The laterodorsal tegmentum is essential for burst firing of ventral tegmental area dopamine neurons D. J. Lodge* and A. A. Grace Departments of Neuroscience, Psychiatry, and Psychology, University of Pittsburgh, Pittsburgh, PA 15260 Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved February 8, 2006 (received for review December 12, 2005) In response to behaviorally salient stimuli, dopamine (DA) neurons colocalized (19, 20). Given the significant and divergent afferent fire in bursts. Burst firing induces a large transient increase in input from the PPTg and LDTg nuclei to the VTA and their synaptic DA and is regarded as the functionally relevant mode of potential role in the generation of burst firing, the aim of this transmission that signals reward and modulates goal-directed study was to examine the effect of activation and inactivation of behavior. DA neuron burst firing is dynamically regulated by these afferent inputs on the activity of the population of DA afferent inputs, and it is not present in vitro because of severing of neurons that are located in the VTA. afferent processes. However, what afferents are requisite for burst firing in vivo is not known. Here, we show that tonic input from the Results laterodorsal tegmental nucleus (LDTg) is required for glutamate- The localization of injection sites throughout the mesopontine elicited burst firing in ventral tegmental area DA neurons of tegmentum are shown in Fig. 1. Modulation of the LDTg was anesthetized rats. Also, after LDTg inactivation, DA neurons fire as done pharmacologically by drug infusion to provide a stable they do in vitro (i.e., as pacemakers); even direct glutamate appli- alteration in activity levels. The specificity and selectivity of cation fails to cause them to burst fire under these conditions. infusions were shown by comparing the actions of NMDA or These data show that the LDTg is critical to normal DA function, baclofen͞muscimol with that of cholinergic agonists and an- and thus, pathology within this region may lead to aberrant DA tagonist that have been reported to modulate LDTg output by signaling. modulation of inhibitory autoreceptors (21, 22). Therefore, activation of M2-like receptors by the cholinergic agonist electrophysiology ͉ laterodorsal tegmental nucleus ͉ firing pattern ͉ carbachol will decrease LDTg output and produce an effect limbic system equivalent to that produced by baclofen͞muscimol-induced inactivation. Conversely, antagonism of tonically activated esolimbic dopamine (DA) neurotransmission is mediated autoreceptors by the muscarinic receptor antagonist scopol- Mby the following two independent mechanisms: extrasyn- amine will increase LDTg output, producing an effect that is aptic (or ‘‘tonic’’) DA is mediated by basal DA neuron activity equivalent to that produced by NMDA-induced activation. ␮ and regulated by presynaptic inputs, and transient (or ‘‘phasic’’) Rats that received control vehicle infusions (0.5 l of Dul- ϭ DA release is mediated by burst firing (1, 2). Thus, DA neuron becco’s PBS; n 6 rats; 50 neurons) into the LDTg exhibited an Ϯ burst firing induces a large transient increase in synaptic DA (3) average of 1.12 0.1 spontaneously active DA neurons per electrode track that fired at an average rate of 4.5 Ϯ 0.3 Hz and and is considered to be the functionally relevant signal sent to Ϯ postsynaptic sites to encode reward prediction or indicate in- with 26 4% of action potentials fired in bursts (Fig. 2 A–C), which is consistent with the findings in refs. 2 and 16. Intra-LDTg centive salience (1, 4–6). ␮ ␮ ϭ DA neurons that are recorded in vivo typically display distinct infusion of NMDA (0.75 g per 0.5 l; n 6 rats; 78 neurons) or the muscarinic receptor antagonist scopolamine (50 ␮g per patterns of activity, including an irregular firing rate ranging ␮ ϭ Ϸ 0.5 l; n 6 rats; 71 neurons) to excite LDTg neurons resulted NEUROSCIENCE from 2 to 10 Hz (average, 4 Hz) with spontaneous burst events Ϸ Ͻ (7). The transition from irregular single-spike firing to burst in a significant ( 2-fold; P 0.05) increase in DA neuron firing depends on an excitatory amino acid because activation of population activity (defined as number of spontaneously active glutamatergic afferents or direct microiontophoretic application DA neurons observed per electrode track; Fig. 2A) without of glutamate induces burst firing in DA neurons in vivo (2, 8, 9). significantly affecting average burst firing or firing rate relative Also, direct application of the competitive NMDA receptor to control (Fig. 2 B and C). antagonists (Ϯ)2-amino,5-phosphonopentanoic acid or (Ϯ)4-(3- Surprisingly, inactivation of the LDTg by coinfusion of the phosphonopropyl)-2-piperazine carboxylic acid potently block GABAA and GABAB agonists muscimol and baclofen, respec- tively (0.2 ␮g each per 0.5 ␮l; n ϭ 6 rats; 52 neurons) or the spontaneous burst firing (10, 11). Therefore, NMDA receptor ␮ ␮ ϭ stimulation by glutamate has a primary role in regulating burst cholinergic agonist carbachol (4 g per 0.5 l; n 6 rats; 47 firing in vivo. However, glutamate alone is not sufficient to neurons) resulted in a small but significant reduction in firing rate (Fig. 2B) but a dramatic reduction in burst firing by 80–90% mediate burst firing. Thus, DA neurons recorded from mesen- ͞ cephalic slices obtained from adult rats, in which afferent input (Fig. 2C). After baclofen muscimol inactivation of the LDTg, has been severed, display a regular pacemaker firing pattern (12) significant burst firing of midbrain DA neurons was observed and cannot be made to fire in bursts in response to glutamate only in 4 of 52 neurons. Also, the characteristics of the bursts remaining after baclofen͞muscimol inactivation was altered in agonist administration or alterations in membrane potential that there was a decrease in the average number of spikes per (12–15). There are several prominent inputs to the ventral tegmental area (VTA) that arrive from the tegmentum. The pedunculo- Conflict of interest statement: No conflicts declared. pontine tegmental nucleus (PPTg) is one such input, a gluta- This paper was submitted directly (Track II) to the PNAS office. ͞ matergic cholinergic neuronal group that has been shown to Abbreviations: DA, dopamine; ISI, interspike interval; LDTg, laterodorsal tegmental nu- drive burst firing in VTA neurons (2, 16). Another input arises cleus; PPTg, pedunculopontine tegmental nucleus; VTA, ventral tegmental area; A͞P, from the laterodorsal tegmental nucleus (LDTg) and associated anterior͞posterior; PFC, prefrontal cortex. CH6 cell group (17, 18). This input contains glutamate as well *To whom correspondence should be addressed. E-mail: [email protected]. as cholinergic and GABAergic components, many of which are © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510715103 PNAS ͉ March 28, 2006 ͉ vol. 103 ͉ no. 13 ͉ 5167–5172 Downloaded by guest on September 28, 2021 Given the prominent role for glutamatergic transmission in the regulation of burst firing in vivo, we examined the effect of the simultaneous activation of the PPTg (a glutamatergic͞ cholinergic region that potently induces burst firing in VTA DA neurons) (2, 16) and LDTg inactivation. Activation of the PPTg by NMDA infusion (0.75 ␮g per 0.5 ␮l) reliably induces a substantial increase in burst firing (to Ϸ45%) (2, 16). However, after inactivation of the LDTg, activation of the PPTg (n ϭ 6 rats; 48 neurons) failed to significantly affect any parameter of DA neuron activity compared with LDTg inactivation alone (Fig. 2D), suggesting that the decreased burst firing observed after LDTg inactivation is not simply a reflection of a decrease in glutamatergic tone. Direct application of glutamate is known to potently increase DA neuron burst firing in vivo but not in vitro. During glutamate microiontophoresis (Ϫ2.5 to Ϫ40 nA), there was a substantial Fig. 1. Localization of cannula placements throughout the PPTg and LDTg. increase in the firing rate and burst firing of individual DA The shaded area represents the range of intracranial cannula tip placements. neurons recorded in control animals (n ϭ 6 rats; 10 neurons; Fig. Numbers beside each plate represent the approximate A͞P distance from 4 A and B), which is consistent with the findings in ref. 9. After bregma. LDTg inactivation by baclofen͞muscimol (n ϭ 11; 26 neurons), the microiontophoretic application of glutamate still produced a significant increase in DA neuron firing rate with a similar burst (control, 3.62 Ϯ 0.04; baclofen͞muscimol, 2.69 Ϯ 0.07; P Ͻ potency to that seen in control animals (Fig. 4C). However, 0.05). In addition to the changes in burst firing, DA neurons recorded after baclofen͞muscimol administration showed a glutamate microiontophoresis did not increase burst firing in DA highly regular pacemaker-like firing pattern, which was defined neurons after LDTg inactivation (Fig. 4D). Results are sum- by a significant decrease in the coefficient of variation (CoV) of marized in histogram form in Fig. 4 E and F. It is important to the interspike interval (ISI) (CoV: control, 0.72 Ϯ 0.06; ba- note that burst firing was observed͞induced in 3 of 26 neurons, clofen͞muscimol, 0.32 Ϯ 0.03; P Ͻ 0.05) (Fig. 3). Pacemaker which is consistent with the number of neurons exhibiting firing is commonly observed in DA neurons in vitro, but it is baseline bursting after LDTg inactivation alone. Control appli- rarely observed in vivo. cation of saline (Ϫ10 nA) did not significantly alter DA neuron Fig. 2. LDTg manipulations alter the activity states of VTA DA neurons. NMDA (0.75 ␮g per 0.5 ␮l), baclofen͞muscimol (0.2 ␮g each per 0.5 ␮l), carbachol (4 ␮g per 0.5 ␮l), scopolamine (50 ␮g per 0.5 ␮l), or vehicle (Dulbecco’s PBS) were injected into the LDTg, and the activity of spontaneously active VTA DA neurons was examined.