Bayesian approach for sample size determination, illustrated with Soil Health Card data of Andhra Pradesh (India).

A crucial decision in designing a spatial sample for soil survey is the number of sampling locations required to answer, with sufficient accuracy and precision, the questions posed by decision makers at different levels of geographic aggregation. In the Indian Soil Health Card (SHC) scheme, many thousands of locations are sampled per district. In this paper the SHC data are used to estimate the mean of a soil property within a defined study area, e.g., a district, or the areal fraction of the study area where some condition is satisfied, e.g., exceedence of a critical level. The central question is whether this large sample size is needed for this aim. The sample size required for a given maximum length of a confidence interval can be computed with formulas from classical sampling theory, using a prior estimate of the variance of the property of interest within the study area. Similarly, for the areal fraction a prior estimate of this fraction is required. In practice we are uncertain about these prior estimates, and our uncertainty is not accounted for in classical sample size determination (SSD). This deficiency can be overcome with a Bayesian approach, in which the prior estimate of the variance or areal fraction is replaced by a prior distribution. Once new data from the sample are available, this prior distribution is updated to a posterior distribution using Bayes' rule. The apparent problem with a Bayesian approach prior to a sampling campaign is that the data are not yet available. This dilemma can be solved by computing, for a given sample size, the predictive distribution of the data, given a prior distribution on the population and design parameter. Thus we do not have a single vector with data values, but a finite or infinite set of possible data vectors. As a consequence, we have as many posterior distribution functions as we have data vectors. This leads to a probability distribution of lengths or coverages of Bayesian credible intervals, from which various criteria for SSD can be derived. Besides the fully Bayesian approach, a mixed Bayesian-likelihood approach for SSD is available. This is of interest when, after the data have been collected, we prefer to estimate the mean from these data only, using the frequentist approach, ignoring the prior distribution. The fully Bayesian and mixed Bayesian-likelihood approach are illustrated for estimating the mean of log-transformed Zn and the areal fraction with Zn-deficiency, defined as Zn concentration <0.9 mg kg -1, in the thirteen districts of Andhra Pradesh state. The SHC data from 2015-2017 are used to derive prior distributions. For all districts the Bayesian and mixed Bayesian-likelihood sample sizes are much smaller than the current sample sizes. The hyperparameters of the prior distributions have a strong effect on the sample sizes. We discuss methods to deal with this. Even at the mandal (sub-district) level the sample size can almost always be reduced substantially. Clearly SHC over-sampled, and here we show how to reduce the effort while still providing information required for decision-making. R scripts for SSD are provided as supplementary material.

Agricultural labor, COVID-19, and potential implications for food security and air quality in the breadbasket of India.

To contain the COVID-19 pandemic, India imposed a national lockdown at the end of March 2020, a decision that resulted in a massive reverse migration as many workers across economic sectors returned to their home regions. Migrants provide the foundations of the agricultural workforce in the 'breadbasket' states of Punjab and Haryana in Northwest India.There are mounting concerns that near and potentially longer-term reductions in labor availability may jeopardize agricultural production and consequently national food security. The timing of rice transplanting at the beginning of the summer monsoon season has a cascading influence on productivity of the entire rice-wheat cropping system. To assess the potential for COVID-related reductions in the agriculture workforce to disrupt production of the dominant rice-wheat cropping pattern in these states, we use a spatial ex ante modelling framework to evaluate four scenarios representing a range of plausible labor constraints on the timing of rice transplanting. Averaged over both states, results suggest that rice productivity losses under all delay scenarios would be low as compare to those for wheat, with total system productivity loss estimates ranging from 9%, to 21%, equivalent to economic losses of USD $674 m to $1.48 billion. Late rice transplanting and harvesting can also aggravate winter air pollution with concomitant health risks. Technological options such as direct seeded rice, staggered nursery transplanting, and crop diversification away from rice can help address these challenges but require new approaches to policy and incentives for change.

Indian agriculture, air pollution, and public health in the age of COVID.

Emerging evidence supports the intuitive link between chronic health conditions associated with air pollution and the vulnerability of individuals and communities to COVID-19. Poor air quality already imposes a highly significant public health burden in Northwest India, with pollution levels spiking to hazardous levels in November and early December when rice crop residues are burned. The urgency of curtailing the COVID-19 pandemic and mitigating a potential resurgence later in the year provides even more justification for accelerating efforts to dramatically reduce open agricultural burning in India.

Taking the climate risk out of transplanted and direct seeded rice: Insights from dynamic simulation in Eastern India.

Rice productivity in Eastern Indo-Gangetic plains (EIGP) is extremely low, in part due to the prevailing practice of cultivating long-duration transplanted rice under rainfed conditions which leads to water stress and significant yield losses in many seasons. Rice establishment alternatives such as direct seeded rice (DSR) require less water at planting but also are accompanied by climate risks that constrain adoption. For both conventional transplanted and DSR systems, successfully addressing climate-based production risks may provide a strong basis for sustainable rice intensification in EIGP. In this ex ante study of rice yield and yield variability, the APSIM cropping system model was used to evaluate the efficacy of risk-reducing management practices in both transplanted and DSR systems. Simulations were conducted with 44 years (1970-2013) of historical weather data from central Bihar, India. Results confirm that the prevailing farmer practice of transplanting long-duration cultivars under rainfed conditions (fTR) often results in delayed transplanting and the use of older seedlings, leading to low (median 1.6 t ha-1) and variable (Standard deviation (SD) 2.1 t ha-1) rice yields. To improve the fTR system, simulations suggest that adoption of medium-duration hybrid rice (3.2 t ha-1), provision of supplemental post-establishment irrigation (3.2 t ha-1), or transplanting appropriately aged seedlings (3.4 t ha -1) can double yields as single interventions while, in the case of supplemental irrigation, significantly reducing inter-annual production variability. Additional gains are achievable when interventions are layered: supplemental irrigation paired with medium-duration hybrids increased median rice yields to 4.6 t ha-1 with much lower variability (SD 1.0 t ha-1). In these improved systems where irrigation is used to transplant the crop, simulations revealed the importance of timely planting: high and stable yields are achievable for long-duration cultivars when transplanting is completed by 2 August with this window of opportunity extending to 16 August for medium-duration hybrids. In rainfed DSR systems, the potential pay-offs from single interventions were even higher with medium-duration hybrids resulting in a median yield of 4.5 t ha-1 against 1.8 t ha-1 with long-duration cultivars. For irrigated DSR systems, an optimum sowing window of early to mid-June was identified which resulted in higher and more stable yields with lower water requirements. Simulation results suggest several risk-reducing intensification pathways that can be selectively matched to farmer risk preferences and investment capabilities within the target region in EIGP.

Mu opioid receptor localization in the basolateral amygdala: An ultrastructural analysis.

Receptor binding studies have shown that the density of mu opioid receptors (MORs) in the basolateral amygdala is among the highest in the brain. Activation of these receptors in the basolateral amygdala is critical for stress-induced analgesia, memory consolidation of aversive events, and stress adaptation. Despite the importance of MORs in these stress-related functions, little is known about the neural circuits that are modulated by amygdalar MORs. In the present investigation light and electron microscopy combined with immunohistochemistry was used to study the expression of MORs in the anterior basolateral nucleus (BLa). At the light microscopic level, light to moderate MOR-immunoreactivity (MOR-ir) was observed in a small number of cell bodies of nonpyramidal interneurons and in a small number of processes and puncta in the neuropil. At the electron microscopic level most MOR-ir was observed in dendritic shafts, dendritic spines, and axon terminals. MOR-ir was also observed in the Golgi apparatus of the cell bodies of pyramidal neurons (PNs) and interneurons. Some of the MOR-positive (MOR+) dendrites were spiny, suggesting that they belonged to PNs, while others received multiple asymmetrical synapses typical of interneurons. The great majority of MOR+ axon terminals (80%) that formed synapses made asymmetrical (excitatory) synapses; their main targets were spines, including some that were MOR+. The main targets of symmetrical (inhibitory and/or neuromodulatory) synapses were dendritic shafts, many of which were MOR+, but some of these terminals formed synapses with somata or spines. All of our observations were consistent with the few electrophysiological studies which have been performed on MOR activation in the basolateral amygdala. Collectively, these findings suggest that MORs may be important for filtering out weak excitatory inputs to PNs, allowing only strong inputs or synchronous inputs to influence pyramidal neuronal firing.

Extrinsic origins of the somatostatin and neuropeptide Y innervation of the rat basolateral amygdala.

The amygdalar basolateral nuclear complex (BLC) is a cortex-like structure that receives inputs from many cortical areas. It has long been assumed that cortico-amygdalar projections, as well as inter-areal intracortical connections, arise from cortical pyramidal cells. However, recent studies have shown that GABAergic long-range nonpyramidal neurons (LRNP neurons) in the cortex also contribute to inter-areal connections. The present study combined Fluorogold (FG) retrograde tract tracing with immunohistochemistry for cortical nonpyramidal neuronal markers to determine if cortical LRNP neurons project to the BLC in the rat. Injections of FG into the BLC produced widespread retrograde labeling in the cerebral hemispheres and diencephalon. Triple-labeling for FG, somatostatin (SOM), and neuropeptide Y (NPY) revealed a small number of FG+/SOM+/NPY+ neurons and FG+/SOM+/NPY- neurons in the lateral entorhinal area, amygdalopiriform transition area, and piriform cortex, but not in the prefrontal and insular cortices, or in the diencephalon. In addition, FG+/SOM+/NPY+ neurons were observed in the amygdalostriatal transition area and in a zone surrounding the intercalated nuclei. About half of the SOM+ neurons in the lateral entorhinal area labeled by FG were GABA+. FG+ neurons containing parvalbumin were only seen in the basal forebrain, and no FG+ neurons containing vasoactive intestinal peptide were observed in any brain region. Since LRNP neurons involved in corticocortical connections are critical for synchronous oscillations that allow temporal coordination between distant cortical regions, the LRNP neurons identified in this study may play a role in the synchronous oscillations of the BLC and hippocampal region that are involved in the retrieval of fear memories.

GABAergic somatostatin-immunoreactive neurons in the amygdala project to the entorhinal cortex.

The entorhinal cortex and other hippocampal and parahippocampal cortices are interconnected by a small number of GABAergic nonpyramidal neurons in addition to glutamatergic pyramidal cells. Since the cortical and basolateral amygdalar nuclei have cortex-like cell types and have robust projections to the entorhinal cortex, we hypothesized that a small number of amygdalar GABAergic nonpyramidal neurons might participate in amygdalo-entorhinal projections. To test this hypothesis we combined Fluorogold (FG) retrograde tract tracing with immunohistochemistry for the amygdalar nonpyramidal cell markers glutamic acid decarboxylase (GAD), parvalbumin (PV), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and the m2 muscarinic cholinergic receptor (M2R). Injections of FG into the rat entorhinal cortex labeled numerous neurons that were mainly located in the cortical and basolateral nuclei of the amygdala. Although most of these amygdalar FG+ neurons labeled by entorhinal injections were large pyramidal cells, 1-5% were smaller long-range nonpyramidal neurons (LRNP neurons) that expressed SOM, or both SOM and NPY. No amygdalar FG+ neurons in these cases were PV+ or VIP+. Cell counts revealed that LRNP neurons labeled by injections into the entorhinal cortex constituted about 10-20% of the total SOM+ population, and 20-40% of the total NPY population in portions of the lateral amygdalar nucleus that exhibited a high density of FG+ neurons. Sixty-two percent of amygdalar FG+/SOM+ neurons were GAD+, and 51% were M2R+. Since GABAergic projection neurons typically have low perikaryal levels of GABAergic markers, it is actually possible that most or all of the amygdalar LRNP neurons are GABAergic. Like GABAergic LRNP neurons in hippocampal/parahippocampal regions, amygdalar LRNP neurons that project to the entorhinal cortex are most likely involved in synchronizing oscillatory activity between the two regions. These oscillations could entrain synchronous firing of amygdalar and entorhinal pyramidal neurons, thus facilitating functional interactions between them, including synaptic plasticity.

Noradrenergic innervation of pyramidal cells in the rat basolateral amygdala.

The basolateral nuclear complex of the amygdala (BLC) receives dense noradrenergic/norepinephrine (NE) inputs from the locus coeruleus that play a key role in modulating emotional memory consolidation. Knowledge of the extent of synapse formation by NE inputs to the BLC, as well as the cell types innervated, would contribute to an understanding of how NE modulates the activity of the BLC. To gain a better understanding of NE circuits in the BLC, dual-label immunohistochemistry was used at the light and electron microscopic levels in the present study to analyze NE axons and their innervation of pyramidal cells in the anterior subdivision of the basolateral amygdalar nucleus (BLa). NE axons and BLa pyramidal cells were labeled using antibodies to the norepinephrine transporter (NET) and Ca(2+)/calmodulin-dependent protein kinase (CaMK), respectively. Dual localization studies using antibodies to NET and dopamine-beta-hydroxylase (DBH) revealed that virtually all NE axons and varicosities expressed both proteins. The BLa exhibited a medium density of NET+ fibers. Ultrastructural analysis of serial section reconstructions of NET+ axons revealed that only about half of NET+ terminals formed synapses. The main postsynaptic targets were small-caliber CAMK+ dendritic shafts and spines of pyramidal cells. A smaller number of NET+ terminals formed synapses with unlabeled cell bodies and dendrites. These findings indicate that the distal dendritic domain of BLa pyramidal cells is the major target of NE terminals in the BLa, and the relatively low synaptic incidence suggests that diffusion from non-synaptic terminals may be important for noradrenergic modulation of the BLa.

Medial prefrontal cortical innervation of the intercalated nuclear region of the amygdala.

The projections of the infralimbic area (IL) of the medial prefrontal cortex to the intercalated nuclei (ICNs) of the amygdala are thought to form a critical component of the forebrain circuitry for fear extinction. Despite the importance of these projections, there have been no focussed anatomical studies that have investigated the extent of IL inputs to different portions of the ICN complex. The present investigation used anterograde tract tracing in the rat to study the projections of the ventromedial PFC, including the IL, to the ICNs and surrounding amygdalar regions. Immunohistochemistry for the µ-opioid receptor (MOR) was used to identify the ICNs. At rostral levels of the amygdala there was a very dense projection to a far lateral portion of the capsular subdivision of the central nucleus (CLC) located between the main and medial ICNs, but only very light projections to these ICNs and the lateral ICNs. This distinct portion of the CLC receiving strong IL inputs was termed the capsular infralimbic target zone (CITZ), and was MOR-negative. Likewise, at more caudal levels of the amygdala, IL projections to the medial, lateral, and dorsal ICNs were light to moderate compared with projections to adjacent portions of the basolateral amygdala and amygdalostriatal transitional area. These findings suggest that the putative role of the IL-to-ICN connection in fear inhibition may be mediated by light to moderate projections from the IL to the medial ICN, and that the CITZ may be an equally important amygdalar target for this function.

Neuronal localization of M2 muscarinic receptor immunoreactivity in the rat amygdala.

Muscarinic cholinergic neurotransmission in the amygdala is critical for memory consolidation in emotional/motivational learning tasks, but little is known about the neuronal distribution of different receptor subtypes. Immunohistochemistry was used in the present investigation to localize the m2 receptor (M2R). Differential patterns of M2R-immunoreactivity (M2R-ir) were observed in the somata and neuropil of the various amygdalar nuclei. Neuropilar M2R-ir was strongest in rostral portions of the basolateral nuclear complex (BLC). M2R-positive (M2R+) somata were seen in low numbers in all nuclei of the amygdala. Most M2R+ neurons associated with the BLC were in the lateral nucleus and external capsule. These cells were nonpyramidal neurons that contained glutamatic acid decarboxylase (GAD), somatostatin (SOM), and neuropeptide Y (NPY), but not parvalbumin (PV), calretinin (CR), or cholecystokinin (CCK). Little or no M2R-ir was observed in GAD+, PV+, CR+, or CCK+ axons in the BLC, but it was seen in some SOM+ axons and many NPY+ axons. M2R-ir was found in a small number of spiny and aspiny neurons of the central nucleus that were mainly located along the lateral and ventral borders of its lateral subdivision. Many of these cells contained SOM and NPY. M2R+ neurons were also seen in the medial nucleus, including a distinct subpopulation of neurons that surrounded its anteroventral subdivision. The latter neurons were negative for all neuronal markers analyzed. The intercalated nuclei (INs) were associated with two types of large M2R+ neurons, spiny and aspiny. The small principal neurons of the INs were M2R-negative. The somata and dendrites of the large spiny neurons, which were actually found in a zone located just outside of the rostral INs, expressed SOM and NPY, but not GAD. These findings indicate that acetylcholine can modulate a variety of discrete neuronal subpopulations in various amygdalar nuclei via M2Rs, especially neurons that express SOM and NPY.

Postsynaptic targets of GABAergic basal forebrain projections to the basolateral amygdala.

Recent studies indicate that the basolateral amygdala, like the neocortex and hippocampus, receives GABAergic inputs from the basal forebrain in addition to the well-established cholinergic inputs. Since the neuronal targets of these inputs have yet to be determined, it is difficult to predict the functional significance of this innervation. The present study addressed this question in the rat by employing anterograde tract tracing combined with immunohistochemistry at the light and electron microscopic levels of analysis. Amygdalopetal axons from the basal forebrain mainly targeted the basolateral nucleus (BL) of the amygdala. The morphology of these axons was heterogeneous and included GABAergic axons that contained vesicular GABA transporter protein (VGAT). These axons, designated type 1, exhibited distinctive large axonal varicosities that were typically clustered along the length of the axon. Type 1 axons formed multiple contacts with the cell bodies and dendrites of parvalbumin-containing (PV+) interneurons, but relatively few contacts with calretinin-containing and somatostatin-containing interneurons. At the ultrastructural level of analysis, the large terminals of type 1 axons exhibited numerous mitochondria and were densely packed with synaptic vesicles. Individual terminals formed broad symmetrical synapses with BL PV+ interneurons, and often formed additional symmetrical synapses with BL pyramidal cells. Some solitary type 1 terminals formed symmetrical synapses solely with BL pyramidal cells. These results suggest that GABAergic neurons of the basal forebrain provide indirect disinhibition, as well as direct inhibition, of BL pyramidal neurons. The possible involvement of these circuits in rhythmic oscillations related to emotional learning, attention, and arousal is discussed.

Parvalbumin-immunoreactive neurons and GABAergic neurons of the basal forebrain project to the rat basolateral amygdala.

The basal forebrain (BF) contains a diffuse array of cholinergic and non-cholinergic neurons that project to the cerebral cortex and basolateral nuclear complex of the amygdala (BLC). Previous studies have shown that the GABAergic subpopulation of non-cholinergic corticopetal BF neurons selectively innervates cortical interneurons. Although several investigations in both rodents and primates have indicated that some BF neurons projecting to the BLC are non-cholinergic, there have been no studies that have attempted to identify the neurochemical phenotype(s) of these neurons. The present study combined Fluorogold retrograde tract tracing with immunohistochemistry for two markers of BF GABAergic neurons, parvalbumin (PV) or glutamic acid decarboxylase (GAD), to determine if a subpopulation of BF GABAergic cells projects to the BLC. Injections of Fluorogold confined to the rat BLC, and centered in the basolateral nucleus, produced extensive retrograde labeling in the ventral pallidum and substantia innominata regions of the BF. Although the great majority of retrogradely labeled neurons were not double-labeled, about 10% of these neurons, located mainly along the ventral aspects of the fundus striati and globus pallidus, exhibited immunoreactivity for PV or GAD. The results of this investigation contradict the long-held belief that there is no extra-amygdalar source of GABAergic inputs to the BLC, and indicate that the cortex-like BLC, in addition to the cortex proper, receives inhibitory inputs from the basal forebrain.

Immunohistochemical characterization of parvalbumin-containing interneurons in the monkey basolateral amygdala.

Interneurons expressing the calcium-binding protein parvalbumin (PV) are a critical component of the inhibitory circuitry of the basolateral nuclear complex (BLC) of the mammalian amygdala. These neurons form interneuronal networks interconnected by chemical and electrical synapses, and provide a strong perisomatic inhibition of local pyramidal projection neurons. Immunohistochemical studies in rodents have shown that most parvalbumin-positive (PV+) cells are GABAergic interneurons that co-express the calcium-binding protein calbindin (CB), but exhibit no overlap with interneuronal subpopulations containing the calcium-binding protein calretinin (CR) or neuropeptides. Despite the importance of identifying interneuronal subpopulations for clarifying the major players in the inhibitory circuitry of the BLC, very little is known about these subpopulations in primates. Therefore, in the present investigation dual-labeling immunofluorescence histochemical techniques were used to characterize PV+ interneurons in the basal and lateral nuclei of the monkey amygdala. These studies revealed that 90-94% of PV+ neurons were GABA+, depending on the nucleus, and that these neurons constituted 29-38% of the total GABAergic population. CB+ and CR+ interneurons constituted 31-46% and 23-27%, respectively, of GABAergic neurons. Approximately one quarter of PV+ neurons contained CB, and these cells constituted one third of the CB+ interneuronal population. There was no colocalization of PV with the neuropeptides somatostatin or cholecystokinin, and virtually no colocalization with CR. These data indicate that the neurochemical characteristics of the PV+ interneuronal subpopulation in the monkey BLC are fairly similar to those seen in the rat, but there is far less colocalization of PV and CB in the monkey. These findings suggest that PV+ neurons are a discrete interneuronal subpopulation in the monkey BLC and undoubtedly play a unique functional role in the inhibitory circuitry of this brain region.

Dopaminergic innervation of interneurons in the rat basolateral amygdala.

The basolateral nuclear complex of the amygdala (BLC) receives a dense dopaminergic innervation that plays a critical role in the formation of emotional memory. Dopamine has been shown to influence the activity of BLC GABAergic interneurons, which differentially control the activity of pyramidal cells. However, little is known about how dopaminergic inputs interface with different interneuronal subpopulations in this region. To address this question, dual-labeling immunohistochemical techniques were used at the light and electron microscopic levels to examine inputs from tyrosine hydroxylase-immunoreactive (TH+) dopaminergic terminals to two different interneuronal populations in the rat basolateral nucleus labeled using antibodies to parvalbumin (PV) or calretinin (CR). The basolateral nucleus exhibited a dense innervation by TH+ axons. Partial serial section reconstruction of TH+ terminals found that at least 43-50% of these terminals formed synaptic junctions in the basolateral nucleus. All of the synapses examined were symmetrical. In both TH/PV and TH/CR preparations the main targets of TH+ terminals were spines and distal dendrites of unlabeled cells. In sections dual-labeled for TH/PV 59% of the contacts of TH+ terminals with PV+ neurons were synapses, whereas in sections dual-labeled for TH/CR only 13% of the contacts of TH+ terminals with CR+ cells were synapses. In separate preparations examined in complete serial sections for TH+ basket-like innervation of PV+ perikarya, most (76.2%) of TH+ terminal contacts with PV+ perikarya were synapses. These findings suggest that PV+ interneurons, but not CR+ interneurons, are prominent synaptic targets of dopaminergic terminals in the BLC.

Neuronal localization of 5-HT type 2A receptor immunoreactivity in the rat basolateral amygdala.

Although it is well established that there are alterations in type 2A 5-HT receptors (5-HT2ARs) in the basolateral nuclear complex of the amygdala (BLC) in several neuropsychiatric disorders, very little is known about the neuronal localization of these receptors in this brain region. Single-labeling and dual-labeling immunohistochemical techniques were utilized in the rat to address this question. Three different 5-HT2AR antibodies were used, each producing distinct but overlapping patterns of immunostaining. Two of three 5-HT2AR antibodies mainly stained pyramidal projection neurons in the BLC. The third antibody only stained pyramidal cells in the dorsolateral subdivision of the lateral amygdalar nucleus. With one of the antibodies, the most intensely stained neurons were a population of large nonpyramidal neurons whose morphology and distribution closely resembled those shown in previous studies to project to the mediodorsal thalamic nucleus (MD). This was confirmed in the present study using a technique that combined 5-HT2AR immunohistochemistry with fluorogold retrograde tract-tracing. Two of three 5-HT2AR antibodies stained large numbers of parvalbumin-containing interneurons in the BLC. One of these two antibodies also stained a subpopulation of somatostatin-containing neurons. None of the 5-HT2AR antibodies stained significant numbers of the other two main interneuronal subpopulations, the large cholecystokinin-positive neurons or the small interneurons that exhibit extensive colocalization of calretinin and cholecystokinin. Since each of the three antibodies was raised against a distinct immunizing antigen, they may recognize different conformations of 5-HT2AR in different neuronal domains. The expression of 5-HT2ARs in pyramidal cells and parvalbumin-positive interneurons in the BLC is consistent with the results of previous electrophysiological studies, and suggests that 5-HT may produce excitation of several neuronal populations in the BLC via 5-HT2ARs.

A novel subpopulation of 5-HT type 3A receptor subunit immunoreactive interneurons in the rat basolateral amygdala.

The amygdalar basolateral nuclear complex (BLC) has very high levels of the 5-HT type 3 receptor (5-HT(3)R). Previous studies have reported that 5-HT(3)R protein in the BLC is expressed in interneurons and that 5-HT(3)R mRNA is coexpressed with GABA and certain neuropeptides or calcium-binding proteins in these cells. However, there have been no detailed descriptions of the distribution of 5-HT(3)R+ neurons in the rat amygdala, and no quantitative studies of overlap of neurons expressing 5-HT(3)R protein with distinct interneuronal subpopulations in the BLC. The present investigation employed dual-labeling immunohistochemistry using antibodies to the 5-HT-3A receptor subunit (5-HT(3A)R) and specific interneuronal markers to address these questions. These studies revealed that there was a moderate density of nonpyramidal 5-HT(3A)R+ neurons in the BLC at all levels of the amygdala. In addition, immunostained cells were also seen in anterior portions of the cortical and medial nuclei. Although virtually all 5-HT(3A)R+ neurons in the BLC were GABA+, very few expressed neuropeptide or calcium-binding protein markers for individual subpopulations. The main interneuronal marker expressed by 5-HT(3A)R+ neurons was cholecystokinin (CCK), but only 8-16% of 5-HT(3)R+ neurons in the BLC, depending on the nucleus, were CCK+. Most of these CCK+/5-HT(3A)R+ double-labeled neurons appeared to belong to the subpopulation of large type L CCK+ interneurons. Very few 5-HT(3A)R+ neurons expressed calretinin, vasoactive intestinal peptide, or parvalbumin, and none expressed somatostatin or calbindin. Thus, the great majority of neurons expressing 5-HT(3A)R protein appear to constitute a previously unrecognized subpopulation of GABAergic interneurons in the BLC.

Differential expression of Kv3.1b and Kv3.2 potassium channel subunits in interneurons of the basolateral amygdala.

The expression of Kv3.1 and Kv3.2 voltage-gated potassium channel subunits appears to be critical for high-frequency firing of many neuronal populations. In the cortex these subunits are mainly associated with fast-firing GABAergic interneurons containing parvalbumin or somatostatin. Since the basolateral nuclear complex of the amygdala contains similar interneurons, it is of interest to determine if these potassium channel subunits are expressed in these same interneuronal subpopulations. To investigate this issue, peroxidase and dual-labeling fluorescence immunohistochemistry combined with confocal laser scanning microscopy was used to determine which interneuronal subpopulations in the basolateral nuclear complex of the rat amygdala express Kv3.1b and Kv3.2 subunits. Antibodies to parvalbumin, somatostatin, calretinin, and cholecystokinin were used to label separate subsets of basolateral amygdalar interneurons. Examination of immunoperoxidase preparations suggested that the expression of both channels was restricted to nonpyramidal interneurons in the basolateral amygdala. Somata and proximal dendrites were intensely-stained, and axon terminals arising from presumptive basket cells and chandelier cells were lightly stained. Immunofluorescence observations revealed that parvalbumin+ neurons were the main interneuronal subpopulation expressing the Kv3.1b potassium channel subunit in the basolateral amygdala. More than 92-96% of parvalbumin+ neurons were Kv3.1b+, depending on the nucleus. These parvalbumin+/Kv3.1b+ double-labeled cells constituted 90-99% of all Kv3.1b+ neurons. Parvalbumin+ neurons were also the main interneuronal subpopulation expressing the Kv3.2 potassium channel subunit. More than 67-78% of parvalbumin+ neurons were Kv3.2+, depending on the nucleus. However, these parvalbumin+/Kv3.2+ double-labeled cells constituted only 71-81% of all Kv3.2+ neurons. Most of the remaining neurons with significant levels of the Kv3.2 subunit were somatostatin+ interneurons. These Kv3.2-containing somatostatin+ interneurons constituted 27-50% of the somatostatin+ population, depending on the nucleus in question. These data suggest that both fast-firing and burst-firing parvalbumin+ interneurons in the basolateral amygdala express the Kv3.1b subunit. The significance of Kv3.2 expression in some parvalbumin+ and somatostatin+ interneurons remains to be determined.

Do competition and herbivory alter the internal nitrogen dynamics of birch saplings?

Deciduous trees recycle nitrogen within their tissues. The aim of this study was to test the hypothesis that reductions in plant growth, caused by competition and herbivory, reduce the sink strength for N during autumn nutrient withdrawal, and reduce the storage capacity and hence the amount of N remobilized in the following spring. We used (15)N-labelled fertilizer to quantify N uptake, leaf N withdrawal and remobilization. Betula pubescens saplings were grown with either Molinia caerulea or Calluna vulgaris, and subjected to simulated browsing damage. Competition reduced B. pubescens leaf N withdrawal and remobilization, with C. vulgaris having a greater effect than M. caerulea. However, simulated browsing had no significant effect on sapling N dynamics. The patterns of leaf N withdrawal and remobilization closely followed sapling dry mass. We conclude that the effect of competition on sapling mass reduces their N-storage capacity. This reduces sink strength for leaf N withdrawal and the source strength for remobilized N. The ability of saplings to compensate for browsing damage removed any potential effect of browsing on N dynamics.

Quadrilateral space syndrome: a case study and review of the literature.

Quadrilateral space syndrome is an uncommon injury. The true prevalence is unknown because of a lack of literature and possible misdiagnosis. Prevalence may increase as knowledge of the syndrome increases. The case is presented of a recreational triathlete who had a spontaneous onset of quadrilateral space syndrome. The diagnosis was made by physical examination and confirmed with magnetic resonance imaging. A conservative, yet aggressive rehabilitation programme resulted in functional improvement within six weeks. Results have been maintained for eight weeks.

Localization of the CB1 type cannabinoid receptor in the rat basolateral amygdala: high concentrations in a subpopulation of cholecystokinin-containing interneurons.

The neuronal localization of the CB1 cannabinoid receptor in the rat basolateral amygdala was studied using peroxidase and fluorescence immunohistochemical techniques. All nuclei of the basolateral amygdala contained a large number of lightly stained pyramidal neurons and a small number of more intensely stained non-pyramidal neurons. Most of the latter cells had medium-sized to large multipolar somata and three to four aspiny dendrites, but some exhibited smaller oval somata. The axon initial segments of some of these non-pyramidal neurons exhibited large swollen varicosities in colchicine-injected animals, suggesting that much of the CB1 receptor protein is transported down the axons of these cells. Double-labeling studies using immunofluorescence histochemistry combined with confocal laser scanning microscopy revealed that the great majority of non-pyramidal neurons with CB1 receptor immunoreactivity belonged to a cholecystokinin-containing subpopulation. Whereas none of the other subpopulations of non-pyramidal neurons (exhibiting immunoreactivity for calretinin, parvalbumin, or somatostatin) expressed high levels of CB1 receptor immunoreactivity, a small percentage of these cells exhibited low levels of immunoreactivity. The results indicate that cannabinoids may modulate the activity of pyramidal projection neurons as well as a subpopulation of cholecystokinin-containing non-pyramidal neurons in the basolateral amygdala. Previous studies indicate that most of the latter are inhibitory interneurons that utilize GABA as a neurotransmitter. The intense staining of the cholecystokinin-containing interneurons and the evidence that large amounts of CB1 receptor protein are transported down the axons of these cells suggests that, as in the hippocampus, cannabinoids may inhibit the release of GABA from the axon terminals of these neurons.