Laura M. Grant

Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system.

The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 1013) or varying titers (2.2 x 1012, 1.0 x 1013, 5.9 x 1013, or 1.0 x 1014) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 1014 titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 1013 titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 1013 and 1.0 x 1014 rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 1013 rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.

Early identification and treatment of communication and swallowing deficits in Parkinson disease.

Parkinson disease (PD) is a complex, progressive, neurodegenerative disorder that leads to a wide range of deficits including fine and gross sensorimotor impairment, autonomic dysfunction, mood disorders, and cognitive decline. Traditionally, the focus for diagnosis and treatment has been on sensorimotor impairment related to dopamine depletion. It is now widely recognized, however, that PD-related pathology affects multiple central nervous system neurotransmitters and pathways. Communication and swallowing functions can be impaired even in the early stages, significantly affecting health and quality of life. The purpose of this article is to review the literature on early intervention for communication and swallowing impairment in PD. Overarching themes were that (1) studies and interpretation of data from studies in early PD are limited; (2) best therapy practices have not been established, in part due to the heterogeneous nature of PD; and (3) as communication and swallowing problems are pervasive in PD, further treatment research is essential.

Dopamine D1 and D2 receptor antagonism effects on rat ultrasonic vocalizations.

Voice disorders manifest in the early stages of Parkinson disease (PD), suggesting the vulnerability of the laryngeal sensorimotor system to mild alterations in dopamine signaling. Previous research has demonstrated that manipulations of central dopamine result in acoustic changes in rat ultrasonic vocalization (USV) and selective manipulation of receptor subtypes results in dose dependent changes in call rate and complexity. However, no study has specifically focused on the influence of dopamine receptor subtypes on acoustic features of USV production. This study examined the influence of D1 and D2 receptor subtypes on voluntary laryngeal sensorimotor control (USV) and gross whole-body involvement. Rat USV acoustics and catalepsy descent time were analyzed following the administration of selective D1 and D2 receptor antagonists in isolation and in combination, and a vehicle control. Results support the hypothesis that degradations of the acoustic signal would be most severe following combined receptor antagonism (D1+D2) compared with D1 or D2 receptor antagonism alone, and the vehicle (saline) condition. In addition, results indicate that selective D1 receptor antagonism alters acoustic parameters to a greater extent than D2 receptor antagonism. Thus, dopamine receptor subtypes appear to influence acoustic parameters to different degrees. Catalepsy descent time was longest following combined dopamine receptor antagonism but was also significantly increased with selective D1 or D2 antagonism. Together, these results support the potentially different contributions receptor subtypes play in cranial and limb sensorimotor control.

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations. The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training. To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 μg of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging). Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1). An ultrasonic microphone mounted above the males home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Evidence for early and progressive ultrasonic vocalization and oromotor deficits in a PINK1 gene knockout rat model of Parkinson's disease

Parkinsons disease (PD) is a progressive neurodegenerative disease that leads to a wide range of motor and nonmotor deficits. Specifically, voice and swallow deficits manifest early, are devastating to quality of life, and are difficult to treat with standard medical therapies. The pathological hallmarks of PD include accumulation of the presynaptic protein α‐synuclein (αSyn) as well as degeneration of substantia nigra dopaminergic neurons. However, there is no clear understanding of how or when this pathology contributes to voice and swallow dysfunction in PD. The present study evaluates the effect of loss of function of the phosphatase and tensin homolog‐induced putative kinase 1 gene in rats (PINK1–/–), a model of autosomal recessive PD in humans, on vocalization, oromotor and limb function, and neurodegenerative pathologies. Behavioral measures include ultrasonic vocalizations, tongue force, biting, and gross motor performance that are assayed at 2, 4, 6, and 8 months of age. Aggregated αSyn and tyrosine hydroxylase immunoreactivity (TH‐ir) were measured at 8 months. We show that, compared with wild‐type controls, PINK1–/– rats develop 1) early and progressive vocalization and oromotor deficits, 2) reduced TH‐ir in the locus coeruleus that correlates with vocal loudness and tongue force, and 3) αSyn neuropathology in brain regions important for cranial sensorimotor control. This novel approach of characterizing a PINK1–/– genetic model of PD provides the foundational work required to define behavioral biomarkers for the development of disease‐modifying therapeutics for PD patients.

Vocalization Deficits in Mice Over-Expressing Alpha-Synuclein, a Model of Pre-Manifest Parkinson's Disease

Communication and swallowing deficits are common in Parkinsons disease (PD). Evidence indicates that voice and speech dysfunction manifest early, prior to motor deficits typically associated with striatal dopamine loss. Unlike deficits in the extremities, cranial sensorimotor deficits are refractory to standard dopamine-related pharmacological and surgical interventions, thus the mechanisms underlying vocal deficits are unclear. Although neurotoxin models have provided some insight, they typically model nigrostriatal dopamine depletion and are therefore limited. Widespread alpha-synuclein (aSyn) pathology is common to familial and sporadic PD, and transgenic mouse models based on aSyn overexpression present a unique opportunity to explore vocalization deficits in relation to extrastriatal, nondopaminergic pathologies. Specifically, mice overexpressing human wild-type aSyn under a broad neuronal promoter (Thy1-aSyn) present early, progressive motor and nonmotor deficits starting at 2-3 months, followed by parkinsonism with dopamine loss at 14 months. We recorded ultrasonic vocalizations from Thy1-aSyn mice and wild-type (WT) controls at 2-3, 6-7, and 9 months. Thy1-aSyn mice demonstrated early, progressive vocalization deficits compared with WT. Duration and intensity of calls were significantly reduced and call profile was altered in the Thy1-aSyn mice, particularly at 2-3 months. Call rate trended toward a more drastic decrease with age in the Thy1-aSyn mice compared with WT. Alpha-synuclein pathology is present in the periaqueductal gray and may underlie the manifestation of vocalization deficits. These results indicate that aSyn overexpression can induce vocalization deficits at an early age in mice and provides a new model for studying the mechanisms underlying cranial sensorimotor deficits and treatment interventions for PD.

Changes in Rat 50-kHz Ultrasonic Vocalizations During Dopamine Denervation and Aging: Relevance to Neurodegeneration.

Vocal communication is negatively affected by neurodegenerative diseases, such as Parkinson disease, and by aging. The neurological and sensorimotor mechanisms underlying voice deficits in Parkinson disease and aging are not well-understood. Rat ultrasonic vocalizations provide a unique behavioral model for studying communication deficits and the mechanisms underlying these deficits in these conditions. The purpose of this review was to examine the existing literature for methods using rat ultrasonic vocalization with regard to the primary disease pathology of Parkinson disease, dopamine denervation, and aging. Although only a small amount of papers were found for each of these topics, results suggest that both shared and unique acoustic deficits in ultrasonic vocalizations exist across conditions and that these acoustic deficits are due to changes in either dopamine signaling or denervation and in aging models changes to the nucleus ambiguus, at the level of the neuromuscular junction, and the composition of the vocal folds in the larynx. We conclude that ultrasonic vocalizations are a useful tool for studying biologic mechanisms underlying vocal communication deficits in neurodegenerative diseases and aging.

Relationships among rat ultrasonic vocalizations, behavioral measures of striatal dopamine loss, and striatal tyrosine hydroxylase immunoreactivity at acute and chronic time points following unilateral 6-hydroxydopamine-induced dopamine depletion.

Voice deficits in Parkinson disease (PD) emerge early in the disease process, but do not improve with standard treatments targeting dopamine. Experimental work in the rat shows that severe and chronic unilateral nigrostriatal dopamine depletion with 6-OHDA results in decreased intensity, bandwidth, and complexity of ultrasonic vocalizations. However, it is unclear if mild/acute dopamine depletion, paralleling earlier stages of PD, results in vocalization deficits, or to what degree vocalization parameters are correlated with other dopamine-dependent indicators of lesion severity or percent of tyrosine hydroxylase (%TH) loss. Here, we assayed ultrasonic vocalizations, forelimb asymmetry, and apomorphine rotations in rats with a range of unilateral dopamine loss resulting from 6-OHDA or vehicle control infusions to the medial forebrain bundle at acute (72 h) and chronic (4 weeks) time points post-infusion. The %TH loss was evaluated at 4 weeks. At 72 h, forelimb asymmetry and %TH loss were significantly correlated, while at 4 weeks, all measures of lesion severity were significantly correlated with each other. Call complexity was significantly correlated with all measures of lesion severity at 72 h but only with %TH loss at 4 weeks. Bandwidth was correlated with forelimb asymmetry at both time points. Duration was significantly correlated with all dopamine depletion measures at 4 weeks. Notably, not all parameters were affected universally or equally across time. These results suggest that vocalization deficits may be a sensitive index of acute and mild catecholamine loss and further underscores the need to characterize the neural mechanisms underlying vocal deficits in PD.

Noradrenergic receptor modulation influences the acoustic parameters of pro-social rat ultrasonic vocalizations.

Rats produce high rates of ultrasonic vocalizations (USVs) in social situations; these vocalizations are influenced by multiple neurotransmitter systems. Norepinephrine (NE) plays a significant role in vocalization biology; however, the contribution of NE to normal, prosocial vocal control has not been well established in the rat. To address this, we used NE adrenoceptor agonists (Cirazoline, Clonidine) and antagonists (Prozasin, Atipamezole, Propranolol) to quantify the contribution of specific alpha-1, alpha-2, and beta NE receptors to USV parameters in male Long Evans rats during seminaturalistic calling. We found that multiple USV acoustic variables (intensity, bandwidth, duration, peak frequency, and call profile) are modified by alterations in NE signaling. Very generally, agents that increased NE neurotransmission (Atipamezole) or activated alpha-1 receptors (Cirazoline), led to an increase in intensity and duration, respectively. Agents that decreased NE neurotransmission (Clonidine) or blocked alpha-1 receptors (Prazosin) reduced call rate, intensity, and bandwidth. However, the beta-receptor antagonist, Propranolol, was associated with increased call rate, duration, and intensity. Limb motor behaviors were largely unaffected by any drug, with the exception of Clonidine. Higher doses of Clonidine significantly reduced gross motor, grooming, and feeding behavior. These results confirm the involvement of NE transmission in vocal control in the rat, and suggest that this USV model is useful for studying the neuropharmacology of behavioral measures that may have implications for disease states, such as Parkinson’s disease.