Research

John Walkey (December 2019)

The relationship between electrical brain stimulation and rat vocalizations.

Introduction: Research has found that rats make two categories of ultrasonic vocalizations. Burgdorf et al. (2011) based on a review of the research, states that 22 kHz vocalizations represent negative affect and 50 kHz vocalizations represent positive emotion.

How do rats use 50 kHz vocalizations? Burke et al. (2017) found rats emit more 50 kHz in anticipation of a play partner, suggesting that vocalizations are used for social communication. Knutson et al. (1998) observed that rats emit more 50 kHz USVs during rough-and-tumble play.

Burgdorf et al. (2008) reported 50 kHz vocalizations occur during mating, play, and aggression. He showed that some USVs in rats, such as the flat category, are not associated with reward but the frequency modulated (FM) category of calls (trill, step, etc.) is. Also, of some interest is that male rats emit more 50 kHz calls before ejaculation than after. Brudzynski (2009) reviewed research that looked at play and tickling, both appetitive behaviors, and both were strongly associated with increased 50 kHz vocalizations. Evidence suggests that a whole range of positive events such as tickling, sex, food, play, and stimulant drugs cause rats to emit 50 kHz calls as a way of broadcasting positive affect. Panksepp and Burgdorf (2003) controversially suggested that 50 kHz calls are used to broadcast positive emotions to conspecifics and even went so far as to suggest that that FM 50 kHz USVs are equivalent to human laughter. Brudzynski (2019) suggests that 22 kHz rat vocalizations represent the human equivalent of crying.

Conspicuously absent from current research has been research to identify if specific categories of 50 kHz vocalizations are associated with positive events. Types and timing of calls associated with food reward, drug reward, and with mating was reviewed by Wohr et al. (2007/2008). They report that 50 kHz call patterns occur during or in anticipation of juvenile play, tickling, mating, food consumption, electrical self-stimulation of the brain, and addictive drugs. Simola (2015) reported that 50 kHz call pattern frequency increases in anticipation of many drugs of abuse. White and Barfield (1990) found that both male and female rats emit ultrasonic vocalizations during copulation and suggested that these calls serve as signals to communicate with a prospective mate. Wohr and Schwarting (2013) identified two broad types of 50 kHz vocalizations. Rats in isolation produced 50 kHz flat calls and rats searching for a play partner emitted FM 50 kHz. These observations were tested via playback recordings to show the FM calls induced social approach behavior.

Burke, et al (2017) have stated that it is, “plausible that call types have specific meaning”. Flat calls are the least frequent and trill type calls constitute over 75% of calls. Their research showed that specific call categories were emitted during specific behaviors while rats were waiting for a play partner. This provides evidence that individual calls types have specific meaning. Burgdorf et al. (2008) identified specific calls (trills and step calls) positively correlated with appetitive behaviors of mating, play, and aggression. Specific types of calls have been associated with play and anticipation of play (Knutson, 1998). Wright et al. (2010) identified 14 categories of ultrasonic vocalizations where the subtypes were affected by social context, amphetamine dose, and time within session. The acoustic characteristics of call subtypes were notably stable. The authors suggested that their research demonstrated that the vocalizations emitted by rats served both social inter-rat communication as well as the affective state of the subjects.

To understand the behavioral significance of rat ultrasonic vocalizations, one would need to know both the identity of specific calls as well as their precise timing with respect to a target behavior.  None of the studies, done to date, have provided a clear indication of which and when 50 kHz vocalization patterns occur in appetitive situations relative to rewarding events.

Rat 50 kHz calls are associated with dopamine level changes in the central nervous system. Systemic dopamine agonists or reuptake inhibitors cause an increase in 50 kHz frequency modulated (FM) calls. Dopamine release in the nucleus accumbens is associated with 50 kHz USVs in rats (Burgdorf et al., 2007). Affective experience in rats is expressed relative to the mesolimbic dopaminergic system (Mu et al., 2009). Their research showed that rats bred for higher production of 50 kHz USVs exhibit higher levels of cocaine induced sensitization than rats selectively bred for lower levels of USVs. Ahrens et al. (2009) found that 50-kHz FM USVs are increased by repeated intravenous amphetamine but flat calls were not increased. The elevated number of FM calls, specifically trill calls, compared to flat calls suggests a positive association with dopaminergic levels because amphetamine is a strong dopamine reuptake inhibitor. Further support for a dopamine-vocalization link comes from the fact that 50-kHz USVs are increased over time by repeated amphetamine injections in parallel with increased signs of behavioral activation. Simola et al. (2012) found that methylphenidate, another dopamine reuptake inhibitor, increased the number of 50-kHz USVs emitted by rats but MDMA, morphine, and nicotine did not. Their research shows that stimulants are more influential in eliciting 50 kHz vocalizations in rats than are other drugs. Simola et al. (2010) showed that caffeine does not cause an increase the number of 50 kHz USVs in rats which is evidence that dopamine specifically is not, and not just behavioral activation is key to vocalizations. Further supporting the association between dopamine and 50 kHz vocalizations in rats, Brudzynski et al. (2012) injected Quinpirole, a D2/D3 dopamine agonist, into the shell of the nucleus accumbens and found that it increased 50 kHz vocalizations.

Other studies have shown that decreasing dopamine via antagonists or selective destruction of dopaminergic cells causes a decrease in 50 kHz vocalizations in rats. Burgdorf (2007) showed that both the dopamine D1/D2 antagonist flupenthixol and 6-hydroxydopamine (6-OHDA) lesions reduced 50 kHz vocalizations. In addition, Ciucci (2009) showed that two rat Parkinson’s models, one produced by unilateral infusions of 6-OHDA and the other with a dose of haloperidol (dopamine antagonist), both produce fewer FM calls and more flat calls.

One key locus for the effect of dopamine on vocalizations in the nucleus accumbens.  Burgdorf et al. (2001) demonstrated that microinjections of amphetamine into the nucleus accumbens greatly increased the number of 50 kHz USVs emitted by rats. This effect was further localized to the nucleus accumbens shell rather than core.  This latter finding was confirmed by Thompson et al. (2006), again using microinjections of amphetamine.  These findings are important because they localize the effect of dopamine on vocalizations.

The 50 kHz vocalizations of peer rats can elicit dopamine release in nucleus accumbens. Willuhn et al. (2014) tested the hypothesis that 50 kHz USVs, but not 22 kHz USVs, would elicit nucleus accumbens dopamine release by analysis of subjects who were presented with 50 or 22 kHz playback vocalizations. Their findings show that dopamine release in nucleus accumbens is associated with 50 kHz USVs, but not with the alarming 22 kHz USVs, which supports the idea that the two USV types are processed by separate brain regions.

Dopamine is released both tonically and phasically, with the former often associated with motivation and the latter with learning. Schultz & Dickinson (2000) have suggested that VTA neurons encode reward prediction error. The dopamine reward response is greater the greater the discrepancy between the expected reward and the reward actually received. Initially, in a classical conditioning paradigm, the reward elicits an increased phasic dopamine response (unexpected reward, positive prediction error). During learning, the repeated pairing of the stimulus and the reward elicits an increased dopamine response to the stimulus, which is the first indicator of the upcoming reward amount. Meanwhile, the VTA response to the reward itself decreases because, once the cue stimulus is known, the amount of reward is known and hence there is not reward prediction error. 

Another line of research demonstrates tonic release of dopamine in the ventral striatum tied to both the expectation of reward and response vigor.  Howe et al. (2013) showed that dopamine signals ramp up in rats in expectation of attaining reward. Tonic activity is related to the delay to the reward as well as the size of the reward. They suggest that the tonic dopamine levels are indicative of maintaining a motivational state. Salamone et al. (2016) reviewed research that shows that dopamine is required for effort-based reward choices. Nucleus accumbens lesions lead to lack of willingness to pursue reward in face of physical effort. This idea supports the idea that dopamine levels in nucleus accumbens are related to response vigor. In a recent attempt to reconcile the roles of tonic and phasic dopamine, Berke (2018) pointed out that phasic, reward-prediction signals are seen in the firing of VTA cells whereas measurements of tonic dopamine are typically done in the ventral striatum using methods such as microdialysis.  This raises the possibility that the phasic signal is carried broadly by VTA neuronal projections but tonic dopamine in the striatum is caused by pre-synaptic modulation of the dopaminergic VTA terminals in the striatum.  Hence, dopamine may serve both as a teaching signal for learning as well as a motivational signal based on tonic levels in the ventral striatum.

Electrical stimulation of the medial forebrain bundle is positively reinforcing likely because it stimulates dopamine release. Pioneering work in the study of electrical stimulation of the brain (Olds, 1956) showed that when midbrain nuclei and fiber pathways are electrically stimulated rats prefer stimulation over basic survival drives such as eating and mating. Gallistel et al. (1981) showed that electrical stimulation of the medial forebrain bundle in the rat is both positively reinforcing and motivating. They investigated and described the properties of neural tissue in the medial forebrain bundle whose excitation results in reinforcing and motivating effects in the rat.

Carlson (2009) used microdialysis and found that electrical stimulation of the medial forebrain bundle caused the release of dopamine from the nucleus accumbens. Coenen (2018) analyzed DTI images from human subjects and determined projections from the MFB reward system connect to the PFC and OFC. Wise (1980) reports that the ascending dopamine systems are in highest concentration in the MFB, through self-stimulation the ventral tegmental area and the substantia nigra areas were high is dopamine-containing cells.

As one might expect based on the strong evidence for a link between dopamine and vocalizations, electrical brain stimulation has been linked to increased vocalizations (Burgdorf et al. 2000). On a fixed-time (FT20) schedule, rats were given electrical stimulation of the VTA and the effect was that 50 kHz vocalizations ramped up in anticipation of stimulation. The ramp was seen in anticipation of stimulation as the rats will self-stimulate and the self-simulation can be cued with light for stimulation. This suggests that a conditioning stimulus can be classically conditioned to elicit a positive USV behavior in the absence of the stimulus.

Theory: A specific type of 50 kHz vocalization (to be identified) will increase in frequency depending on tonic dopamine levels in the ventral striatum. 

Prediction: Specific 50 kHz vocalization will increase in occurrence as the time to an expected reward decreases. 

Glickman & Schiff (1967) suggest that reinforcement evolved as a mechanism to insure species-typical responses to appropriate stimuli. Evidence was reviewed which suggests that such response sequences are organized in the brain stem of the mammal. From an evolutionary point of view, the USVs produced by rats, signal positive affective situations associated midbrain neural activities and expressed through brainstem systems.

This thesis seeks to explore the relationship between dopamine and vocalizations by closely examining the temporal relationship between rat 50 kHz vocalizations and MFB stimulation.

Three experimental phases:

1. Uncued stimulation on a fixed-time schedule with a delay of 20 seconds between stimulations
2. Based on Pavlovian conditioning, an unpredictable light will predict the onset of stimulation after a brief delay
3. An instrumental conditioning test where an unpredictable cue will initiate a series of two responses leading to stimulation

The dependent variable is the rate of 50 kHz vocalizations and we will be testing whether vocalizations are time-locked in any way to the time of reward.  An escalation of vocal counts leading up to reward would be consistent with a link between dopamine in ventral striatum and 50 kHz vocalizations.  Vocalizations occurring immediately after reward would be consistent with a link between the phasic, reward-prediction error signal and vocalizations.  Further, if vocalizations are tied to the offset of reward (and hence perhaps tied to a reward-prediction error signal) we would also predict that, just as with the VTA dopamine cell firing rates, during learning, post-reward vocalizations will decrease while vocalizations tied to the offset of the first cue predictive of reward will increase. 

Each experimental stage adds a new feature, allowing better separation of the relationship between reward, cues, and instrumental actions to obtain reward:

• the first condition has no cues and the only predictive information is the periodic timing of the stimulation.  The first condition is also a direct replication of an earlier study by Burgdorf et al., (2000), providing an important point of comparison
• the second condition allows determination if cues predictive of reward can also elicit vocalizations
• the third condition provides a longer sequence of events, allowing better testing of whether vocalizations are tied to primary reward and/or the cues and actions which predict reward.

This thesis will also examine the specific calls associated with rewarding brain stimulation.

50 kHz calls have been linked to reward (Burgdorf et al., 2000 & 2011). Brudzynski et al., 2015 has even suggested that “Emission of 50 kHz vocalizations is signaling to conspecifics a hedonic, positive emotional state labeled in human terms as joy, pleasure, or euphoria”.

This raises the issue of exactly which call is associated with reward, a question which has not been clearly addressed. Many studies have suggested that FM calls are the ones most associated with reward (Brudzynski, 2009; Wright et al., 2010; Simola et al., 2012).

There are many different calls rats make which would be classified as FM. Most of the calls, except the flats and shorts, are in the 50 kHz frequency, hence this distinction is limited and not very informative (Ahrens et al., 2009).

By comparing the specific reward-associated calls from MFB stimulation, to those reported in studies of other types of reward, we will be able to address the larger question, is there a specific call, or group of calls, associated with reward.

Testing Chamber Design Considerations:  Why have nose-pokes that signal reward availability?  We don’t want the animal to dwell next to a single port.  Instead, we want him to be watching and waiting for one of the two to become active, then he must make his way over to that port and poke.  This gives us a delay between cue onset and the time when he makes the response during which we can look for specific vocalizations.

Figure 1. Lighted nose-poke switches (A, B, C) housed in a plexiglass operant chamber which is inside a sound-proof exterior chamber. Above the box are an ultrasonic microphone (W), Video recording camera (X), commutator to connect electrodes to subject allowing free movement (Y), and a LED light and beeper to signal when a trial starts and ends (Z). Top down view.

Electrode Conformation

After behavioral testing the subjects will be perfused and histology will be performed with brain tissue stained with cresyl violet to identify the location of simulation electrodes.

Vocalization Results

Vocalizations will be scored using the DeepSqueak software in Matlab to identify call categories. Frequency modulated call patterns (trills, and trill with jumps, etc.) will be plotted using pre-event time histogram (PETH) in Matlab. This histogram will show the rate and timing of the vocalization patterns in relation to the stimulation event times.

Future Directions

Dopamine agonists and antagonists could be used to confirm the role of dopamine, exploration of events tied to 22 kHz calls using a fear conditioning paradigm.

References

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