Cellular composition and circuit organization of the locus coeruleus of adult mice
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Evaluation Summary:
Recent studies of the brainstem locus coeruleus (LC) noradrenaline system have demonstrated a partially modular organization in which specific classes of neurons can serve distinct functions or exhibit module-specific co-activity. However, how noradrenaline cell classes function in a modular way is not clear. The authors have accomplished a technical feat by recording up to eight LC neurons at once using ex-vivo, multi-patch recordings. In doing so, two empirically-derived classes of LC neurons were identified and the analysis of electrical coupling between these neurons established some principles of local circuit communication occurring preferentially within the defined cell classes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The locus coeruleus (LC) houses the vast majority of noradrenergic neurons in the brain and regulates many fundamental functions, including fight and flight response, attention control, and sleep/wake cycles. While efferent projections of the LC have been extensively investigated, little is known about its local circuit organization. Here, we performed large-scale multipatch recordings of noradrenergic neurons in adult mouse LC to profile their morpho-electric properties while simultaneously examining their interactions. LC noradrenergic neurons are diverse and could be classified into two major morpho-electric types. While fast excitatory synaptic transmission among LC noradrenergic neurons was not observed in our preparation, these mature LC neurons connected via gap junction at a rate similar to their early developmental stage and comparable to other brain regions. Most electrical connections form between dendrites and are restricted to narrowly spaced pairs or small clusters of neurons of the same type. In addition, more than two electrically coupled cell pairs were often identified across a cohort of neurons from individual multicell recording sets that followed a chain-like organizational pattern. The assembly of LC noradrenergic neurons thus follows a spatial and cell-type-specific wiring principle that may be imposed by a unique chain-like rule.
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Author Response
Reviewer #1 (Public Review):
This is timely and foundational work that links cellular neurophysiology with extracellular single-unit recordings used to study LC function during behavior.
The strengths of this paper include:
- Providing an updated assessment of LC cell morphology and cell types since much of the prior work was completed in the late 1970s and early to mid-1980s.
- Connecting LC cell morphology with membrane properties and action potential shape.
- Showing that neurons of the same type have electrical coupling
Collectively, these findings help to link LC neuron morphology and firing properties with recent work using extracellular recordings that identify different types of LC single units by waveform shape.
Another strength of this work is that it addresses recent findings suggesting the LC neurons …
Author Response
Reviewer #1 (Public Review):
This is timely and foundational work that links cellular neurophysiology with extracellular single-unit recordings used to study LC function during behavior.
The strengths of this paper include:
- Providing an updated assessment of LC cell morphology and cell types since much of the prior work was completed in the late 1970s and early to mid-1980s.
- Connecting LC cell morphology with membrane properties and action potential shape.
- Showing that neurons of the same type have electrical coupling
Collectively, these findings help to link LC neuron morphology and firing properties with recent work using extracellular recordings that identify different types of LC single units by waveform shape.
Another strength of this work is that it addresses recent findings suggesting the LC neurons may release glutamate by showing that, at least within the LC, there is no local glutamatergic excitatory transmission.
Weaknesses:
The authors also propose to test the role of single LC neuron activity in evoking lateral inhibition, as well as proposing that electrical coupling between LC cell pairs is organized into a train pattern. The former point is based on a weak premise and the latter point has weak support in their data given the analyses performed.
Point 1: lateral inhibition in the LC
The authors write in the abstract that "chemical transmission among LC noradrenergic neurons was not detected" and this was a surprising claim given the wealth of prior evidence supporting this in vitro and in vivo (Ennis & Aston-Jones 1986. Brain Res 374, 299-305; Aghajanian, Cedarbaum & Wang 1977. Brain Res 136, 570-577; Cedarbaum & Aghajanian. 1978 Life Sci 23, 1383-1392).
Huang et al. 2007 (Huang et al. 2007. Proc National Acad Sci 104, 1401-1406) showed that local inhibition in the LC is highly dependent on the frequency of action potentials, such that local release requires multiple APs in short succession and then requires some time for the hyperpolarization to appear (even over 1 sec). This work suggests that it is not a "concentration issue" per se, rather it is just that a single AP will not cause local NE release in the LC. Although the authors did try 5APs at 50Hz this may not be enough to generate local NE release according to this prior work. A longer duration may be needed. Additionally, although the authors incubated the slices with a NET inhibitor, that will not increase volume transmission unless there is actually NE release, which may have not happened under the conditions tested. In sum, there is no reason to expect that a single AP from one neuron would cause an immediate (within the 100 msec shown in Fig 3B) hyperpolarization of a nearby neuron. Therefore, the premise of the experiment that driving one neuron to fire one AP (or even 5AP's at 50Hz in some) is not an actual test of lateral inhibition mediated by NE volume neurotransmission in the LC. Strong claims that "chemical transmission...was not detected" require substantial support and testing of a range of AP frequencies and durations. Given the wealth of evidence supporting lateral inhibition of the LC, this claim seems unwarranted.
We thank the reviewers for their constructive comments and interpretations of the data regarding lateral inhibition. In fact, we were fully aware of the prior wealth of data supporting the existence of lateral inhibition and have discussed possible reasons for the absence of lateral inhibition in our dataset. Now both reviewers provided additional potential explanations for this absence. The most plausible explanation appears to be that α2AR-mediated lateral inhibition is a population phenomenon, which would not be readily detected at the single-cell level in in vitro conditions. As reviewers suggested, we may need to vary spike frequency and timing to identify optimal spiking parameters (or stimulating multiple LC neurons at one time) to detect this phenomenon in slices. Alternatively, we could employ other approaches (optogenetic or chemogenetic approach) to activate a group of neurons at one time to evoke this phenomenon, as a recent preprint paper demonstrated (Line 528-535). All these are excellent suggestions, but it may take more than six months to complete these experiments since we need to train another person from scratch for LC recordings (the first author graduated from the program and has left the lab). We thus chose to remove most of the data (about α2AR-mediated lateral inhibition) from the paper in the revision, as the reviewers suggested. We do plan to further explore this interesting topic in our next study.
Point 2: Train-like connection pattern
Demonstrating that connected cell pairs often share a common member is an important demonstration of a connection motif in the LC. However, a "train" connection implies that you can pass from A to B to C to D (and in reverse). However, the authors do not do an analysis to test whether this occurs. Therefore, "train" is not an appropriate term to describe the interesting connection motif that they observed.
In fact, writing A↔B↔C in the paper implies a train without direct support for that form of electrical transmission. For example, in Fig. 6C, it is clear that cell 6 is coupled to cell 1 and that cell 6 is also coupled to cell 8. In both cases, the connection is bilateral. Using the author's formatting of A↔B↔C , would correspond with Cell 6 being B and cells 1 and 8 being A and C (or vice versa). However, writing A↔B↔C implies a train, whereas one can instead draw this connection pattern where B is a common source:
A C
. .
. .
B
An analysis showing that spikes in A can pass through B and later appear in C is necessary to support the use of "train". The example in Fig. 6C argues against train at least for this one example.
Although the analysis is possible to do with the authors' substantial and unique data set, it should be also noted that prior work on putative electrical coupling in extracellular recordings from rat LC showed that trains among 3 single units occurred at an almost negligible rate because out of 12 rats "Only 1 triplet out of 22,100 possible triplet patterns (0.005%) was found in one rat and 4 triplets out of 1,330 possible triplet patterns (0.301%) were found in the other rat." and moreover patterns beyond 3 units were never observed (Totah et al 2018. Neuron 99, 1055-1068.e6). We thank the reviewer for this astute argument and agree that the word “train-like connection” assumes a physiological, functional relationship A→B→C which the data do not show. Therefore, we now term these connections as “chain-like” to indicate the structural nature of the connection, which we believe leaves no room for the interpretation that there is a functional, physiological connection among the three neurons. In fact, we have discussed this issue as a first-order vs second-order coupling issue in our original manuscript (Line 632-639), and concluded that electrical signals hardly pass through the second-order gap junctions in LC, that is, in those two connections sharing the same partner like A↔B↔C (here A and C are not directly connected, but coupled in the second-order), spikes in A hardly pass-through B and later appear in C (Line 632-639).
Reviewer #2 (Public Review):
McKinney et al set out to better understand local circuit organization within the mouse locus coeruleus (LC). To do so, the authors achieved the technical feat of performing multiple, simultaneous whole-cell recordings (up to 8 LC neurons at once). This approach gives the authors a powerful and relatively high throughput means of assessing LC neuronal activity and potentially its rate of interconnectedness. In addition to recording from these cells, many were also filled with biocytin to recover their morphology. Using traditional reconstruction approaches the authors identified two morphological classes of LC neurons, fusiform(FF) and multipolar (MP). Although the selection of these classes was biased from previous literature, the authors used machine classification to rigorously demonstrate that these classes indeed exist. From there, the electrical properties of these distinct LC neurons were compared and a number of distinct action potential properties were identified between the two groups. Although firing in response to injected current showed that the FF class could maintain a higher firing rate, basal firing was not explicitly compared as the cells were prevented from firing upon entering whole-cell. The authors next explored the extent to which local chemical transmission occurs within the LC. Although there is evidence of glutamatergic transmission from LC neurons, the authors did not directly observe any evidence of local glutamate release from these neurons. This effect might be expected given the severing of axons in the slice preparation. Somewhat less expected is the author's claim that they could not find evidence of local NE release via alpha2 adrenergic receptor activation. This lack of evidence might well arise because this phenomenon does not occur, but it also remains possible that we do not have sufficient understanding of volume transmission to properly detect a change, particularly in whole-cell current clamp. The evidence that alpha2-mediated hyperpolarization is intact is somewhat adjacent to the concept as the concentrations of NE and clonidine used to show this robust suppression of firing is well above what is likely physiologically released by these neurons. One thing the authors do not consider is that the slice orientation (horizontal vs. coronal) greatly alters local glutamatergic input to the point that glutamate-mediated phasic bursts often do not occur in horizontal slices.
A major strength of the multi-patch approach used here is the ability to identify electrical connections between LC neurons. While gap junction-coupling has long been established in these neurons, multiple reports suggest that this coupling is decreased as the animal matures into adulthood. Here the authors provide clear evidence for a stable rate of electrical coupling well into adulthood. This approach also gives the authors the relatively unique ability to look for second-order connections between LC neurons and the amount of coupling was elegantly used to model how the LC might wire together more broadly. Although this approach is very powerful and likely at the edge of what is physically possible for whole-cell recordings in this brain structure it still likely undersamples LC local circuitry and biases investigations to be relatively close to one another spatially. While the authors rightfully consider the intersoma distance (ISD), the longest the gets in these studies is still smaller than most anatomical axes of the LC. This is an important limitation because the electrical coupling between FF-FF and FF-MP both appear to increase as ISD increases, suggesting more coupling could be occurring in distal dendrites. Furthermore, if coupling is occurring in distal dendrites it may be harder to detect as shunting in these distal dendrites could prevent signal detection.
This work is timely and important to the LC field which is on the precipice of having a greater understanding of heterogeneity based on a number of different principles, and this work adds local circuit dynamics as one of these principles. It will be important for the field to see how different efferent anatomical modules align with the cell types and circuit properties identified here.
We appreciate the reviewer’s constructive comments and suggestions.
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Evaluation Summary:
Recent studies of the brainstem locus coeruleus (LC) noradrenaline system have demonstrated a partially modular organization in which specific classes of neurons can serve distinct functions or exhibit module-specific co-activity. However, how noradrenaline cell classes function in a modular way is not clear. The authors have accomplished a technical feat by recording up to eight LC neurons at once using ex-vivo, multi-patch recordings. In doing so, two empirically-derived classes of LC neurons were identified and the analysis of electrical coupling between these neurons established some principles of local circuit communication occurring preferentially within the defined cell classes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
Recent studies of the brainstem locus coeruleus (LC) noradrenaline system have demonstrated a partially modular organization in which specific classes of neurons can serve distinct functions or exhibit module-specific co-activity. However, how noradrenaline cell classes function in a modular way is not clear. The authors have accomplished a technical feat by recording up to eight LC neurons at once using ex-vivo, multi-patch recordings. In doing so, two empirically-derived classes of LC neurons were identified and the analysis of electrical coupling between these neurons established some principles of local circuit communication occurring preferentially within the defined cell classes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
This is timely and foundational work that links cellular neurophysiology with extracellular single-unit recordings used to study LC function during behavior.
The strengths of this paper include:
1. Providing an updated assessment of LC cell morphology and cell types since much of the prior work was completed in the late 1970s and early to mid-1980s.
2. Connecting LC cell morphology with membrane properties and action potential shape.
3. Showing that neurons of the same type have electrical couplingCollectively, these findings help to link LC neuron morphology and firing properties with recent work using extracellular recordings that identify different types of LC single units by waveform shape.
Another strength of this work is that it addresses recent findings suggesting the LC neurons may release glutamate …
Reviewer #1 (Public Review):
This is timely and foundational work that links cellular neurophysiology with extracellular single-unit recordings used to study LC function during behavior.
The strengths of this paper include:
1. Providing an updated assessment of LC cell morphology and cell types since much of the prior work was completed in the late 1970s and early to mid-1980s.
2. Connecting LC cell morphology with membrane properties and action potential shape.
3. Showing that neurons of the same type have electrical couplingCollectively, these findings help to link LC neuron morphology and firing properties with recent work using extracellular recordings that identify different types of LC single units by waveform shape.
Another strength of this work is that it addresses recent findings suggesting the LC neurons may release glutamate by showing that, at least within the LC, there is no local glutamatergic excitatory transmission.
Weaknesses:
The authors also propose to test the role of single LC neuron activity in evoking lateral inhibition, as well as proposing that electrical coupling between LC cell pairs is organized into a train pattern. The former point is based on a weak premise and the latter point has weak support in their data given the analyses performed.Point 1: lateral inhibition in the LC
The authors write in the abstract that "chemical transmission among LC noradrenergic neurons was not detected" and this was a surprising claim given the wealth of prior evidence supporting this in vitro and in vivo (Ennis & Aston-Jones 1986. Brain Res 374, 299-305; Aghajanian, Cedarbaum & Wang 1977. Brain Res 136, 570-577; Cedarbaum & Aghajanian. 1978 Life Sci 23, 1383-1392).Huang et al. 2007 (Huang et al. 2007. Proc National Acad Sci 104, 1401-1406) showed that local inhibition in the LC is highly dependent on the frequency of action potentials, such that local release requires multiple APs in short succession and then requires some time for the hyperpolarization to appear (even over 1 sec). This work suggests that it is not a "concentration issue" per se, rather it is just that a single AP will not cause local NE release in the LC. Although the authors did try 5APs at 50Hz this may not be enough to generate local NE release according to this prior work. A longer duration may be needed. Additionally, although the authors incubated the slices with a NET inhibitor, that will not increase volume transmission unless there is actually NE release, which may have not happened under the conditions tested. In sum, there is no reason to expect that a single AP from one neuron would cause an immediate (within the 100 msec shown in Fig 3B) hyperpolarization of a nearby neuron. Therefore, the premise of the experiment that driving one neuron to fire one AP (or even 5AP's at 50Hz in some) is not an actual test of lateral inhibition mediated by NE volume neurotransmission in the LC. Strong claims that "chemical transmission...was not detected" require substantial support and testing of a range of AP frequencies and durations. Given the wealth of evidence supporting lateral inhibition of the LC, this claim seems unwarranted.
Point 2: Train-like connection pattern
Demonstrating that connected cell pairs often share a common member is an important demonstration of a connection motif in the LC. However, a "train" connection implies that you can pass from A to B to C to D (and in reverse). However, the authors do not do an analysis to test whether this occurs. Therefore, "train" is not an appropriate term to describe the interesting connection motif that they observed.In fact, writing A↔B↔C in the paper implies a train without direct support for that form of electrical transmission. For example, in Fig. 6C, it is clear that cell 6 is coupled to cell 1 and that cell 6 is also coupled to cell 8. In both cases, the connection is bilateral. Using the author's formatting of A↔B↔C , would correspond with Cell 6 being B and cells 1 and 8 being A and C (or vice versa). However, writing A↔B↔C implies a train, whereas one can instead draw this connection pattern where B is a common source:
A C
. .
. .
BAn analysis showing that spikes in A can pass through B and later appear in C is necessary to support the use of "train". The example in Fig. 6C argues against train at least for this one example.
Although the analysis is possible to do with the authors' substantial and unique data set, it should be also noted that prior work on putative electrical coupling in extracellular recordings from rat LC showed that trains among 3 single units occurred at an almost negligible rate because out of 12 rats "Only 1 triplet out of 22,100 possible triplet patterns (0.005%) was found in one rat and 4 triplets out of 1,330 possible triplet patterns (0.301%) were found in the other rat." and moreover patterns beyond 3 units were never observed (Totah et al 2018. Neuron 99, 1055-1068.e6).
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Reviewer #2 (Public Review):
McKinney et al set out to better understand local circuit organization within the mouse locus coeruleus (LC). To do so, the authors achieved the technical feat of performing multiple, simultaneous whole-cell recordings (up to 8 LC neurons at once). This approach gives the authors a powerful and relatively high throughput means of assessing LC neuronal activity and potentially its rate of interconnectedness. In addition to recording from these cells, many were also filled with biocytin to recover their morphology. Using traditional reconstruction approaches the authors identified two morphological classes of LC neurons, fusiform(FF) and multipolar (MP). Although the selection of these classes was biased from previous literature, the authors used machine classification to rigorously demonstrate that these …
Reviewer #2 (Public Review):
McKinney et al set out to better understand local circuit organization within the mouse locus coeruleus (LC). To do so, the authors achieved the technical feat of performing multiple, simultaneous whole-cell recordings (up to 8 LC neurons at once). This approach gives the authors a powerful and relatively high throughput means of assessing LC neuronal activity and potentially its rate of interconnectedness. In addition to recording from these cells, many were also filled with biocytin to recover their morphology. Using traditional reconstruction approaches the authors identified two morphological classes of LC neurons, fusiform(FF) and multipolar (MP). Although the selection of these classes was biased from previous literature, the authors used machine classification to rigorously demonstrate that these classes indeed exist. From there, the electrical properties of these distinct LC neurons were compared and a number of distinct action potential properties were identified between the two groups. Although firing in response to injected current showed that the FF class could maintain a higher firing rate, basal firing was not explicitly compared as the cells were prevented from firing upon entering whole-cell. The authors next explored the extent to which local chemical transmission occurs within the LC. Although there is evidence of glutamatergic transmission from LC neurons, the authors did not directly observe any evidence of local glutamate release from these neurons. This effect might be expected given the severing of axons in the slice preparation. Somewhat less expected is the author's claim that they could not find evidence of local NE release via alpha2 adrenergic receptor activation. This lack of evidence might well arise because this phenomenon does not occur, but it also remains possible that we do not have sufficient understanding of volume transmission to properly detect a change, particularly in whole-cell current clamp. The evidence that alpha2-mediated hyperpolarization is intact is somewhat adjacent to the concept as the concentrations of NE and clonidine used to show this robust suppression of firing is well above what is likely physiologically released by these neurons. One thing the authors do not consider is that the slice orientation (horizontal vs. coronal) greatly alters local glutamatergic input to the point that glutamate-mediated phasic bursts often do not occur in horizontal slices.
A major strength of the multi-patch approach used here is the ability to identify electrical connections between LC neurons. While gap junction-coupling has long been established in these neurons, multiple reports suggest that this coupling is decreased as the animal matures into adulthood. Here the authors provide clear evidence for a stable rate of electrical coupling well into adulthood. This approach also gives the authors the relatively unique ability to look for second-order connections between LC neurons and the amount of coupling was elegantly used to model how the LC might wire together more broadly. Although this approach is very powerful and likely at the edge of what is physically possible for whole-cell recordings in this brain structure it still likely undersamples LC local circuitry and biases investigations to be relatively close to one another spatially. While the authors rightfully consider the intersoma distance (ISD), the longest the gets in these studies is still smaller than most anatomical axes of the LC. This is an important limitation because the electrical coupling between FF-FF and FF-MP both appear to increase as ISD increases, suggesting more coupling could be occurring in distal dendrites. Furthermore, if coupling is occurring in distal dendrites it may be harder to detect as shunting in these distal dendrites could prevent signal detection.
This work is timely and important to the LC field which is on the precipice of having a greater understanding of heterogeneity based on a number of different principles, and this work adds local circuit dynamics as one of these principles. It will be important for the field to see how different efferent anatomical modules align with the cell types and circuit properties identified here.
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