantly increased the duration of forelimb airstepping relative to subjects receiving the vehicle for prazosin 43
(p’s<.01). Subjects receiving the medium prazosin dose engaged in more forelimb airstepping than subjects receiving the low prazosin dose (p<.05). The medium (p<01) and high (p<.05) prazosin doses significantly increased the duration of atypical forelimb
stepping relative to subjects receiving the vehicle for prazosin. The medium prazosin dose significantly increased the duration of 1 -limb airstepping (data not shown) relative to subjects receiving the vehicle for prazosin (p<.01), the low prazosin dose (p<.01) and the high prazosin dose (p<.05).
In subjects receiving l-DOPA, subjects receiving the low prazosin dose engaged
in significantly more atypical air-stepping while subjects receiving the high prazosin dose
engaged in significantly less atypical air-stepping than subjects receiving the vehicle for prazosin (p’s<.01). Subjects in the high prazosin dose group engaged in significantly less
atypical air-stepping than subjects in the medium dose group and subjects in the medium
dose group engaged in less atypical air-stepping than subjects in the low dose group
(p’s<.01). Subjects in the medium (p<.05) and high (p<.01) prazosin dose groups were
significantly more inactive than subjects receiving the vehicle for prazosin. This effect
was not apparent in subjects that received the same doses of prazosin followed by the
vehicle for l-DOPA, however, suggesting that prazosin did not sedate the subjects. All
subjects receiving l-DOPA were less inactive than subjects receiving the vehicle for L-
DOPA (p’s<.01), with the exception of subjects receiving the high prazosin dose. There
were no significant effects on nonstereotyped activity. 44
Prazosin / L-DOPA - PD 10
In PD 10 rat pups, l-DOPA preceded by the vehicle for prazosin induced diagonal progression, 3 -limb airstepping, atypical air- stepping and extension/tremor. When L-
DOPA was preceded by prazosin, it also induced airswimming, forelimb airstepping and forelimb flexion. The data are not shown for forelimb airstepping, which occurred for
less than a minute in all experimental groups. The significant main effects and/or interactions for each quantified behavior were as follows.
Brief episodes of 3-limb airstepping were induced by L-DOPA (F=4.8, df=l,63, p=.029), with no effect by prazosin and no interaction. l-DOPA also induced extension/tremor (F=15.7, df=l,63, p<.001) and forelimb flexion (F=6.3, df=l,63, p=.013), with no effects by prazosin and no interactions. There were significant main effects by prazosin and l-DOPA and significant interactions between them for each of the following behaviors: total airstep, diagonal progression, forelimb airstepping, airswimming, atypical air-stepping and relaxed inactivity. The significant interactions
reflect the fact that prazosin affected the durations of these behaviors when it preceded L-
DOPA, but not when it preceded the vehicle for l-DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA.
All three prazosin doses significantly decreased the durations of total airstep and diagonal progression relative to subjects receiving the vehicle for prazosin (p’s<.01).
Subjects in the medium prazosin dose group engaged in significantly less total airstep and diagonal progression (p’s<.01) than subjects in the low prazosin dose group. The medium prazosin dose significantly increased the duration of forelimb airstepping relative 5
45 to subjects receiving the vehicle for prazosin, the low prazosin dose and the high prazosin dose (p’s<.01). The low prazosin dose significantly increased the duration of airswimming relative to subjects receiving the vehicle for prazosin, the medium prazosin dose and the high prazosin dose (p’s<.01).
Subjects receiving the low prazosin dose engaged in significantly more atypical air-stepping (p<.01) while subjects receiving the high prazosin dose engaged in significantly less (p<.05) atypical air-stepping than subjects receiving the vehicle for prazosin. Subjects in the high prazosin dose group engaged in significantly less atypical air-stepping than subjects in the medium and low prazosin dose groups (p’s<.01). The high prazosin dose significantly increased the duration of relaxed inactivity relative to the vehicle for prazosin, the low prazosin dose and the medium prazosin dose (p’s<.01).
There were no significant main effects on nonstereotyped activity, but there was a significant interaction. The duration of nonstereotyped activity was significantly longer in the medium prazosin dose group than in subjects receiving the vehicle for prazosin or the high prazosin dose (p’s<.01).
Prazosin / l-DOPA - PD 1
In PD 15 rat pups, l-DOPA preceded by the vehicle for prazosin induced diagonal progression, 3-limb airstepping, airswimming, galloping, atypical air-stepping and extension/tremor. The data are not shown for galloping, which occurred for less than a
minute in all experimental groups. l-DOPA significantly induced airswimming (F=5.5, df=l,65, p=.02), galloping (F=8.1, df=l,65, p=.006) and extension/tremor (F=10.0, df=l,65, p=.002), with no effects by prazosin and no interactions. There were significant 46 main effects by prazosin and l-DOPA and significant interactions between them for each of the following behaviors: total airstep, diagonal progression and 3-limb airstepping.
For each of these behaviors, the interaction occurred because the prazosin effect was evident when prazosin preceded L-DOPA, but not when prazosin preceded the vehicle for l-DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA.
All three prazosin doses significantly decreased the durations of total airstep and diagonal progression relative to subjects receiving the vehicle for prazosin (p’s<.01).
Subjects in the medium prazosin dose group engaged in significantly less total airstep and diagonal progression (p’s<.01) than subjects in the low prazosin dose group. Subjects in the high dose group engaged in significantly less total airstep (p<.05) than subjects in the medium dose group. Subjects receiving the low prazosin dose engaged in significantly more 3-limb stepping than subjects receiving the vehicle for prazosin, the medium prazosin dose or the high prazosin dose (p’s<.01). The high prazosin dose decreased the duration of 3-limb stepping relative to subjects receiving the vehicle for prazosin (p<.05).
There were significant main effects by prazosin (F=2.9, df=3,65, p=.043) and L-
DOPA (F=29.1, df=l ,65, p<.001) on the duration of atypical air-stepping, with no interaction. l-DOPA increased the duration of atypical air-stepping relative to its vehicle while prazosin decreased the duration of this behavior. There were significant main effects by prazosin (F=l 3.8, df=3,65, p<.001) and l-DOPA (F=30.0, df=l,65, p<.001) on the duration of relaxed inactivity, with no interaction. l-DOPA decreased the duration of
relaxed inactivity relative to its vehicle. Prazosin increased the duration of relaxed 47
inactivity when it preceded either L-DOPA or its vehicle. There were no significant main effects by prazosin or l-DOPA on the duration of nonstereotyped activity, but there was a significant interaction. The medium (p<.01) and high (p<.05) prazosin doses decreased the duration of nonstereotyped activity in subjects than received the vehicle for l-DOPA but not in subjects that received L-DOPA.
Prazosin / L-DOPA - PD 20
In PD 20 rat pups, l-DOPA preceded by the vehicle for prazosin induced diagonal progression, galloping and atypical air-stepping. When preceded by prazosin, l-DOPA also induced 3-limb airstepping and airswimming. There were significant main effects by prazosin and l-DOPA and significant interactions between them for each of the following behaviors: total airstep, diagonal progression, 3-limb airstepping, airswimming and galloping. The significant interactions reflect the fact that prazosin affected the durations
of these behaviors when it preceded l-DOPA, but not when it preceded the vehicle for L-
DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA.
The high prazosin dose decreased the duration of total airstep relative to the vehicle for prazosin, the low prazosin dose and the medium prazosin dose (p’s<.01). All three prazosin doses decreased the durations of diagonal progression and galloping relative to subjects receiving the vehicle for prazosin (p’s<.01). Subjects receiving the low prazosin dose engaged in significantly more 3-limb stepping than subjects receiving the vehicle for prazosin (p<.01), the medium prazosin dose (p<.05) or the high prazosin dose (p<.01). The low and medium prazosin doses (p’s<.01) increased the duration of 48 airswimming relative to subjects receiving the vehicle for prazosin. There were significant main effects by l-DOPA on the durations of atypical air-stepping (F=32.3, df=l,74, p<.001) and relaxed inactivity (F=45.8, df=l,74, p<.001). Subjects receiving L-
DOPA engaged in significantly more atypical air-stepping and significantly less relaxed inactivity than subjects receiving the vehicle for l-DOPA. There were no significant effects on the duration of nonstereotyped activity.
Idazoxan / l-DOPA - PD 5
Subjects of all ages administered idazoxan or its vehicle followed by the vehicle for l-DOPA alternated between episodes of nonstereotyped activity and relaxed inactivity. l-DOPA preceded by the vehicle for idazoxan induced significant durations of
air-stepping at all ages. The primary L-DOPA-induced air-stepping gait was diagonal progression. At PD 15, l-DOPA also induced 3-limb airstepping and airswimming.
The mean durations of each of the typical air-stepping gaits quantified following
idazoxan and l-DOPA administration to PD 5, 10, 15 and 20 rat pups are presented in
Figure 5. Because air-stepping was not significantly induced by idazoxan followed by the vehicle for l-DOPA, the means are presented only for those subjects that received L-
DOPA. The mean durations of atypical air-stepping and the other non-airstepping behaviors are presented in Table 2. F values, degrees of freedom and p values are reported in the appendix for all behaviors in which there was a significant interaction between idazoxan and l-DOPA.
In PD 5 rat pups, L-DOPA preceded by the vehicle for idazoxan significantly
induced diagonal progression, atypical air-stepping and extension/tremor. There were 49
35 Bl Total Airstep ©U Total Airstep Diagonal Progression L/kJ Diagonal Progression 30
I 1 3-Limb Airstep
25
C c 20 2
.9 15
10
JE .125 0.5 0.0 .0125 .05 0.2 0.0 .03125
Idazoxan Dose (mg/kg) with 100 mg/kg L— DOPA Idazoxan Dose (mg/kg) with 100 mg/kg L— DOPA
a) Postnatal Day 5 b) Postnatal Day 10
30 25 Sill Total Airstep ISJ Total Airstep LZVJ Diagonal Progression L/\J Diagonal Progression Airstep 25 - I | 3-Limb Airswim 1 1 1 1 II
20 - 15
X 10 - X X X 5 - X
•• St <• LJ 0.2 2.0 0.0 0.02 0.0 .0625 .25 1.0 Idazoxan Dose (mg/kg) with 100 mg/kg L— DOPA Idazoxan Dose (mg/kg) with 100 mg/kg L-DOPA
c) Postnatal Day 15 d) Postnatal Day 20
Figure 5. Mean durations and standard deviations of typical air-stepping gaits quantified following idazoxan and l-DOPA administration. Note the different time scales at the different ages. Asterisks denote significant differences from vehicle (0.0 mg/kg) subjects. Crosses denote significant differences from next lower idazoxan dose. *=p<.05; **=p<.01; +=p<.05; ++=p<.01
a) PD 5; b) PD 10; c) PD 15; d) PD 20 Hi ( « .. »
50
s J- behaviors o D
t C/3 _o Tb" t * 4-J + * +w" t O rn in NO CN l 1 +1 1 1 1 +1 +1 +1 + + 1 + + +1 + +1 + o o o tremor It 00 l> rn NO CN CN vq ON r—' Nonstereotyped o d ON 43 T3 & no 00 00 vd in NO ON 00 II 0> II S-4 d * NO cd G * mg/kg in in 00 rt O NO m NO cn o in in ON © G t- 00 rd in r- NO ON N- 00 ON 00 o i— O 1 1 ~b +1 +1 +1 + 1 + 1 + +1 +1 +1 + +1 +1 *4— inactivity » V Relaxed so oo in CN N; ON m 00 r^ 00 1 CN 1 +1 + +1 cd CU t~~ ; NO in ON rd in vd in CN 00 d d O d II “H *"* r- T-' CN r CN CN CN CN 0D * with 0) Tl **" G-t V, O 3 g o 2 in g 00 idazoxan ^ ) in CN m c3 "o U affected the durations of these behaviors when it preceded l-DOPA, but not when it preceded the vehicle for l-DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA. In subjects receiving l-DOPA, the medium and high idazoxan doses significantly decreased the durations of total airstep and diagonal progression relative to subjects receiving the vehicle for idazoxan (p’s<.01). Subjects in the medium idazoxan dose group engaged in less total air-stepping and diagonal progression than subjects in the low dose group (p’s<.01). Subjects in the high idazoxan dose group engaged in less total air- stepping and diagonal progression than subjects in the medium dose group (p’s<.01). The medium and high idazoxan doses significantly increased the duration of extension/tremor relative to subjects receiving the vehicle for idazoxan and the low idazoxan dose (p’s<.01). Atypical air-stepping was significantly induced by l-DOPA when it was preceded by the vehicle for idazoxan (p<.05) and when it was preceded by the high idazoxan dose (pc.Ol) but not when preceded by the low or medium idazoxan doses. In subjects receiving l-DOPA, the duration of atypical air-stepping was significantly longer in the high idazoxan dose group than in the medium or low idazoxan dose groups (p’s<.01). There were significant main effects by idazoxan (F=4.2, df=3,56, p=.009) and l-DOPA (F=629.8, df=l,56, p<.001) on the duration of relaxed inactivity. L-DOPA decreased 52 while idazoxan increased the duration of relaxed inactivity. There was a significant main effect by idazoxan on the duration of nonstereotyped activity, with a significant interaction. In subjects given l-DOPA, those receiving the high idazoxan dose engaged in more nonstereotyped activity than subjects receiving the vehicle for idazoxan, the low idazoxan dose or the medium dose (p’s<.01). Idazoxan / L-DOPA - PD 10 In PD 10 rat pups, L-DOPA preceded by the vehicle for idazoxan significantly induced diagonal progression, atypical air-stepping and extension/tremor. There was a significant main effect by l-DOPA on the duration of extension/tremor (F=40.8, df=l,60, pc.OOl), with no effect by idazoxan and no interaction. There were significant main effects by idazoxan and l-DOPA and significant interactions between them for each of the following behaviors: total airstep, diagonal progression and atypical air-stepping. The significant interactions reflect the fact that idazoxan affected the durations of these behaviors when idazoxan preceded l-DOPA, but not when it preceded the vehicle for L- DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA. All three idazoxan doses significantly decreased the duration of total airstep and diagonal progression relative to subjects receiving the vehicle for idazoxan (p’s<.01). Subjects in the medium idazoxan dose group engaged in significantly less total airstep and diagonal progression (p’s<.01) than subjects in the low idazoxan dose group. Subjects in the high idazoxan dose group engaged in significantly less total airstep and diagonal progression (p’s<.01) than subjects in the medium dose group. The high 53 idazoxan dose significantly decreased the duration of atypical air-stepping relative to the vehicle for idazoxan, the low idazoxan dose and the medium idazoxan dose (p’s<.01). There were significant main effects by idazoxan (F=8.0, df=3,60, p<.001) and L- DOPA (F=246.8, df=T,60, p<.001) on the duration of relaxed inactivity. L-DOPA decreased while idazoxan increased the duration of relaxed inactivity. There was a significant main effect by l-DOPA on the duration of nonstereotyped activity and a significant interaction between them. l-DOPA increased the duration of nonstereotyped activity in subjects receiving the medium and high idazoxan doses (p’s<.01). Idazoxan / l-DOPA - PD 15 In PD 15 rat pups, l-DOPA preceded by the vehicle for idazoxan significantly induced diagonal progression, 3-limb airstepping, airswimming, atypical air-stepping and extension/tremor. There were significant main effects by l-DOPA on the durations of 3- limb stepping (F=8.0, df=l,56, p=.006), airswimming (F=4.0, df=l,56, p=.046), atypical air-stepping (F=27.5, df=l,56, p<.001) and extension/tremor (F=l 1.0, df=l,56, p=.002), with no effects by idazoxan and no interactions. There were significant main effects by idazoxan and l-DOPA and significant interactions between them for total airstep and diagonal progression. In both cases, the interactions occurred because the idazoxan effect was evident when idazoxan preceded l-DOPA, but not when it preceded the vehicle for L-DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA. The low (p’s<.05), medium (p’s<.01) and high (p’s<.01) idazoxan doses decreased the durations of total airstep and diagonal progression relative to subjects receiving the vehicle for idazoxan. Subjects receiving the medium idazoxan dose 54 engaged in significantly less total airstep and diagonal progression than subjects receiving the low idazoxan dose (p’s<.01). There were significant main effects by idazoxan (F=9.4, df=3,56, p<.001) and L- DOPA (F=95.2, df=l,56, p<.001) on the duration of relaxed inactivity, with no interaction. l-DOPA decreased while idazoxan increased the duration of relaxed inactivity. There were no main effects on the duration of nonstereotyped activity, but there was a significant interaction. Subjects receiving the high dose of idazoxan followed by the vehicle for l-DOPA were significantly less active than subjects receiving both vehicles (p<-05) and significantly less active than subjects receiving the high idazoxan dose followed by l-DOPA (p<.05). Idazoxan / l-DOPA - PD 20 In PD 20 rat pups, l-DOPA preceded by the vehicle for idazoxan significantly induced diagonal progression and atypical air-stepping. There were significant main effects by idazoxan and l-DOPA and significant interactions between them for total airstep, diagonal progression and atypical air-stepping. The significant interactions reflect the fact that idazoxan affected the durations of these behaviors when idazoxan preceded l-DOPA, but not when it preceded the vehicle for l-DOPA. The following pairwise differences thus occurred only in subjects that received l-DOPA. The low (p’s<.05), medium (p’s<.01) and high (p’s<.01) idazoxan doses decreased the duration of total airstep and diagonal progression relative to subjects receiving the vehicle for idazoxan. Subjects receiving the medium idazoxan dose engaged in less total airstep and diagonal progression than subjects receiving the low 55 idazoxan dose (p’s<.01). Subjects receiving the high idazoxan dose engaged in less total airstep and diagonal progression than subjects receiving the medium idazoxan dose (p’s<.05). Subjects receiving the medium idazoxan dose engaged in more atypical air- stepping than subjects receiving the vehicle for idazoxan (p<.01), the low idazoxan dose (p<.05) or the high idazoxan dose (p<.01). There was a significant main effect by l-DOPA on the duration of relaxed inactivity (F=T09.7, df=l,66, p<.001), with no idazoxan effect or interaction. l-DOPA decreased the duration of relaxed inactivity. There was a significant main effect by L- DOPA on the duration of nonstereotyped activity, along with a significant interaction. L- DOPA increased the duration of nonstereotyped activity when it was preceded by the medium or high idazoxan doses (p’s<.01) but not when preceded by the low idazoxan dose or the vehicle for idazoxan. CHAPTER 4 DISCUSSION AND CONCLUSIONS Experiment 1A: Agonists Administered Separately Cirazoline (a receptor agonist! j_ Cirazoline’s effects in PD 5-20 rat pups in the air-stepping paradigm were consistent with previous research demonstrating that a noradrenergic receptor t stimulation does not elicit locomotor activity. The a, agonist phenylephrine administered systemically to 6-day-old rat pups did not induce overground locomotion [19]. Although phenylephrine does not cross the blood brain barrier in adult animals, it is possible that phenylephrine penetrated the immature blood brain barrier at PD 6 and stimulated central adrenoceptors. In the present study, the a, agonist cirazoline induced air-stepping in some subjects, but the mean duration of air-stepping was not significantly longer than in control subjects. Similarly, the a, noradrenergic receptor agonist methoxamine administered intrathecally to spinal-transected adult cats induced brief bouts of hindlimb treadmill stepping in a few subjects but did not induce hindlimb stepping in most of the subjects. In most subjects, however, methoxamine increased hindlimb extensor muscle activity, thus increasing the cats’ ability to stand. After methoxamine administration, the cats’ joints appeared stiffer and there were spontaneous movements of the tail [27], 56 57 Like methoxamine administered to spinal-transected cats, the ot[ receptor agonist cirazoline elicited a very pronounced extension of the limbs at all ages in the air-stepping paradigm. When these pups were handled, the extended limbs exerted noticeable resistance against the hand that held them. Cirazoline also caused the younger subjects, whose tails ordinarily hung limp beneath them, to extend their tails horizontally or vertically. Older subjects held their tails up whether they received cirazoline or its vehicle. This cirazoline-induced “tail extension” may be similar to the methoxamine- induced tail movements in spinal-transected cats. The pronounced limb extension elicited by cirazoline was similar to the “extension/tremor” elicited by L-DOPA in the air-stepping paradigm, usually immediately prior to an episode of air-stepping. Unlike L-DOPA-induced extension/tremor, cirazoline-induced limb extension did not precede air-stepping episodes and was generally not accompanied by a tremor. However, since a selective a, noradrenergic receptor agonist elicited such pronounced limb extension, it seems likely that a! noradrenergic receptor stimulation contributes to L-DOPA-induced extension/tremor, as well. It seems unlikely that this contribution is exclusive, however, since preventing l-DOPA’ s conversion to dopamine or dopamine’s conversion to noradrenaline does not affect the duration of L-DOPA-induced extension/tremor [5,88]. Very pronounced limb extension can also be elicited in rat pups under more natural circumstances. Oral milk infusions cause a “stretch response” in rat pups attached to the nipples of their anesthetized dam [54], At PD 5 and 10, this stretch response is characterized by a very pronounced extension of all four limbs that lifts the pups off the 58 ground and pushes them away from the dam. This stretch response is also elicited by oral milk infusions in 22 hour maternal and food deprived rat pups, along with locomotion and other diffuse motor behaviors [52]. Perhaps the limb extension, tail extension, hindlimb flexion and 4-limb flexion elicited by cirazoline in the air-stepping paradigm reflect a similar form of diffuse behavioral activation. UK-14.304 (a^receptor agonist! Like noradrenergic a2 receptor agonists administered to cats with spinal transections, which have repeatedly been shown to induce hindlimb treadmill stepping, UK- 14,304 elicited very brief episodes of diagonal progression at all four ages in the air- stepping paradigm. With the exception of the PD 10 age group, the mean duration of UK-14,304-induced air-stepping was significantly longer than the mean duration of air- stepping in control subjects. Neither extension/tremor nor tail extension were elicited by UK-14,304 at any age. While significantly different from subjects receiving the vehicle for UK- 14,3 04, the UK-14,304-induced air-stepping in rat pups in the present study was extremely short- lasting. There are a number of possible explanations for the brevity of the UK- 14,304- induced air-stepping. The simplest explanation is that UK- 14,304 is a very short-acting drug. This seems unlikely, however, given that UK- 14,304 elicited much longer-lasting air-stepping when preceded by cirazoline. Another possibility is that the procedures utilized to test subjects in the air- stepping paradigm are not conducive toward maintaining a2 agonist-induced air-stepping 59 once it has been initiated. Unlike subjects on a treadmill, which receive continuous afferent input from a moving substrate, the only afferent input received by rat pups suspended in the air is provided by the harness and by feedback from their own movements. The fact that the limbs hang pendant in the air-stepping paradigm also affects the starting limb position and the position of the limbs as they step in isolation from a supporting substrate. It seems possible that the afferent feedback in the air- stepping paradigm is not sufficient or does not mimic the more natural overground situation closely enough to maintain UK-14,304-induced air-stepping. In support of this possibility, the less selective a2 agonist clonidine induces much longer durations of overground locomotion than air-stepping in PD 10 rat pups [116]. Afferent input from the supporting substrate may thus facilitate clonidine-induced locomotor behavior in the PD 10 rat pups. Thus, in the younger rat pups, a relative lack of afferent stimulation in the air- stepping paradigm may limit the duration of a2 agonist-induced air-stepping. This seems unlikely in the PD 20 rat pups, however. The same supporting surface that facilitates clonidine-induced stepping in the younger rat pups has an opposite effect at PD 20. In PD 20 rat pups, clonidine induces much longer durations of air-stepping than it does overground locomotion [116]. In fact, clonidine elicits catalepsy in PD 20 subjects tested overground [93,116]. A more likely explanation for the brevity of the UK-14,304-induced air-stepping in the older rats is that UK- 14,3 04, like other a2 agonists, decreased neural activity in the locus coeruleus, thus decreasing the synthesis and release of noradrenaline. This 60 possibility also extends to the younger rat pups. UK- 14,304 substantially increased the duration of relaxed inactivity at PD 5, 10 and 15. These very long durations of inactivity may reflect a sedative effect resulting from UK-14,304’s suppression of neural activity in the locus coeruleus and, perhaps, other brain regions. It is also possible that, in the absence of tonic activity at other noradrenergic receptor subtypes or at receptors for other neurotransmitters, selective a2 receptor activation is not sufficient to maintain air-stepping for long periods of time. As a first attempt to address this possibility, it is useful to compare the effects of UK- 14,304 with the effects of the less selective a2 agonist clonidine. In PD 5 and 10 pups, UK-14,304’s effects in the air-stepping paradigm were similar to those of the less selective a2 agonist clonidine [116]. Like clonidine, UK- 14,3 04 induced very brief episodes of air-stepping in the younger pups. At PD 20, however, clonidine induced much longer durations of airswimming [116] than the more selective a2 noradrenergic receptor agonist UK- 14,3 04 utilized in the present research. It thus seems possible that the longer-duration clonidine- induced airswimming at PD 20 resulted from clonidine’ s combined activation of both its this and a2 noradrenergic receptor subtypes. Admittedly, on own, comparison between the effects of clonidine and UK- 14,304 is very weak evidence from which to speculate about differences between the locomotor effects of selective a2 receptor will activation versus mixed a! and a2 receptor activation. However, this issue be addressed more completely in the discussion of the effects of combined cirazoline and UK- 14,304 administration and in the discussion of the effects of a! noradrenergic receptor blockade on L-DOPA-induced air-stepping. 61 Experiment IB: Agonists Administered in Combination At all ages, the cq agonist cirazoline followed by the a2 agonist UK-14,304 induced air-stepping that was much longer-lasting than the air-stepping induced by either separately. cirazoline or UK-14,304 administered The effectiveness of either a, or a2 receptor activation was thus determined by whether or not the other receptor subtype was activated. It is not surprising that broader receptor activation more effectively induced locomotion in the present research than selective activation of one receptor subtype. There are many examples of more effective elicitation of locomotion when multiple receptor types are stimulated. An obvious example is provided by L-DOPA, a general catecholamine receptor agonist that more effectively induces air-stepping than either of the selective a noradrenergic receptor agonists in the present research. Similarly, L- DOPA or combined direct dopamine and noradrenaline receptor agonists more effectively induced overground locomotion in rat pups than dopamine or noradrenaline receptor agonists administered separately [63]. In PD 5 rat pups with spinal transections, combined serotonin and catecholamine receptor activation more effectively induced hindlimb air-stepping than selective activation of either serotonin or catecholamine receptors [81]. In the in vitro preparation of the neonatal rat spinal cord, combined application of NMDA and dopamine or NMDA and serotonin more effectively induced hindlimb airstepping and/or fictive locomotion than NMDA, dopamine or serotonin applied separately to the spinal cord bath [21,22,56,68,59,71,72,75]. 62 It is, however, somewhat surprising that even combined a! and a2 noradrenergic receptor activation was sufficient to induce air-stepping. This is surprising because previous research has demonstrated that dopamine receptor activation is necessary for L- DOPA-induced air-stepping in PD 5 rat pups [101]. However, dopamine receptor activation by endogenously-released dopamine was not prevented in the present research. Thus, the fact that a noradrenergic receptor activation is sufficient to induce air-stepping does not exclude the possibility that a certain level of dopamine release (or any other neurotransmitter, for that matter) is necessary for air-stepping. In fact, it is entirely possible that dopamine receptor antagonists would be as disruptive to the air-stepping induced by the direct noradrenergic agonists as they are to L-DOPA-induced air-stepping. Dopamine’s contribution to air-stepping may also be indicated by the fact that the noradrenergic agonist combination induced air-stepping that was somewhat different from the air-stepping induced by l-DOPA. As previously described, diagonal progression is the predominant air-stepping gait induced by l-DOPA at PD 5, 10, 15 and 20. L-DOPA-induced airswimming at PD 15 and galloping at PD 20 occur much less frequently and with much more variability than diagonal progression. In the present study, the a noradrenergic receptor agonist combination induced diagonal progression at all ages, but diagonal progression was the predominant gait only at PD 5. The diagonal progression induced by the direct noradrenergic receptor agonists was noticeably slower than L-DOPA-induced diagonal progression. At PD 5, the direct noradrenegic receptor agonists induced much more atypical air-stepping than l-DOPA. 63 At PD 10, 15 and 20, the noradrenergic agonists induced airswimming much more consistently than l-DOPA. The primary difference between l-DOPA and the direct noradrenergic receptor agonist combination is that l-DOPA ultimately causes the activation of dopamine, a noradrenergic and (5 noradrenergic receptors while cirazoline and UK- 14,304 selectively activate only a noradrenergic receptors. Thus, the slower stepping, the longer duration of atypical air-stepping at PD 5 and the longer durations of airswimming at PD 10, 15 and 20 following administration of the direct noradrenergic agonists may be due to the absence of P noradrenergic and/or dopaminergic receptor activation. It is also possible that the different topography of air-stepping induced by the direct noradrenergic receptor agonists relative to L-DOPA-induced air-stepping was specific to the particular doses of each agonist selected for combined administration at each age. In pilot experiments, varying doses of each agonist were combined with varying doses of the other agonist and the dose combinations that induced the closest approximation of L-DOPA-induced air-stepping were selected. It is possible, however, that the agonist doses selected for combined administration in the present study were not optimal. It is also possible that the chosen time course between the two agonist injections was not optimal. Perhaps the air-stepping would have been more like L-DOPA-induced air-stepping if 10 or 30 minutes, rather than 20 minutes, had elapsed between the cirazoline and UK- 14,304 injections. Direct comparisons between the air-stepping induced by combined administration of the noradrenergic agonists and L-DOPA-induced air-stepping may thus be somewhat arbitrary. 64 Regardless of the specific selection of agonist doses at each age, it is significant its that the same a2 agonist dose that did not induce air-stepping when administered on own induced such long durations of air- stepping when preceded by an a agonist. In t spinal-transected cats on a treadmill, intrathecal administration of the a agonist ( methoxamine produced only slight improvements (decreased knee sag and increased stepping spinal- extensor tonus) in effective their because of the facilitatory transected cats, a2 agonists may be more on own effects of the afferent input from the moving treadmill belt. However, it is not realistic for spinal cord-injured humans to rely on continuous afferent input from a moving treadmill belt to locomote. In the present research, a! receptor activation seems to have provided an alternate facilitatory influence that enabled sustained a2 agonist-induced locomotion. This finding has potential clinical significance if future research in spinal- transected rat pups determines that cirazoline has similar facilitatory effects on a2 agonist-induced locomotion in the absence of continuous afferent input from a moving treadmill belt. Experiment 2: Antagonists / l-DOPA At all ages, the high dose of the a, receptor antagonist prazosin and the high dose idazoxan almost completely blocked diagonal progression, of the a2 receptor antagonist the predominant L-DOPA-induced air-stepping gait. Both antagonists dose-dependently decreased the duration of “total airstep,” the sum of all typical air-stepping gaits, at all activation is necessary for l-DOPA- ages. Thus, both a! and a2 noradrenergic receptor 65 induced air-stepping at PD 5, 10, 15 and 20. While the antagonists had similar effects on diagonal progression and the total duration of air-stepping, however, they had very different effects on other air-stepping gaits. At all ages, the a2 noradrenergic receptor antagonist idazoxan dose-dependently decreased the duration of diagonal progression, but did not change the topography of the L-DOPA-induced air-stepping. The a, antagonist prazosin also decreased the duration of diagonal progression, but induced alternate air-stepping gaits in its place. Prazosin preadministration caused l-DOPA to induce different gaits at different ages. At PD 5, L- DOPA preceded by all prazosin doses induced forelimb stepping. The low prazosin dose doubled the duration of atypical air-stepping at PD 5 and 1 0, induced airswimming at PD 10 and increased the duration of 3-limb air-stepping at PD 15 and 20. At PD 20, all prazosin doses substantially increased the duration of L-DOPA-induced airswimming. Each of these alternate air-stepping gaits may indicate varying levels of disruption of the typical L-DOPA-induced air-stepping pattern. At PD 5 and 10, low levels of a, receptor blockade interfered with the dorsiflexed posture or the coordination between limbs, thus changing the L-DOPA-induced diagonal progression into atypical air- stepping. At PD 5, the low prazosin dose also blocked the stepping of the hindlimbs, thus changing the diagonal progression into forelimb stepping. The medium dose disrupted hindlimb stepping even more than the low dose. The selective disruption of hindlimb stepping at PD 5 may reflect the rostrocaudal pattern of development of the spinal cord and its descending inputs. At PD 0, forelimb air-stepping is the predominant L-DOPA-induced air-stepping gait. The stepping of the 66 forelimbs but not the hindlimbs at PD 0 may reflect the fact that the forelimbs are controlled by the more mature and more densely innervated cervical spinal cord while the hindlimbs are controlled by the less mature lumbar spinal cord. Similarly, at PD 5, the less mature lumbar spinal pattern generators may be more susceptible to disruption than the more mature cervical spinal pattern generators controlling the forelimbs. The forelimb stepping was induced by combined prazosin and l-DOPA administration only at PD 5, perhaps because the lumbar cord and its descending inputs mature substantially thereafter, enabling the lumbar cord to “catch up” with the cervical cord. At PD 15 and 20, the low prazosin dose briefly uncoupled one limb from the other three, inducing 3-limb air-stepping. It seems possible that this gait was another example of slightly disrupted diagonal progression. It is much less intuitive to interpret the airswimming at PD 10, 15 and 20 as a “disrupted” version of diagonal progression, however. The airswimming gait is typically not induced by l-DOPA until PD 15. This gait was so named because the position of the forelimbs and the pattern of limb activity so strongly resemble the adult swimming pattern. Rat pups placed in water also do not adopt this adult-like swimming pattern until PD 18-21 [16,23], suggesting that this gait occurs only in older subjects. The fact that prazosin preadministration caused l-DOPA to induce airswimming earlier in ontogeny than l-DOPA alone slightly erodes the view that airswimming is a gait that occurs only in older subjects. In another recent study, preadministration of the NMDA receptor antagonist MK-801 also caused l-DOPA to induce airswimming earlier in ontogeny than l-DOPA alone [82], In fact, MK-801 caused l-DOPA to induce 67 airswimming as early as PD 5. Airswimming is thus a gait that is clearly not limited to older subjects. Like diagonal progression and galloping, airswimming is a highly stereotyped and coordinated air-stepping gait. It is thus very difficult to explain an increased, rather than decreased, occurrence of airswimming following a, noradrenergic receptor blockade. McEwen et al. [82] proposed that the increased L-DOPA-induced airswimming following NMDA receptor blockade, along with other effects on stepping, indicated a role for glutamate acting at NMDA receptors in the coupling between limbs. Different gaits were interpreted as different patterns of limb coupling and transitions between gaits as changes in limb coupling. Based on the present results, it seems reasonable to propose a similar role for noradrenaline acting at a, receptors in the coupling between limbs. In fact, the effects of a! noradrenergic receptor blockade and NMDA glutamate receptor blockade may be interrelated. As previously mentioned, glutamate-evoked lumbar motoneuron firing rate is increased by microiontophoretic application of noradrenaline or the a noradrenergic receptor agonist phenyleprine in adult rats t [121,122]. NMDA evokes noradrenaline release in the adult rat spinal cord [73,1 10]. The fact that NMDA glutamate receptor blockade and a, noradrenergic receptor blockade have such similar effects on L-DOPA-induced air-stepping suggests that these interactions at the synaptic level are behaviorally meaningful. Thus, prazosin’s dose-dependent blockade of diagonal progression and the different gaits induced by combined prazosin and l-DOPA administration may reflect progressively higher levels of a receptor varying degrees of disruption caused by t 68 blockade. Total blockade of diagonal progression and other air-stepping gaits by the high prazosin dose was the most severe form of disruption, followed by selective disruption of hindlimb stepping by all three prazosin doses at PD 5. Following the low prazosin dose, the increased incidence of atypical air-stepping, 3-limb air-stepping and/or airswimming at PD 5, 10 and 15 may reflect subtle alterations in the coupling between limbs. However, at PD 20, the prazosin-induced gait change was not so subtle. Preadministration of the low and medium prazosin doses caused l-DOPA to induce substantial levels of airswimming at PD 20. While these doses almost completely blocked diagonal progression, they had no effect on the total duration of air-stepping. Thus, the low and medium prazosin doses caused a very obvious transition from L- DOPA-induced diagonal progression to L-DOPA-induced airswimming. It is hard to rationalize this profound change as a “subtle” disruption of limb coupling. A better understanding of this gait change at PD 20 may be derived from a careful consideration of the synaptic activity that remains, not the synaptic activity that has been blocked in this experiment. l-DOPA is converted to dopamine and noradrenaline and, following a noradrenergic receptor blockade, the catecholamine neurotransmitters t noradrenergic receptors. activate dopamine receptors, p noradrenergic receptors and a2 The L-DOPA-induced airswimming at PD 20 following prazosin administration strongly resembled clonidine-induced airswimming at this age, suggesting that noradrenaline’s this behavior. actions at a2 receptors underlie Noradrenaline acting at p receptors exerts a “gating” effect in many brain regions, decreasing spontaneous background activity and increasing the influence of sensory 69 inputs and afferent inputs from other brain regions [118]. Noradrenaline also modulates afferent input to the spinal cord, presumably via its very dense population of a2 noradrenergic receptors in the dorsal horn. Numerous experiments have demonstrated that the central pattern generator for locomotion can be reset by stimulation of sensory afferents to the spinal cord [30,39,47,58,59,66], The change in gait from diagonal progression to airswimming may reflect noradrenaline’s actions at a2 and/or (3 receptors in the spinal cord and brain, substantially increasing the influence of sensory inputs on the central pattern generator and initiating a change in gait. Interestingly, rat pups suspended in the air, like subjects placed in water, do not receive continuous afferent input from a supporting substrate. Thus, airswimming is the most appropriate gait for the environment of the subjects and the increased occurrence of this gait may reflect the increased influence of sensory input on the central pattern generator for locomotion. Although the airswimming induced by prazosin combined with l-DOPA resembled clonidine-induced airswimming, it cannot be concluded that this behavior was is entirely mediated by noradrenaline’s actions at a2 receptors. Clonidine not a selective noradrenergic receptor agonist particularly selective a2 agonist and the more a2 UK- 14,304 did not significantly induce airswimming at PD 20, although statistically non- significant durations of airswimming were elicited. UK-14,304 preceded by the a] agonist cirazoline, however, did significantly induce airswimming. Also, the longest duration of L-DOPA-induced airswimming at PD 20 occurred following the lowest prazosin dose, when partial a, receptor activation was most likely preserved. Of course, noradrenaline’s actions at (3 receptors and dopamine’s actions at its receptors may also 70 play a role, but it appears that a certain level of a, receptor activation contributes to the occurrence of airswimming. Thus, perhaps a particular ratio of activity between a, and a2 noradrenergic receptors is optimal for sensorimotor integration and the occurrence of airswimming. The neural activity of the locus coeruleus is sporadic in younger pups but matures into the tonic, adultlike pattern by PD 20 [84], Perhaps blocking a subset of receptors that is ordinarily tonically activated provides its own “gating” mechanism, decreasing spontaneous background activity and increasing the influence of sensory inputs. Thus, combined activation of a2 noradrenergic receptors and a particular level of activation of a noradrenergic receptors might provide the optimal conditions for resetting of the t central pattern generator by afferent input. l-DOPA administration following partial receptor blockade provides this ratio of a receptor subtype activation, as does clonidine. Though not statistically significant, brief durations of airswimming were elicited by the a! agonist cirazoline at PD 15 and 20 administered and by the a2 agonist UK-14,304 at PD 20. When these two agonists were in combination, they significantly induced airswimming at PD 10, 15 and 20, suggesting that a particular ratio of activity at these receptor subtypes increased the likelihood of airswimming. To test this hypothesis that varying the ratio of activation of these two receptor subtypes affects the occurrence of airswimming, it would be worthwhile to administer varying doses of cirazoline in combination with varying doses of UK-14,304. Under most of the conditions described above, much longer durations of airswimming were induced in older pups than in younger pups. The increased occurrence 71 of airswimming across ontogeny may reflect the progressive maturation of sensorimotor integration. Due to this maturation of sensorimotor integration, conditions that increase the influence of sensory input may more easily reset the central pattern generator in older pups than in younger pups, thus more consistently inducing airswimming in the older pups. Just as l-DOPA’s behavioral effects following a! noradrenergic receptor blockade may provide valuable information about the role of a2 noradrenergic receptor activation in locomotion, l-DOPA’s behavioral effects following a2 noradrenergic receptor blockade may provide similar clues about the role of a, noradrenergic receptor activation in locomotion. The fact that the a2 noradrenergic antagonist idazoxan simply blocked L- DOPA-induced air-stepping without changing its topography suggests that a! noradrenergic receptor activation, along with P noradrenergic and dopamine receptor activation, is not sufficient to induce air-stepping of any form. However, idazoxan preadministration caused L-DOPA to induce significantly more extension/tremor in PD 5 rat pups than l-DOPA alone. At this same age, the high prazosin dose blocked l-DOPA- induced extension/tremor. Thus, an a, antagonist blocks extension/tremor and l-DOPA induces significantly with the more extension/tremor following a2 noradrenergic receptor blockade. Combined previously-described effects of the a! noradrenergic receptor agonist cirazoline on limb extension, these results suggest that a, noradrenergic receptor activation causes limb extension. Given that a certain level of a, receptor activation is necessary for l-DOPA- 72 induced air-stepping, perhaps the limb extension elicited by dj receptor activation is a necessary prerequisite for air-stepping. Summary and Conclusions for L- Although both and a2 noradrenergic receptor activation was necessary DOPA-induced air- stepping in PD 5-20 rat pups, selective activation of either or a2 receptors was only minimally effective at inducing air-stepping. Combined activation of effective as but was effective at d[ and a2 receptors was not as l-DOPA, much more inducing air-stepping than selective activation of either a receptor subtype. Each of the a noradrenergic receptor subtypes played a different role in air-stepping, roles that appeared to be slightly modified during ontogeny. Activation of receptors elicited a pronounced limb extension that may be necessary for the occurrence of air-stepping. This receptor subtype is most likely not the only contributor to this behavior, however. Activation of the receptors of other descending systems, such as the serotonin or dopamine system, might provide a similar descending drive that would substitute for the a, noradrenergic receptor activation in the specific present investigation. Activation of a2 receptors seemed to provide a more trigger for the initiation of air-stepping. However, a2 receptor activation may have initiated air-stepping indirectly by modulating the influence of incoming sensory information, by modulating the influence of descending signals to the spinal cord and by modulating afferent input to brain regions implicated in locomotion. 73 As the rat pups mature, particularly between PD 1 5 and 20, a slight modification of the roles of the receptor subtypes appears to take place. Activation of both receptor subtypes is still necessary for the occurrence of air-stepping, but the air-stepping may be more heavily influenced and easily modified by afferent input. Evidence for similar ontogenetic changes in the neuromodulatory influences of octopamine on the flight circuitry of the locust and of serotonin on the spinal locomotor network of Xenopus [37] suggest that such changes in neuromodulatory influences on rhythmic networks across ontogeny are not uncommon. In the absence of pharmacological intervention, there is a similar fine-tuning of the overground locomotor abilities of rat pups during this same ontogenetic period, a fine-tuning that has also been interpreted in terms of the maturation of sensorimotor integration [28,1 19]. Interactions between the two a noradrenergic receptor subtypes may also have a changing impact on behavior as the pups mature. For example, either increasing the activity of a! noradrenergic receptors from basal levels or decreasing the activity of receptors from unusually high levels increased the occurrence of the airswimming gait in the present research, particularly at PD 20. The increased incidence of airswimming activation, occured only in the presence of simultaneous a, and a2 noradrenergic receptor suggesting that the ratio of activity at these two receptor subtypes becomes increasingly important as the pups mature. A number of recent investigations have demonstrated that combining locomotor receptor stimulation contributes to the recovery of training with a2 noradrenergic locomotor abilities in cats with spinal transections [10,26] and spinal cord-injured 74 humans [12,40]. The results of the present research suggest that noradrenergic receptor activation modulates the locomotor effects of a2 noradrenergic receptor activation in intact rat pups. Future research should thus investigate the influence of interactions between these two receptor subtypes on locomotion in rat pups with spinal transections, with particular emphasis on the effects of afferent input. 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APPENDIX STATISTICS TABLES Table A1 - Cirazoline followed by UK-14,304 F values, degrees of freedom and p values for behaviors with significant drug interactions Age Behavior Cirazoline UK- 14,3 04 Interaction F df P F df P F df P PD 5 Diagonal progression 226.2 1,28 .001 268 1,28 .001 222.8 1,28 .001 Forelimb gallop 12.5 1,28 .001 12.5 1,28 .001 12.5 1,28 .001 Atypical airstep 97.2 1,28 .001 104 1,28 .001 97.2 1,28 .001 Tail extension 28.7 1,28 .001 18 1,28 .001 18 1,28 .001 Forelimb flexion 10.3 1,28 .003 10.3 1,28 .003 10.3 1,28 .003 Relaxed inactivity 134.9 1,28 .001 6.3 1,28 .017 93.7 1,28 .001 Nonstereotyped activity 12.5 1,28 .001 89.9 1,28 .001 20.2 1,28 .001 PD 10 Airswim 14.7 1,30 .001 16.1 1,30 .001 14.6 1,30 .001 Atypical airstep 4.2 1,30 .048 4.9 1,30 .033 4.3 1,30 .044 Extension/tremor 29.3 1,30 .001 10.3 1,30 .003 10.4 1,30 .003 Forelimb flexion 8.9 1,30 .005 8.6 1,30 .006 8.6 1,30 .006 Relaxed inactivity 110.2 1,30 .001 15.7 1,30 .001 27.5 1,30 .001 Nonstereotyped activity 6.3 1,30 .017 15.4 1,30 .001 20.4 1,30 .001 PD 15 Diagonal progression 8.9 1,27 .006 9.7 1,27 .004 19.8 1,27 .001 Airswim 12.2 1,27 .002 12.4 1,27 .001 12.2 1,27 .002 Gallop 11.7 1,27 .002 11.7 1,27 .002 15.8 1,27 .001 Forelimb flexion 6.4 1,27 .016 6.4 1,27 .016 6.4 1,27 .016 Relaxed inactivity 106.2 1,27 .001 24.2 1,27 .001 72.6 1,27 .001 Nonstereotyped activity .01 1,27 91.3 1,27 .001 20.1 1,27 .001 PD 20 Airswim 16.4 1,29 .001 12.6 1,29 .001 11.8 1,29 .002 Atypical airstep 12.4 1,29 .001 15.7 1,29 .001 6.12 1,29 .018 Forelimb flexion 5.3 1,29 .027 5.3 1,29 .027 5.3 1,29 .027 86 87 Table A2 - Prazosin / l-DOPA F values, degrees of freedom and p values for behaviors with significant drug interactions Age Behavior Prazosin L-DOPA Interaction F df P F df P F df P PD 5 Total airstep 21.9 3,55 .001 198 1,55 .001 21.8 3,55 .001 Diagonal progression 64 3,55 .001 173.8 1,55 .001 64 3,55 .001 Forelimb airstep 4.3 3,55 .009 29.9 1,55 .001 4.3 3,55 .009 One-limb airstep 3.1 3,55 .031 11.8 1,55 .001 3.1 3,55 .031 Atypical airstep 17.6 3,55 .001 88.1 1,55 .001 17.6 3,55 .001 Atypical forestep 2.8 3,55 .049 12.4 1,55 .001 22.5 3,55 .049 Extension/tremor 6.5 3,55 .001 62.1 1,55 .001 17.2 3,55 .001 Relaxed inactivity 11.5 3,55 .001 143.5 1,55 .001 12.8 3,55 .001 PD 10 Total airstep 18.2 3,63 .001 71.5 1,63 .001 18.3 3,63 .001 Diagonal progression 18 3,63 .001 57.6 1,63 .001 18.1 3,63 .001 Airswim 4.4 3,63 .007 9.6 1,63 .003 4.4 3,63 .007 Forelimb airstep 3.6 3,63 .017 4.3 1,63 .040 3.8 3,63 .015 Atypical airstep 6.8 3,63 .001 45.4 1,63 .001 6.8 3,63 .001 Relaxed inactivity 21.9 3,63 .001 63.1 1,63 .001 10.9 3,63 .001 Nonstereotyped activity 2.4 3,63 .075 2.3 1,63 .130 5 3,63 .003 PD 15 Total airstep 29.2 3,65 .001 109.3 1,65 .001 22.8 3,65 .001 Diagonal progression 37.4 3,65 .001 88.4 1,65 .001 26.3 3,65 .001 3 -limb airstep 12.7 3,65 .001 33.8 1,65 .001 12.4 3,65 .001 Nonstereotyped activity .8 3,65 2.3 1,65 .129 4.5 3,65 .006 PD 20 Total airstep 3.5 3,74 .020 57.4 1,74 .001 3 3,74 .033 Diagonal progression 18.2 3,74 .001 34.5 1,74 .001 14.7 3,74 .001 3 -limb airstep 3.1 3,74 .033 24.7 1,74 .001 3.1 3,74 .032 Airswim 3.7 3,74 .015 22.9 1,74 .001 4.1 3,74 .009 Gallop 3.7 3,74 .015 4.2 1,74 .040 3.8 3,74 .013 88 Table A3 - Idazoxan / l-DOPA F values, degrees of freedom and p values for behaviors with significant drug interactions Age Behavior Idazoxan L-DOPA Interaction F df P F df P F df p PD 5 Total airstep 35.8 3,56 .001 453 1,56 .001 35.8 3,56 .001 Diagonal progression 37.1 3,56 .001 463.3 1,56 .001 37.1 3,56 .001 Atypical airstep 3.1 3,56 .034 19.9 1,56 .001 3.1 3,56 .034 Extension/tremor 6.3 3,56 .001 136.1 1,56 .001 7.9 3,56 .001 Nonstereotyped activity 10.7 3,56 .001 .1 1,56 16.1 3,56 .001 PD 10 Total airstep 21.5 3,60 .001 112.4 1,60 .001 21.5 3,60 .001 Diagonal progression 16.1 3,60 .001 92.9 1,60 .001 16.1 3,60 .001 Atypical airstep 3.0 3,60 .039 28.1 1,60 .001 3 3,60 .039 Nonstereotyped activity .36 3,60 30.7 1,60 .001 7.1 3,60 .001 PD 15 Total airstep 10.5 3,56 .001 41.5 1,56 .001 9.7 3,56 .001 Diagonal progression 9.5 3,56 .001 38.9 1,56 .001 8.8 3,56 .001 Nonstereotyped activity .08 3,56 2.1 1,56 .149 4.2 3,56 .009 PD 20 Total airstep 14.0 3,66 .001 52.9 1,66 .001 9.2 3,66 .001 Diagonal progression 14.2 3,66 .001 59.4 1,66 .001 10.3 3,66 .001 Atypical airstep 4.4 3,66 .007 24.2 1,66 .001 4.4 3,66 .007 Nonstereotyped activity 1.8 3,66 .162 52.9 1,66 .001 7.8 3,66 .001 BIOGRAPHICAL SKETCH Lynette Leith Taylor was bom in Fort Bragg, North Carolina on November 8, 1968. She graduated from Shades Valley High School in Birmingham, Alabama in 1986. From 1986 to 1990, she majored in biology and minored in English at the University of Alabama at Tuscaloosa, where she received the degree of Bachelor of Science in 1990. She attended the University of Alabama at Birmingham as a non-degree graduate student from 1990 to 1991. In August of 1991, she began graduate school at the University of Florida as a student in the psychobiology area of the Department of Psychology. She was awarded the degree of Master of Science by the University of Florida in December, 1994 and the degree of Doctor of Philosophy in May, 1999. 89 I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Carol Van Hartesveldt, Chairman Professor of Psychology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Michael J. Meldrum Associate Professor of Pharmacodynamics I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Neil E. Rowland Professor of Psychology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Donald J. Stehamver Professor of Psychology This dissertation was submitted to the Graduate Faculty of the Department of Psychology in the College of Liberal Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. May 1 999 Dean, Graduate School